REPLICATION AND PATHOGENESIS OF AN IRIDESCENT

VIRUS IN THE COTTON BOLL WEEVIL

by

CURTIS HENDERSON, B.S.

A DISSERTATION

IN

BIOLOGY

Submitted to the Graduate Faculty of Texas Tech University in

Partial Fulfillment of the Requirements for

the Degree of

DOCTOR OF PHILOSOPHY

Approved

December, 2000

ACKNOWLEDGEMENTS

This work was supported by grants to Dr. Shan Bilimoria from the Texas

Advanced Research Program (ARP), Texas Advanced Technology Program (ATP), the

Institute for Biotechnology at Texas Tech University, and the Vice Provost for Research

at Texas Tech University. In addition to Research Assistantships from the above ATP and

ARP grants, Teaching Assistantships and a Summer Research Award were received from

the Department of Biological Sciences.

I am gratefiil for the support, helpful suggestions, and friendship of my committee

members: Drs. Randy Allen, Michael San Francisco, S. Sridhara, and Hong Zhang.

I thank Dr. Susan D'Costa and Rajeswari Jayraman for their significant

contributions to the apoptosis research; Drs. Zihni Demirbag and Susan D'Costa for their

considerable help with the BIR research; and to Dr. P. K. Lawrence for assistance with

the final stages of the BIR analysis. I would also like to thank Sundus Lodhi and Cynthia

Johnson for their assistance with the viral replication work and Mark Grimson for his

technical assistance. I would also like to thank Dr. Susan San Francisco and Ruwanthi

Wettasinghe for all their help with protein and DNA sequencing, their technical e3q)ertise,

and desire to help in any way possible.

I sincerely thank my family for their love and support. My wife, Tracie, has

supported me with loyalty and love even in the worst of times and has still found the time

and energy to care for our daughters, Mackenzie and Emily. My parents have also given

me love and support, as well as the will and determination to succeed.

I also sincerely thank my advisor, Dr. Shan Bilimoria, for his helpful criticism,

guidance, support, encouragement, and friendship. He has provided me with an excellent

basis and exanqile for my professional career.

m

TABLE OF CONTENTS

ACKNOWLEDGEMENTS u

ABSTRACT be

LIST OF TABLES xi

LISTOFHOURES xii

LIST OF ABBREVIATIONS xiii

CHAPTER

L INTRODUCTION 1

Relevance 1

Literature Review of CIV 2

Taxonomy 3

Stmcture 3

Iridescent Vims Interaction vnth

Insect Hosts 4

Iridovirus Infection in Cell Culture 6

CIV Infectivity and Induction of Inhibition 7

Inhibition by Vimses 8

Apoptosis in Viral Infections II

Protein Kinase Involvement in

Inhibition and Apoptosis 13

BIR Kinase 14

Objectives 15 iv

References 19

n. REPLICATION OF CHILOIREDESCENT VIRUS IN THE COTTON BOLL WEEVIL, ANTHONOMUS GRANDIS, AND THE DEVELOPMENT OF AN INFECTIVITY ASSAY 32

Introduction 32

Materials and Methods 33

Vims Rearing 33

Vims Purification 34

Preparation of Infected Sanples 34

Viral DNA Replication 34

Electron Microscopy 35

Infectivity Assay 35

Dot Blot Analysis 36

Results 37

Evidence of Viral DNA Replication in Pupae 37

Formation of Con^lete Vims Particles 37

Infectivity of Progeny Vims 38

Discussion 39

References 44

III. INDUCTION OF APOPTOSIS AND INHIBITION OF PROTEIN SYNTHESIS BY A VIRION PROTEIN EXTRACT FROM CfflLO IRIDESCENT VIRUS 46

Introduction 46

Material and Methods 4g

V

Cell Line Maintenance 48

Virus Rearing 49

Virus Purification and Quantitation 49

Preparation of Virion Extract 49

Bradford Assay for Determination of

Protein Concentration 50

Apoptosis Assay 50

Endpoint Dilution Assay (Tissue Culture Toxicity

Dose, TCTDso) of Virion Extract 51

DNA Fragmentation Assay 52

Inhibition of Protein Synthesis 53

Results 53

Detection of Apoptotic Morphology 53

Endpoint Dilution Assay (Tissue Culture Toxicity

Dose, TCTDso) of Virion Extract 53

DNA Fragmentation in CF and AG Cells 54

Inhibition of Protein Synthesis 54

Discussion 55

References 65 TV. IDENTinCATION AND CHARACTERIZATION OF A CIV

OPEN READING FRAME WITH HIGH SIMILARITY TO THE VACCINIA VIRUS BIR GENE 69

Introduction 69

Materials and Methods 70

DNA Sequencing and Analysis 70 vi

PCR Air^lification for DNA Sequencing 70

Virus Infection and Passaging 71

RNA Isolation and Northern Blot Analysis 72

Resuhs 72

Anplification and Sequencing of the B1 R-Like ORF 72

Similarity and Analysis of the CIV

Open Reading Frame 73

Northern Analysis ofthe BIR-Like Gene 74

Discussion 74

References 83

V. IDENTMCATION OF KINASE ACTD/ITY IN SOLUBLE EXTRACTS OF CHILO IRIDESCENT VIRUS 85

Introduction 85

Materials and Methods 87

Virus Rearing 87

Virus Extraction and Quantitation 87

OGE Extract Preparation 87

CHAPS Extract Preparation 88

Fast Performance Liquid Chromatography 89

Kinase Assay 89

Amino-Terminal Protein Sequencing 90

Southern Blotting v dth an Oligonucleotide Probe 90

DNA Sequencing of the Candidate Region 91

vii

Results 91

Polypeptide Conqjosition of Soluble Extracts 91

Kinase Activity ofthe OGE and CHAPS Extracts 91

Analysis of FPLC Fractions 92

Polypeptide Sequencing and Application to

Gene Sequencing 92

Discussion 93

References 102

VI. CONCLUSIONS 104

viu

ABSTRACT

The boll weevil is a devastating pest of cotton and with increasing problems

relating to chemical control, it is clear that biological approaches must be developed.

Chilo iridescent virus (CIV) is the only vims known to infect the boll weevil, and research

in our laboratory has shown that CIV induces up to 70% mortality and deformity in

infected insects. The objectives ofthe present study were fourfold. The first objective

was to demonstrate a coirplete replication cycle of CIV in the boll weevil and develop an

infectivity assay. The second objective was to study inhibition of host protein synthesis

and induction of apoptosis caused by CIV virion extracts. The third objective was to

characterize a putative CIV homo log ofthe vaccinia virus BIR gene. The final objective

was to establish protein kinase activity with virion extracts.

Dot blot analysis of infected boll weevils provided stiong evidence of viral DNA

replication. Election microscopy established high levels of complete virus particles in

infected cells. An infectivity assay (using viral DNA replication as indicator) was

developed, and production of infectious progeny vims was demonstrated.

Extracts prepared from CIV severely reduced protein synthesis in treated boll

weevil and budworm cells as detected by SDS-PAGE analysis of labeled amino acid

uptake into cellular proteins. The virion extracts also induced apoptosis in treated cells,

which exhibited typical morphology of apoptosis as well as DNA fragmentatioa

Analysis of CIV DNA revealed the presence of an open reading frame with high

similarity to the vaccinia vims BIR protein kinase gene. The BIR gene has been

inqjlicated in the shutdown of host macromolecular synthesis in cells infected with vaccinia

ix

vims possibly via phosphorylation of ribosomal proteins. Analysis ofthe CIV sequence

revealed the two regions characteristic of protein kinases.

Finally, in vitro assays established protein kinase activity in virion extracts. This

activity was correlated with two polypeptides by FPLC. Our data suggest that CIV is a

promising source of genes for the genetic engineering of boll weevil-resistant cotton

plants.

LIST OF TABLES

1.1 Stmctural viral factors inducing apoptosis 17

1.2 Non-stmctural viral factors inducing apoptosis 18

3.1 TCTDso assay ofCIV extract in CF and AG cells 64

4.1 Similarity con^jarison of the CIV B IR-like polypeptide

to other known genes 82

5.1 Kinase activity ofthe OGE extract and controls 100

5.2 Kinase activity ofthe CHAPS extract and controls 101

XI

LIST OF FIGURES

2.1 Replication of Chilo iridescent vims (CIV) in boU weevil pupae 41

2.2 CIV particle formation in boll weevil pupae 42

2.3 End-point dilution assay and increase in infectious CrV titer in boll weevil pupae 43

3.1 Photomicrography of apoptotic morphology induced by a virion extract of CIV 60

3.2 DNA fra^entation, characteristic of apoptosis, observed in CF cells 61

3.3 DNA fragmentation, characteristic of apoptosis, observed in AG cells 62

3.4 Inhibition of proteui synthesis in CF and AG

cells induced by the virion extract 63

4.1 Primers used for PCR anplification 77

4.2 PCR anqjlification of the putative CW B1R homolog 78

4.3 Mapping and sequencing ofthe BlR-like gene in the CIV genome 79

4.4 Alignment of the CIV B1 R-like protein predicted amino acid sequence with that ofthe vaccinia BIR protein kinase 80

4.5 Expression of CIV B IR-like gene in infections

of 5/?L-^G-3^ cells 81

5.1 Polypeptide conposition of the octylglucoside extract 96

5.2 Polypeptide conqjosition ofthe CHAPS extract 97 5.3 Analysis of FPLC fractions from the

octylglucoside extract 98

5.4 Analysis of FPLC fractions from the CHAPS extract 99

6.1 Working model for induction of apoptosis by CIV 107

xu

LIST OF ABBREVIATIONS

(3-(3-Cholamidopropyl)-dimethylammonio)-1 -propane sul&te CHAPS

African swine fever vims ASFV

Autographa califomica multicapsid nuclear polyhedrosis virus AcMNPV

Cap-binding protein CBP

Chilo iridescent virus CIV

Coimts per minute cpm

Cytopathic effect CPE

Days post infection dpi

Deoxyribonucleic acid DNA

Ethylenediaminetetraacetic acid EDT A

Eukaryotic initiation factor eEF

Fast performance liquid chromotography FPLC

Fetal bovine serum FBS

Frog vims 3 FV3

Hours post infection h p.i.

Infectious unit lU

KUobase kb

Kilodalton kD

Major capsid protein MCP

Map units mu

xiii

Microgram |ig

Microliter |il

Milligram mg

Milliliter ml

MiUimolar mM

Molar M

Multiplicity of infection MOI

Octylglucoside OGE

Open reading frame ORF

Polymerase chain reaction PCR

Post infection p.i.

Ribonucleic acid RNA

RNA-dependant protein kinase PKR

Sodium dodecyl sulfate SDS

Tissue culture infectious dose-50 TCIDso

Tissue culture toxicity dose-50 TCTDso

Trichplusia ni medium formula Hanks TNM-FH

Tris[hydroxymethyl]aminomethane Tris

Ultraviolet UV

Virion-associated protein VAP

Virion host shutoff vhs

xiv

CHAPTER I

INTRODUCTION

Relevance

The boll weevil (Anthonomus grandis Boheman: Coleoptera: Circulionidae) is a

devastating pest of cotton in the Western Hemisphere causing over $300 million in

damages annually in the United States alone [139]. Dependence upon chemical contiol

is becoming increasingly problematic since resistance to pyrethroid insecticides continues

to build [56, 100]. It is also becoming more difficult to develop new chemicals to

combat the development of insecticide resistance [57}. In addition, chemical pesticides

may also harm the natural enemies of pest insects. Furthermore, the requirement for

precise timing of chemical application is difficult to achieve, particularly in developing

nations. Therefore, iimovation for alternative, biological control systems is critical [94,

110].

Several attempts have afready been made to develop biological contiol systems

for the boll weevil, ranging from the mass intioduction of beneficial insects,

development of resistant plants, and combinations of stiategies [1, 80, 88, 97, 132]. The

fungus, Beauveria bassiana, has been used in several field trials with some success [146],

and has even been marketed as a commercial product, Naturalis L®. However, fungal

pathogens will not be as effective in dry areas such as the South Plains of Texas, one of

the major cotton producing areas in the United States. The most popular current

approach to biocontiol is genetic engineering of tiansgenic plants. The delta-endotoxin

1

of Bacillus thuringiensis has been cloned into cotton to produce cultivars resistant to

lepidopteran pests, but this toxin is not effective against coleopteran pests such as the boll

weevil. Another gene of potential utility in developing resistant plants is the cholesterol

oxidase gene firom Streptomyces sp. [33, 58, 59, 112]. In addition, some insect vimses

are currentiy being used as effective biopesticides [75, 95, 96] and the use of viral

pesticides is likely to increase as the potency increases through genetic engineering [92,

104, 127, 131]. (CIV) has been shown to infect the boll weevil [49] and has also been

considered as a possible insect contiol agent for the green rice leafhopper, Nephtotettix

cincticeps,, and the leafhopper Colladonus montanus, in which it causes 99% mortality

[70]. In addition, our laboratory is studying the interaction ofthe cotton boll weevil with

CIV, the only virus known to induce mortality and metamorphic deformity in this host

[149]. This research represents cmcial research toward the development of CIV or its

components for pest contiol.

Literature Review of CIV

CIV was first isolated by Fukaya and Nasu [49] from the rice stem borer, Chilo

suppressalis (Lepidoptera) and naturally occurs in Japan and the United States. CIV is

also the type species for the small insect iridescent vimses (Family: Iridoviridae, Genus:

Iridovirus) [75]. These viruses received their name from the blue-green (small insect

iridovimses) or orange-yellow (large insect iridoviruses) color of heavily infected insects

and viral pellets. The color is due to Bragg diffraction of tightiy packed paracrystalline

arrays of vims particles [75]. Iridoviruses are complex cytoplasmic vimses with AT-

rich, linear, double-stranded DNA genomes (ca. 200 kb) with circular permutation and

2

terminal redundancy [41, 141, 142]. Altiiough basic information of tiiese viruses (such

as stmcture) has been acquired, little is known about the infectivity, repUcation, or

transmission of these vimses.

Taxonomy

CIV is a member ofthe family Iridoviridae, which are icosahedral, cytoplasmic

DNA vimses that can be subdivided into two invertebrate genera and two vertebrate

genera. The invertebrate genera include Chloriridovirus, which are large insect

iridovimses, 180-200 nm face-to-face dnd Iridovirus, the small insect iridovimses BO-

MO nm face-to-face. The vertebrate genera include tiie amphibian viruses, Ranavirus,

and the fish viruses. Goldfish virus-like viruses and Lymphocystivirus. CIV is the type

stiain for the Iridovirus genus.

Structure

Members ofthe genus Iridovirus have an icosahedral outer capsid and an electron

dense core separated by an intemal lipid membrane [75] consisting predominantly of

phospholipid [10, 73, 76] and differing in composition from host membranes [10, 141].

The capsid is largely composed of a single polypeptide unit referred to as the major

capsid protein (MCP). The MCP is approximately 50 kD and comprises 40-45% ofthe

total capsid polypeptide [27, 128]. The MCP forms two stmctures, one of which consists

of a homotrimer held together by hydrogen bonding at the particle surface. The second

stmcture also consists of a homotrimer, but is located beneath the surface layer [27]. As

mentioned previously, the intemal lipid membrane ofthe CIV particle differs in

3

composition from host cell membranes, suggesting that it is synthesized de novo.

Vaccinia vims has also been shown to synthesize a viral membrane, a process that is

facilitated by viral proteins [122]. However, the significance of a specific membrane for

the CIV particle has not been determined. The cential core ofthe CIV particle consists

of viral DNA associated with six proteins in a nucleosome-like stmcture. These proteins

range in size from 12.5 to 81 kD with the 12.5 kD protein being the predominant

member [26].

Iridescent Vims Interaction with Insect Hosts

The first Iridovirus was discovered in the larvafc ofthe crane fly, Tipula paludosa,

by Xeros in 1954 [147]. Since then, numerous other hosts have been identified including

insects from the Diptera (40 spp.), Coleoptera (8 spp.), and Lepidoptera (7 spp.), as well

as non-insect species, such as terrestrial isopods (8 spp.) [142]. However, despite the

potential use of CIV and other iridovimses in pest contiol, little has been discovered

about the ecology and pathogenicity of this genus. The apparent lack of interest in these

vimses was probably due to low incidence of lethal infections and difficulty with

tiansmission. Iridescent vims infections often produce covert infections, which do not

cause death in infected hosts. Much ofthe information available on ecology uses the

iridescent phenomenon as the sole criterion for infection [141]. This characteristic may

not be the most accurate means for determining infection because iridescence appears to

be a trivial characteristic, giving the virus no selective advantage, and ignores the

possibility of covert infections [75]. Very few studies have been performed on the

infectivity of the Iridovirus genus [17, 19, 65].

4

Studies have been performed in an attempt to examine iridovims ecology,

mechanisms of infection, and persistence in host populations [62, 76, 137], but a unifying

theme has yet to be established [75]. Evidence suggests that infection strategies depend

on the biology and life history ofthe respective host, making generalization difficult.

Another unknown factor is how infection is typically initiated. Many possibilities have

been proposed such as cannibalism of infected insects, cuticular lesions, parasitic vectors

(such as nematodes), and intioduction via spiracles. The theory of transmission by

cannibalism seems plausible in light ofthe tiemendous production of progeny virus in

infected insects. Remarkably, 25% (dry weight) of a dead insect may be vims [140]. In

addition to these, horizontal and vertical tiansmission" of virus has been demonstiated in

several systems. When mosquito larvae were infected (by feeding) slightiy before

pupation, the progeny of 19-47% females developed infection [145]. Other studies have

also reported evidence of tiansovarial infection [50, 86, 87].

Several studies have been performed regarding the persistence of iridovimses in

host populations [60, 61, 116, 119]. These seemed to indicate that in general,

iridoviruses replicate best in hosts found in damp areas such as ponds and marshes.

Results also showed that even geographical dispersal of an infected population and host

density within areas of concentiated host population have a bearing on the percentage of

hosts infected [61]. In some systems, infection reached up to 90% in host populations

[116].

Survival times after infection seem to be independent of dose [24] and vary

greatly between insect-virus systems. Survival after per os infection varied from 6 to 45

days in different systems [22, 48, 60, 61, 125, 126], whereas survival times were reduced

5

30-50% when inoculum was intioduced by injection [22, 23]. However, very high doses

resulted in paralysis and death without typical disease development [125, 137]. This

ability is probably due to the cytotoxic effects of iridoviruses [149].

Iridovirus Infection in Cell Culture

Larval-derived CIV has been shown to undergo a productive replication cycle in

several insect cells lines [42]. It was initially believed that Iridoviruses were able to

replicate independently ofthe nucleus, but later experiments using UV-irradiated and

enucleated cells showed that a fimctional nucleus is required at least for FV3 replication

[55]. Further examination showed the presence of viral nucleic acid in the nucleus of

infected cells [54]. FV3 DNA replication is now known to first occur in the nucleus in

units up to twice the genomic size. The DNA is then tiansported into the cytoplasm late

in infection to form large (lOX) concatemers [143]. The formation of concatemers is

most likely a mechanism for overcoming the 3' end-replication problem encoimtered by

viruses with linear, double-stianded DNA genomes. It is imclear at this point if this

DNA replication stiategy is applicable to the insect iridoviruses [79].

Transcription patterns ofthe Iridoviridae have been studied extensively in FV3.

Recently, our laboratory has demonstrated the existence of three distinct classes of viral

tianscripts [18,40] in CFV-infected cell cultures. Similar to other iridoviruses, CIV gene

expression is temporally regulated, and the tianscripts are classified as early, and late.

Early classes are expressed prior to DNA replication, whereas the late class has a

requirement for viral DNA replication [18,40, 144, 148]. The early classes are further

classified according to requirement for de novo protein synthesis. Genes expressed

6

witiiout de novo protein synthesis, are designated immediate early while those requiring

it are designated delayed early. Transfection with naked CIV DNA does not initiate

infection, indicating that a tiansacting protein is required for initiation of infection. In

CIV this putative polypeptide has been referred to as the virus associated polypeptide

(VAP) [27].

CIV Infectivity and Induction of Inhibition

Earlier work by McLaughlin [93] used the phenomenon of iridescence in infected

insects to show that CIV replicates in the boll weevil, Anthonomus grandis. In addition,

the identity of progeny vims was confumed after extraction from infected insects using

antibodies raised to the vims. This work also studied the persistence of CIV in artificial

bait used to infect boll weevils. The results showed that viral replication occurred in the

insect, but did not answer questions regarding the extent or efficiency of replication.

Several years later, Cemtti and Devauchelle reported that CIV induced a rapid inhibition

of DNA, RNA, and protein synthesis in multiple cell lines [25]. This effect was also

shown with a soluble extiact from the vims prepared by repeated freezing and thawing in

the presence of EDTA. Further work was performed on a soluble extract treatment of

virus with high salt and a nonionic detergent [27]. This extiact had several activities

associated with it: inhibition of host macromolecular synthesis, formation of syncitia in

cell culture, and stimulation of immediate early tianscription [27]. However, no further

information regarding inhibition by CIV has been published.

Inhibition by Vimses

In the process of taking over the repHcative machinery of a host cell, vimses often

induce a cytotoxic effect. This effect can be related to reduced macromolecular synthesis

as the gene expression of a virus dominates, but vimses often contain stmctural

polypeptides or express polypeptides tiiat are responsible for this effect. The best-studied

inhibition systems among DNA viruses are foimd in the Adenoviruses, Herpesvimses,

and the Poxviruses. In comparison, inhibition of gene expression in Iridoviruses have

only been studied to a limited extent.

Adenoviruses induce significant inhibition of DNA and protein synthesis in

infected cells, while their effect on RNA synthesis is les^ dramatic. By the time vims

DNA rephcation begins, host DNA synthesis is reduced by more then 90% [68]. There

is some evidence that this inhibition occurs at the level of initiation. Cells in the G phase

of their cycle were inhibited upon adenovims infection, whereas cells already in the S

phase were not [67]. Studies using temperature-sensitive viral mutants have shown that

viral DNA replication is not necessary for the inhibition of cellular DNA synthesis [44,

138]. This suggests that the inhibition is achieved by an early gene product, although

this product has not yet been identified. Inhibition of protein synthesis also reaches

levels of nearly 90% in infected cells, but steady state levels of mRNA are not

significantly reduced [3, 6, 78]. This suggests that infection induces alteration ofthe

translational apparatus and that viral transcripts are somehow able to overcome this

alteration. It was recently discovered that the cap-binding protein (CBP) in host cells

was inactivated by preventing the phosphorylation ofthe eIF-4E component [150].

Although this activity has not yet been isolated, the study indicates that this effect is

8

caused by an event late in the adenovims cycle. Late viral transcripts contain a tripartite

leader that is required for late, but not early, viral translation. This suggests that the

tripartite leader is the factor enabling translation of late viral genes under conditions of

reduced functional CBP levels.

Herpesvirus is able to block the synthesis of host DNA due to an early gene

product [16]. This inhibition occurs in the absence of viral DNA synthesis, but not in the

presence of cycloheximide [15]. The early product responsible for this effect has not yet

been identified. RNA synthesis is inhibited, but to a mild extent, well short of inhibition

levels observed with DNA and protein synthesis [5, 64]. Inhibition of protein synthesis

occurs in a multi-step process. The first step is initiated by the vhs (virion host shut-off)

stmctural polypeptide [45, 107]. The vhs protein degrades both host and early viral

mRNA. While degradation of viral mRNA may seem counter-productive, it may be a

mechanism for switching to late expression. The tianscription ofthe UL 41 gene

encoding the vhs protein is in tum regulated by the product ofthe UL 13 gene [102, 106].

The second step in inhibition of protein synthesis appears to be induced by an early gene

product [103]. Although details have not been established, there is evidence suggesting

that this step may involve inhibition of tianslation initiation [82].

The Poxvirus family is of particular interest to this work since both Poxviruses

and Iridoviruses are cytoplasmic DNA viruses. A majority ofthe research done

concerning inhibition of host gene expression induced by Poxviruses has been with

vaccinia virus. Infection induces rapid inhibition of host DNA synthesis, which is

believed to be the result of virion-associated DNases [111]. Inhibition of protein

synthesis in vaccinia vims-infected cells could be the result of multiple mechanisms.

9

Protein synthesis is reduced approximately 50% by 1 hour post infection [72]. This rapid

shut off suggests tiie role of a stmctiiral polypeptide [13, 99, 124], and surface tubules

have been implicated. In addition small, imtianslated polyA RNAs produced early in

infection [7, 91, 120] have also been implicated. Addition of oUgo dT or polyA binding

protein (PAB) prevented inhibition, suggesting that the shut-off was due to the polyA

RNA [129]. However, an 11 kD phosphoprotein has been isolated from vaccinia vims

that has the ability to inhibit formation ofthe 43 S tianslation initiation complex [14,

108]. Evidence for this is supported by the fact that early inhibition occurs

concomitantly with degradation of polyribosomes [120]. Therefore, the question of what

causes vaccinia vims-induced inhibition of host protein synthesis is still imanswered.

Other studies show that the BIR protein kinase of vaccinia virus phosphorylates

ribosomal proteins [12], and show that this enzyme is involved in the inhibition of host

protein synthesis [81].

Much of what is known about Iridovirus-induced inhibition of gene expression

has come from work with Frog vims 3 (FV3). Although the syntheses of both DNA and

RNA are inhibited, the mechanisms are unknown. Inhibition of protein synthesis in FV3

infection is due to interference with the host tianslational machinery. It has been shown

that FV3 induces phosphorylation ofthe initiation factor, eIF-2a [29, 30]. This

phosphorylation prevents the formation ofthe 43 S pre-initiation complex, thus inhibiting

protein synthesis. Translation of FV3 mRNA has been shown to be more efficient than

host mRNA, suggesting that viral messages outcompete host messages under the

conditions of limited tianslational machinery [30]. It has also been shown that

10

polyribosomes are dismpted in cells infected with FV3 as well as CIV [109, 114, 151].

In addition to dismption ofthe ttanslational complex, host mRNA is degraded in FV3-

infected cells [31], suggesting that this may also play a role in the inhibition of protein

synthesis. Stmctural polypeptides play a role in Iridovirus-induced inhibition of gene

expression, since soluble extiacts prepared from both FV3 and CIV have been shown to

induce inhibition [4, 25, 27, 36]. Interestingly, heat tieatment of FV3 does not prevent

the inhibition activity, suggesting that the inducing factor is either an unusually heat-

stable protein, or is composed of some other heat-stable macromolecule [53].

Conversely, inhibition of protein synthesis by CIV soluble extract is completely

prevented by heating for 30 minutes at 65 °C and drastically reduced after tieatment of

extiact with Proteinase K (Henderson, Jayaraman, D'Costa, and Bilimoria, in

preparation), suggesting protein involvement.

Mechanisms of virus-induced inhibition of gene expression, although driven by

many different products, typically share common themes. Recurrent themes include

degradation of host nucleic acids (FV3, herpesvims, and vaccinia virus) and interference

with host translation initiation complexes (FV3, vaccinia virus, and adenovims).

Although many viral host systems have evolved to include inhibition of host gene

expression, some vimses seem to have evolved the most appropriate common

mechanisms for inhibition.

Apoptosis in Viral Infections

Our laboratory has shown that in addition to inhibiting protein synthesis, soluble

extiacts from CIV particles also induce apoptosis (Henderson, Jayaraman, D'Costa,

11

BiWrnovia, publication in progress) [43, 69], selective cell death in response to a

stimulus. In apoptosis, cellular contents are packaged into discrete portions that are

easily removed from the system. This is in contiast to necrosis, which results in the

haphazard spilling of cellular contents (and toxic compounds) into the interstitial space.

Apoptosis also differs from programmed cell death in that it is induced by a variety of

external stimuli rather than as a result ofthe developmental process of an organism.

Stimuli able to induce apoptosis include, but are not limited to, UV radiation, DNA

damage, cell cycle dismption, and virus infection. Although the apoptotic response

likely developed to prevent the leakage of toxic compounds upon cell death, it is also

able to limit viral infection. By eliminating the infected cell before the viral replication

cycle is complete, the spread of infectious virus is prevented. In tum, some viruses have

evolved genes for preventing apoptosis, thereby allowing viral replication and

dissemination. The apoptosis effect has been established in numerous virus/host systems,

including insect vimses such as the baculovimses. Two gene products from

baculovimses, P35 and lAP (inhibitor of apoptosis), have been shown to prevent

apoptosis [34, 35, 37, 63, 74, 84, 135]. Mutant vimses in which these genes have been

dismpted induce apoptosis upon infection. African swine fever vims (ASFV), related to

Family Iridoviridae, contains genes proposed to inhibit the apoptotic response [115].

One ASFV gene is similar to the cell cycle regulator bcl-2, while the other is

homologous to the lAP gene from baculovimses. The function-of this bcl-2-like gene

has been demonsfrated [21] in ASFV, but the lAP-like protein is apparently non-essential

[101]. Even though two lAP-like genes have been discovered in CIV [8], fimction of

these genes has not been established.

12

Due to the variety of conditions and factors able to induce apoptosis, the

phenomenon must require a high degree of complexity. Virus infection may lead to

apoptosis via a variety of different mechanisms. In some systems, a viral capsid

component is able to induce apoptosis. However, many other viruses induce apoptosis

through a non-stmctural component. Tables 1.1 and 1.2 show viruses causing apoptosis

using stmctural and non-stmctural components, respectively.

The complexity ofthe apoptotic response with regard to the tiemendous variety

of inducing factors can be somewhat better understood after examining the cascade

system governing this response. Typically, the apoptotic response is initiated with

specific cysteine proteases ofthe caspase family [2, 9}. These proteases can be divided

into the subclasses of initiator caspases and effector caspases. Initiator caspases are

involved in signal tiansduction of various pathways, while effector caspases bring about

the apoptotic response, using three distinct mechanisms. The first mechanism is the

cleavage of proteins that normally function in cellular protection of apoptosis. For

example, caspase activated deoxyribonuclease (CAD), is normally present as an inactive

l' ' complex. Caspases are able cleave l' ' , releasing CAD which is then free to cause

DNA fragmentation. A second mechanism of effector caspase function is the collapse of

the rigid nuclear lamina, composed of proteins known as lamins. Cleavage of lamins by

caspases results in the breakdown ofthe lamina and condensation ofthe chromatin. The

third mechanism of effector caspase function is cytoskeletal disassembly via cleavage of

proteins involved in cytoskeletal regulation. For example, caspase cleavage converts a

protein known as gelsolin into a fimctional form that proceeds to fragment actin

filaments [83]. The most notable of these caspase proteases are the ICE-Iike proteases

13

(interleukin-1 beta-converting enzyme), also known as caspase 1. This caspase is

essential in the cascade and is the target of several vimses capable of inhibiting apoptosis

[32,77, 117].

Protein Kinase Involvement in Inhibition and Apoptosis

There is evidence shovdng that the caspase system can be activated indirectly by

interferon [9]. Interferon induced by virus infection in tum induces the production of an

RNA-dependent protein kinase (PKR). PKR phosphorylates the eIF-2a initiation factor,

preventing the formation ofthe tianslation initiation complex, thus stopping protein

synthesis [51]. PKR also fimctions by another mechaiiism, inducing ICE-like proteases

[85]. Many viruses have adapted mechanisms to circumvent the activity of interferon,

usually by inhibiting the activity of PKR [51]. Vaccinia virus prevents apoptosis by

attacking the interferon system through expression of two proteins that prevent eIF-2a

phosphorylation. The K3L gene product has regions of high similarity with the eIF-2a

factor and acts as a pseudosubstiate or decoy. The interferon-induced protein kinase

(PKR) phosphorylates the K3L gene product rather than the initiation factor [39, 123].

The E3L gene product binds dsRNA, preventing the activation of PKR [28, 118]. This

type of phosphorylation activity is not limited to the interferon system. As mentioned

previously. Frog Virus 3 (FV3) infection has also been shown to induce phosphorylation

ofthe eIF-2a factor [29, 30]. The phosphorylation of this factor by FV3 occurs in cell-

free systems [4], suggesting that this is directly due to a viral factor, rather than a signal

tiansduction cascade.

14

BIR Kinase

Another protein kinase that has been implicated in the inhibition of protein

synthesis is the BIR protein of vaccinia vims. Knockout mutants of this gene were

delayed in their ability to induce inhibition of protein synthesis by 8-10 hours [81]. This

gene has also been shown to phosphorylate the Sa and S2 ribosomal proteins [11]. The

exact function of these phosphorylation events is unknown, but it may be involved in the

switch to viral protein synthesis. Interestingly, our laboratory has identified a CIV gene

with high similarity to this gene (Henderson, Demirbag, D'Costa, and Bilimoria,

publication in progress). Typically, as more details are uncovered for mechanisms of

viral inhibition of protein synthesis, the more complicated these mechanisms become.

Therefore, it is possible that CIV inhibits protein synthesis by both eIF-2a

phosphorylation and by a mechanism similar to that of BIR. At this point, these facts

remain unknown for CIV.

Objectives

Previous research has sttongly suggested that CIV replicates in the boll weevil,

but did not examine the efficiency or extent ofthe infection. Other research showed that

infection with CIV and tieatment with a protein extiact from CIV induced the inhibition

of macromolecular synthesis in several cell lines. However, the effect ofthe protein

extiact was not studied in budworm cells (the normal laboratory host) or in boll weevil

cells, a highly important pest species. Research with these highly important cell lines is

15

necessary. Review of current literature, stiongly suggests involvement of a viral-protein

kinase in the inhibition process. In addition, sequencing work performed in our

laboratory had identified a CIV gene with high similarity to the vaccinia vims BIR gene,

which has been implicated in the inhibition of protein synthesis in infected hosts.

Therefore, the specific objectives of this research were to:

1. Examine and confmn complete replication of CIV in boll weevil pupae.

2. Determine if (and to what extent) a CIV virion extiact inhibits protein synthesis

and induces apoptosis in boll weevil and budworm cells.

3. Examine the CIV genome for a complete BlR-like gene.

4. Determine if protein kinase activity is present'in CIV virion extracts and attempt

to isolate the gene for this activity.

16

Table 1.1: Stmctural viral factors inducing apoptosis

Structural component

Capsid proteins

Viral attachment

protein Outer capsid

protein Envelope

glycoproteins Latent

membrane protein

Envelope glycoproteins

Stmctural protein?

Membrane associated

glycoprotein

Surface protein

Envelope protein

Mechanism

?

?

?

?

?

Increased expression of

transglutaminase

?

Bcl-2 like?

?

?

Imbalanced signal

transduction/ blockage of

nuclear transport?

Gene

VP2 [46]

SI

M2 [133] Precursor El and E2 [711

Overexpressed LMPl [90]

gpl20

gpl60 [134]

Homologous witii Bcl-2?

[1051 p25 [130]

Neuraminidase [981

gPr80"^[20]

Virus

Infectious bursal disease

vims Reovims

Sindbis virus

Epstein Ban-virus

HIV

African swine fever virus

Porcine reproductive

and respiratory syndrome vims Influenza vims

Moloney murme

leukemia vims

Family

Bimaviridae

Reoviridae

Togaviridae

Herpesviridae

Retroviridae

ASFV

Arteriviridae

. Ortho-myxoviridae Retroviridae

17

Table 1.2: Non-stmctural viral factors inducing apoptosis

Non­structural component

-

-

?

?

Mechanism

Binds cell cycle regulators

Activation of interferon

Increased Fas expression

Dysregulation of c-myc

expression Caspase

activation Fas mediated

induction? T-cell receptor

(TcR-CD3) mediated induction

Interference with tianslation

Gene

ElA

?

?

?

?

?

9

2A

Virus

Adenovirus [113]

Sendai virus [66]

Murine cytomegalovirus [47]

Epstein Barr vims [121]

Chicken anemia virus [381

Mouse hepatitis virus [1361

Lymphocytic choriomeningitis

virus [89]

Poliovirus [52]

Family

Adenoviridae

Paramyxoviridae

Herpesviridae

Herpesviridae

Circoviridae

Miscellaneous

Arenaviridae

Picomaviridae

18

References

1. Adamcyzk JJ, HoUoway JW, Church GE, Leonard BR, Cjraves IB (1998) Larval survival and development ofthe fall armyworm {Lepidoptera: Noctuidae) on normal and transgenic cotton expressing the Bacillus thuringiensis CrylA(c) delta-endotoxin. JEconEnt91:539-45

2. Ameisen JC (1998) The evolutionary origin and role of programmed cell death in single celled organisms: a new view of executioners, mitochondria, host-pathogen interactions, and the role of death in the process of natural selection. In: Lockshin RA, Zakeri Z and Tilly JL (eds) When Cells Die. Wiley-Liss, New York

3. Anderson CW, Lewis JB, Atidns JF, Gesteland RF (1974) Cell-free syntiiesis of adenovirus 2 proteins programmed by fractionated mRNA: a comparison of polypeptide products and mRNA lengths. Proc Nati Acad Sci USA 80: 2756-60

4. Aubertin AM, Hirth C, Travo C, Nonnenmacher H, Kim A (1973) Preparation and properties of an inhibitory extiact from frog virus 3 particles. J Virol 11: 694-701

5. Aurelian L, Roizman B (1965) Abortive infection of canine cells by HSV. n. The alternative suppression of synthesis of interferon and viral constituents. J Mol Biol 11:539-48

6. Babich A, Feldman LT, Nevins JR, Damell JE, Weinberger C (1983) Effects of adenovirus on metabolism of specific host mRNAs: transport contiol and specific translational domination. Mol Cell Biol 3: 1212-21

7. Bablanian R (1975) Stmctural and functional alterations in cultured cells infected with cytocidal vimses. Prog Med Virol 19: 40-83

8. Bahr U, Tidona CA, Darai G (1997) The DNA sequence of Chilo iridescent vims between the genome coordinates 0.101 and .391; similarities in coding strategy between insect and vertebrate iridoviruses. Vims Genes 15: 235-245

9. Balachandran S, Roberts PC, Kipperman T, Bhalla KN, Compans RW, Archer DR, Barber GN (2000) Alpha/beta interferons potentiate vims-induced apoptosis through activation ofthe FADD/Caspase-8 death signaling pathway. J Virol 74: 1513-23

10. Balange-Orange N, Devauchelle G (1982) Lipid composition of an iridescent vims type 6 (CIV). Arch. Virol. 73: 363

11. Banham AH, Leader DP, Smith GL (1993) Phosphorylation of ribosomal proteins by the vaccinia virus BIR protein kinase. FEBS Lett. 321: 27-31

19

12. Beaud G, Sharif, A., Topa-Masse, A., and Leader, D. P. (1994). J. Gen. Virol. 75, 283-293. (1994) Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the BIR protein kinase and phosphorylates in vitro the viral ssDNA-binding protein. J Gen Virol 75: 283-93

13. Ben-Hamida F, Beaud G (1978) In vitro inhibition of protein synthesis by purified cores from vaccinia vims. Proc Natl Acad Sci USA 75: 175-9

14. Ben-Hamida F, Person A, Beaud G (1983) Solubilization of a protein synthesis inhibitor from vaccinia vims. J Virol 45: 452-5

15. Ben-Porat T, Jean JH, Kaplan AS (1974) Early fimctions of tiie genome of herpesvirus. IV. Fate and tianslation of immediate-early RNA. Virology 50

16. Ben-Porat T, Kaplan AS (1965) Mechanism of inhibition of cellular DNA synthesis by pseudorabies vims. Virology 25: 22-9

17. Bilimoria SL (1975) Iridescent Vims Replication Biology. University of Otago, Dunedin, New Zealand '•

18. Bilimoria SL, D'Costa S (1998) Northern blot analysis and transcriptional mapping of Dazaifii iridescent vims. In: XII Intemational Symposium on Poxviruses, St. Thomas, US Virgin Islands, pp 32

19. Bilimoria SL, Parkinson AJ, Kahnakoff J (1974) Comparative study of 125i_ g^d (^H) acetate-labeled antibodies in detecting iridescent viruses. Applied Microbiology 28: 133-7

20. Bonzon C, Fan H (1999) Moloney murine leukemia virus-induced preleukemic thymic atiophy and enhanced thymocyte apoptosis correlate with disease patiiogenicity. J Virol 73: 2434-41

21. Brun A, Rodriguez F, Escribano JM, Alonso C (1998) Functionality and cell anchorage dependence ofthe African swine fever virus gene A179L, a vfral bcl-2 homolog, in insect cells. J Virol 72: 10227-33

22. Carter JB (1973a) The mode of tiansmission of Tipula iridescent virus. I. Source of infection. J Invertebr Pathol 21: 123-30

23. Carter JB (1973b) The mode of tiansmission of Tipula iridescent vims. II. Route of infection. J Invertebr Patiiol 21: 136-43

24. Carter JB (1974) Tipula iridescent virus infection in teh development stages of Tipula oleracea. J Invertebr Patiiol 24: 271-81

20

25. Cemtti M, Deauvechelle G (1980) Inhibition of host macromolecular synthesis in cells infected with an invertebrate vims. Archives of Virology 63: 297-

26. Cemtti M, Deauvechelle G (1985) Characterization and localization of CIV polypeptides. Virology 145: 123-131

27. Cemtti M, Devauchelle G (1990) Protein composition of Chilo iridescent vims. In: Darai G (ed) Molecular Biology of Iridovimses. Kluwer Academic Press, Boston, pp 81-112

28. Chang HW, Watson JC, Jacobs BL (1992) The E3L gene of vaccinia virus encodes an inhibitor ofthe interferon-induced, double-stianded RNA-dependent protein kiaase. Proc Nati Acad Sci USA 89: 4825-9

29. Chinchar VG, Dholakia. JN (1989) Frog vims 3- induced tiranslational shutoff: activation of an eIF2 kinase in vims mfected cells. Virus Res. 14: 207-224

30. Chinchar VG,YuW (1990) Frog vims 3-induced translational shut-off: Frog virus 3 messages are tianslationally more efficient thatn host and heterologous viral messages under conditions of increased tianslational stiess. Virus Res 16: 363-74

31. Chinchar VG, Yu W (1992) Metabolism of Host and Viral mRNAs in Frog Virus 3-Infected Cells. Virology 186: 435-443

32. Chiou SK, White E (1998) Inhibition of ICE-like proteases inhibits apoptosis and increases virus production during adenovims infection. Virology 244: 108-18

33. Cho HJ, Choi KP, Yamashita M (1995) Introduction and expression of the Streptomyces cholesterol oxidase gene (ChoA), a potent insecticidal protein active against boll weevil larvae into tobacco cells. Apphed Microbiology and Biotechnology 44: 133-138

34. Clem RJ, Fechheimer M, Miller LK (1991) Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254: 1388-90

35. Clem RJ, Miller LK (1994) Contiol of programmed cell death by the baculovirus genes p35 and iap. Molecular and Cellular Biology 14: 5212-5222

36. Cordier O, Aubertin AM, Lopez C, Tondre L (1981) Inhibition de la transduction par le FV3: Action des proteines virales de stmcture solubilisees sur la synthesis proteique in vivo et in vitro. Ann Virol Inst Pasteur 132E: 25-39

37. Crook NE, Clem RJ, Miller LK (1993) An apoptosis-inhibiting baculovims gene with a zinc fmger-like motif J Virol 67: 2168-74

21

38. Danen-van Oorschot AA, van Der Eb AJ, Notebom MH (2000) The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol 74: 7072-8

39. Davies MV, Efroy-Stein O, Jagus R, Moss B, Kaufinan RJ (1992) The vaccinia vims K3L gene product potentiates tianslation by inhibiting double-stianded RNA-activated protein kinase and phosphorylation ofthe alpha subunit ofthe eukaryotic initiation factor 2. J Virol 66: 1943-50

40. D'Costa SM, Yao H, Zhang R, Bilimoria SL (1999) Temporal classification and mapping of Chilo iridescent virus tianscripts. In: 99th General Meeting, ASM, Chicago, IL, pp 624

41. Delius H, Darai G, Flugel RM (1984) DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy. Journal of Virology 49: 609-614

42. Devauchelle G, Attias J, Monnier C, Barray S, Cemtti M, Guerillon J, Orange-Balange N (1985) Chilo iridescent vims. Curr:Top. Microbiol. Immunol. 116: 37-48

43. D'Souza S, Henderson C, Lodhi S, Glass A, Yao H, Bilimoria S (1996) RepUcation and induction of apoptosis by an insect vims pathogenic to the cotton boll weevil. In: SWARM, AAAS, Flagstaff, AZ, pp 23

44. Ensinger M, Ginsberg HS (1972) Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol 10: 328-39

45. Everly DN, Read GS (1997) Mutational analysis ofthe virion host shutoff gene (UL41) of herpes simplex vims (HSV): characterization of HSV type 1 (HSV-l)/HSV-2 chimeras. J. Virol. 71: 7157-7166

46. Fernandez-Arias A, Martinez S, Rodriguez JF (1997) The major antigenic protein of infectious bursal disease vims, VP2, is an apoptotic inducer. J Virol 71: 8014-8

47. Fleck M, Kem ER, Zhou T, Podlech J, Wintersberger W, Edwards CKd, Mountz ID (1998) Apoptosis mediated by Fas but not tumor necrosis factor receptor 1 prevents chronic disease in mice infected with murine cytomegalovirus. J Clin Invest 102: 1431-3

48. Fowler HG (1989) An epizootic iridovims of Orthoptera (Gryllotalpidae: Scaptericus borellii) and its pathogenicity to termites (Isoptera: Cryptotermes). Rev Microbiol 20: 115-20

22

49. Fukaya M, Nasu S (1966) A Chilo Iridescent Virus (CIV) from the rice stem borer, Chilo suppresalis Walker (Lepidoptera, Pyralidae). Applied Entomology and Zoology 1: 69-72

50. Fukuda T, Clark TB (1975) Transmission ofthe mosquito iridescent vims (RMIV) by adult mosquitoes of Aedes taeniorhynchus and their progeny. J Invertebr Pathol 25: 29-46

51. Gale M, Katze MG (1998) Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol. Ther. 78: 29-46

52. Goldstaub D, Gradi A, Bercovitch Z, Grosmaim Z, Nophar Y, Luria S, Sonenberg N, Kahana C (2000) Poliovims 2A protease induces apoptotic cell death. Mol Cell Biol 20: 1271-7

53. Goorha R, Granoff A (1974) Macromolecular synthesis in cells infected by frog vims 3. I. Virus-specific protein synthesis and its regulation. Virology 60: 237-50

54. Goorha R, Murti G, Granoff A, Tirey R (1978) Macromolecular synthesis in cells infected by frog vims 3, VIII: The nucleus is a site for frog virus 3 DNA and RNA synthesis. Virology 49: 86-91

55. Goorha R, Willis DB, Granoff A (1977) Macromolecular synthesis in cells infected by frog virus 3, VI: Frog vims 3 replication is dependent on the cell nucleus. J. Virol. 21: 802-805

56. Graves JB, Leonard BR, Micinski S, Burns G (1991) A three year study of pyrethroid resistance in tobacco budworm in Louisiana: resistance management implications. Southwestern Entomologist: 33-41

57. Graves JB, Leonard BR, Pavloff AM, Burris G, Ratchford K (1989) An update on pyrethroid resistance in tobacco budworm in Louisiana. In: Proceedings ofthe Beltwide Cotton Conferences, pp 877-80

58. Greenplate JT, Corbin DR, Purcell JP (1997) Cholesterol oxidase: a potent boll weevil larvicidal and oostatic agent suitable for tiansgenic cotton development. In: Proceedings ofthe Beltwide Cotton Conferences, pp 877-80

59. Greenplate JT, Duck NB, Pershing JC, Purcell IP (1995) Cholesterol oxidase: an oostatic and larvicidal agent active against the cotton boll weevil, Anthonomus grandis. Entomologia experimentalis et applicata 74: 253-8

60. Grosholz ED (1992) Interaction of intiaspecific, interspecific, and apparent competition with host-pathogen population dynamics. Ecology 73: 507-14

23

61. Grosholz ED (1993) The influence of habitat heterogeneity on host-pathogen population dynamics. Oecologia 96: 347-53

62. Hall DW (1985) Pathobiology of invertebrate icosahedral cytoplasmic deoxyriboviruses (Iridoviridae). In: Maramorosh K and Sherman KE (eds) Viral Insecticides for Biological Contiol. Academic Press, New York, pp 163-96

63. Hawkins CJ, Uren AG, Hacker G, Medcalf RL, Vaux DL (1996) Inhibition of interleukin 1 beta-converting enzyme-mediated apoptosis of mammalian cells by baculovirus IAP. Proc Nati Acad Sci U S A 93: 13786-90

64. Hay J, Koteles GJ, Keir HM, Subak S, H. (1966) Herpesvims-specified ribonucleic acids. Natiire 210: 387-90

65. Henderson CW, Johnson CJ, Lodhi S, Bilimoria SL Replication of Chilo Iridescent Virus in the Cotton Boll Weevil, Anthonomus grandis, and Development of an Infectivity Assay. Arch Virol in press

66. Heylbroeck C, Balachandran S, Servant MJ, DeLuca C, Barber GN, Lin R, Hiscott J (2000) The IRF-3 tianscription factor mediates Sendai virus-induced apoptosis. J Virol 74: 3781-92

67. Hodge LD, Scharff MD (1969) Effect of adenovirus on host DNA synthesis in synchronized cells. Virology 37: 554-64

68. Horwitz MS (1971) Intermediates in the replication of type 2 adenovirus DNA. Virology 8: 675-83

69. Jayaraman R (1999) Induction of apoptosis by an insect iridescent virus in boll weevil and budworm cell culture. Department of Biological Sciences. Texas Tech University, Lubbock

70. Jensen DD, Hukutsara T, Tanada Y (1972) Letiiality of Chilo iridescent virus to Collodonus montanus leafhoppers. J Invertebr Pathol 119: 276-8

71. Joe AK, Foo HH, Kleeman L, Levine B (1998) The transmembrane domains of Sindbis vims envelope glycoproteins induce cell death. J Virol 72: 3935-43

72. Joklik WK, Merigan TC (1966) Concerning the mechanism of action of interferon. Proc Nati Acad Sci USA 56: 558-65

73. Kahnakoff J, Tremaine JH (1968) Physicochemical properties of Tipula iridescent vims. J Virol 2: 738-44

24

74. Kamita SG, Majima K, Maeda S (1993) Identification and characterization ofthe p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis. J Virol 67: 455-63

75. Kelly DC (1985) Insect Iridescent Viruses. Current Topics in Microbiology and Immunology 116: 23

76. Kelly DC, Vance DE (1973) The lipid content of two iridescent viruses. J Gen Virol 21:417-23

77. Kettle S, Alcami A, Khanna A, Ehret R, Jassoy C, Smitii GL (1997) Vaccinia vims serpin B13R (SPI-2) inhibits interleukin-1 beta-converting enzyme and protects vims-infected cells from TNF- and Fas-mediated apoptosis, but does not prevent IL-1 beta-induced fever. J Gen Virol 78: 677-85

78. Khalili K, Weinmann R (1984) Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells. J Mol Biol 175: 453-68

79. Knipe DM, Howley PM, Fields BN (1996) Fields Virology. Lippincott-Raven, New York

80. Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ (1999) Overexpression ofthe Bacillus thuringiensis (Bt) Cry2 Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc Nati Acad Sci USA 96: 1840-5

81. Kovacs GR, Moss B (1998) Regulation of gene expression by the vaccinia vims protein kinase-1. In: Twelfth Intemational Poxvirus Symposium, St.Thomas, United States Virgin Islands, pp 66

82. Laurent AM, Madjar JJ, Greco A (1998) Translational contiol of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that iaitiation of tianslation is the limiting step. J. Gen. Virol. 79: 2765-75

83. Lazebnik Y, Thomberry N (1998) Caspases: enemies within. Science 281: 1312-6

84. Lee JC, Chen HH, Chao YC (1998) Persistent baculovirus infection results from deletion ofthe apoptotic suppressor gene p35. J Virol 72: 9157-65

25

85. Lee SB, Rodriguez D, Rodriguez JR, Esteban M (1997) The apoptosis patiiway triggered by the interferon-induced protein kinase PKR requires the tiiird basic domain, initiates upstieam of Bcl-2, and involves ICE-like proteases. Virology 231: 81-88

86. Linley JR, Nielsen HT (1968a) Transmission ofthe mosquito iridescent vims in Aedes taeniorhynchus. I. Laboratory experiments. J Invertebr Pathol 12: 7-16

87. Linley JR, Nielsen HT (1968b) Transmission ofthe mosquito iridescent vims in Aedes taeniorhynchus. II. Experiments related to transmission in nature. J Inverter Patiiol 12: 17-24

88. Llewellyn D, Cousins Y, Mathews A, Hartweck L, Lyon B (1994) Expression of Bacillus thuringiensis insecticidal protein genes in transgenic crop plants. Agriculture, Ecosystems, and Environment 49: 85-93

89. Lohman BL, Welsh RM (1998) Apoptotic regulation of T cells and absence of immune deficiency in vims-infected gamma interferon receptor knockout mice. J Virol 72: 7815-21

90. Lu JJ, Chen Pf, Hsu TY, Yu WC, Su IJ, Yang CS (1996) Induction of apoptosis in epithelial cells by Epstein-Barr virus latent membrane protein 1. J Gen Virol 77: 1883-92

91. Mbuy GN, Morris RE, Bubel HC (1982) Inhibition of cellular protein synthesis by vaccinia virus surface tubules. Virology 116: 137-47

92. McCutchen BF, Choudary PV, Crenshaw R, al e (1991) Development of a recombinant baculovims expressing an insect-selective neurotoxin: potential for pest contiol. Bio/Technology 9: 848-852

93. McLaughlin RE, Scott HA, Bell MR (1972) Infection of tiie boll weevil by Chilo iridescent virus. J Invertebr Pathol 19: 285-290

94. Metcalf RL, Luckman WH (1982) Intioduction to Pest Management., Second Edition edn. Wiley-Interscience, New York

95. Miller LK (1995) Genetically Engineered Insect Virus Pesticides: Present and Future. Journal of Invertebrate Pathology 65: 211-216

96. Miller LK (1996) The Insect Vimses. In: Fields BN and al e (eds) Virology. Raven Press, New York

26

97. Morales-Ramos JA, Rojas MG, Coleman RJ, King EG (1998) Potention use of in vi>o-reared Catolaccus grandis (Hymenoptera: Pteromalidae) for biological contiol ofthe boll weevil (Coleoptera: Curculionidae). J Econ Ent 91: 101-9

98. Morris SJ, Price GE, Bamett JM, Hiscox SA, Smitii H, Sweet C (1999) Role of neuraminidase in influenza virus-induced apoptosis. J Gen Virol 80: 137-46

99. Moss B (1968) Inhibition of HeLa cell protein synthesis by the vaccinia virion. J Virol 2: 1028-37

100. Murray EE, DeBoer DL (1993). In: Kung S and Wu R (eds) Transgenic Plants. Academic Press, New York, vol 2

101. Neilan JG, Lu Z, Kutish GF, Zsak L, Burrage TG, Borca MV, Carrillo C, Rock DL (1997) A BIR motif containing gene of African swine fever vims, 4CL, ia nonessential for growth in vitro and viral virulence. Virology 230: 252-64

102. Ng TI, Chang YE, Roizman B (1997) Infected cell protein 22 of herpes simplex virus 1 regulates the expression of virion host shutdff gene U(L) 41. Virology 234: 226-34.

103. Nishioka Y, Silverstein S (1978) Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected with HSV-1. J Virol 27: 619-627

104. O'Reilly DR, Miller LK (1992) Improvement of a baculovims pesiticide by deletion of tiie egt gene. Bio/Technology 9: 1086-1089

105. Oura CA, Powell PP, Parkhouse RM (1998) African swine fever: a disease characterized by apoptosis. J Gen Virol 79: 1427-38

106. Overton H, McMillan D, Hope L, Wong-Kai-In P (1994) Production of host shutoff-defective mutants of herpes simplex vims type 1 by inactivation ofthe UL13 gene. Virology 202: 97-106.

107. Pak AS, Everly DN, Knight K, Read GS (1995) The virion host shutoff protein of herpes simplex vims inhibits reporter gene expression in the absence of other viral gene products. Virology 211: 491-506

108. Person-Femandez A, Beaud G (1986) Purification and characterization of a protein synthesis inhibitor associated with vaccinia vims. J Biol Chem 25: 8283-9

109. Petit F, Devauchelle G (1985) Isolation of polysomes from permissive and non-permissive invertebrate cell lines infected with Chilo iridescent virus. Med Microbiol Immunol 174: 67-71

27

110. Pimentel D (1981) Handbook of Pest Management in Agriculture. CRC Press, Boca Raton, FL

111. Pogo GBT, Dales S (1973) Biogenesis of poxviruses: inactivation fo host DNA polymerase by a component ofthe invading inoculum particle. Proc Nati Acad Sci USA 70: 1726-9

112. Purcell JP, Greenplate JT, Jennings MG, Ryerse JS, Pershing JC, Sims SR, Prinsen MJ, Corbin DR, Tran M, D. SR (1993) Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae. Biochem. Biophys. Res. Commxm. 196: 1406-1413

113. Putzer BM, Stiewe T, Parssanedjad K, Rega S, Esche H (2000) ElA is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products. Cell Deatii Differ 7: 177-88

114. Raghow R, Granoff A (1979) Macromolecular synthesis in cells infected by frog virus 3. X. Inhibition of cellular protein synthesis by heat-inactivated virus. Virology 98: 319-27

115. Ramiro-IbanezF, Ortega A, Brun A, Escribano JM, Alonso C (1996) Apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute African swine fever vims infection. J Gen Virol 77: 2209-19

116. Ricou G (1975) Production do Tipula paludosa Meig. En prarie en function de I'humiditie du sol. Rev Ecol Biol Sol 12: 69-89

117. Robertson NM, Zangrilli J, Femandes-Alnemri T, Friesen PD, Litwack G, Alnemri ES (1997) Baculovims P35 inhibits the glucocorticoid-mediated pathway of cell deatii. Cancer Res 57: 43-7

118. Romano PR, Zhang F, Tan SL, Garcia-Barrio M, Katze MG, Dever TE, Hinnebusch AG (1998) Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia vims E3: role of complex formation and the E3 N-terminal domain. Mol Cell Biol 18: 7304-16

119. Rondelaud D, Barthe D (1992) Observations epidemiologiques sur I'iridivirose de Lymnaea tmnculata, moUusque vecteur de Fasciola hepatica. C R Acad Sci Paris Ser 314: 609-12

120. Rosemond-Hombeak H, Moss B (1975) Inhibition of host protein synthesis by vaccinia vims: fate of cell mRNA and synthesis of small poly (A)-rich polyribonucleotides in the presence of actinomycia D. J Virol 16: 34-42

28

121. Ruf DC, Rhyne PW, Yang C, Cleveland JL, Sample JT (2000) Epstein-Barr Vims Small RNAs Potentiate Tumorigenicity of Burkitt Lymphoma Cells Independently of an Effect on Apoptosis. J Virol 74: 10223-8

122. Schmelz M, Sodeik B, Ericsson M, Wolffe EJ, Shida H, Hiller G, Griffitiis G (1994) Assembly of vaccinia virus: the second wrapping cistema is derived from the tians Golgi network. J. Virol. 68: 130-147

123. Sharp TV, Witzel IE, Jagus R (1997) Homologous regions of tiie alpha subunit fo eukaryotic initiation factor 2 (eIF-2a) and the vaccinia virus K3L gene product interact witht he same domain within the dsRNA-activated protein kinase (PKR). Eur J Biochem 250: 85-91

124. Shatkin AJ (1963) Actinomycin D and vaccinia vims infection of HeLa cells. Nature 199: 357-8

125. Sieburth PJ, Camer GR (1987) Infectivity of an iridescent virus for larvae of Anticarsia gemmatalis (Lepidoptera: Noctuidae). J Invertebr Pathol 49: 49-53

126. Sikorowski PP, Tyson GE (1984) Per os transmission of iridescent virus of Heliothis zea (Lepidoptera: Noctuidae). J Invertebr Pathol 44: 97-102

127. Stewart LMD, Hirst M, Ferber ML, Merryweatiier AT, Cayley PJ, Possee RD (1991) Constmction of an improved baculovims insecticide containing an insect-specific toxin gene. Nature 352: 85-88

128. Stohwasser R, Raab K, Schnitzler P, Janssen W, Darai G (1993) Identification of tiie gene encoding the major capsid protein of insect iridescent virus type 6 by polymerase chain reaction. Journal of General Virology 74: 873-879

129. Su MJ, Bablanian R (1990) Polyadenylated RNA sequences from vaccinia virus-infected cells selectively inhibit tianslation in a cell-free system: stmctural properties and mechanism of inhibition. Virology 179: 679-93

130. Suarez P, Diaz-Guerra M, Prieto C, Esteban M, Casfro JM, Nieto A, Ortin J (1996) Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis. J Virol 70: 2876-82

131. Tomalski MD, Miller LK (1991) Insect paralysis by baculovirus-mediated expression of a mite neurotoxin gene. Nature 352: 82-85

29

132. Tumipseed SG (1997) The utilization of natural enemies, viral insecticides and improved information delivery for management of lepidopterous pests developing in transgenic B. t. cotton. In: Agriculture in Concert witii the Environment (ACE) research projects, pp 17

133. Tyler KL, Squier MK, Brown AL, Pike B, Willis D, Oberhaus SM, Demiody TS, Cohen JJ (1996) Linkage between reovims-induced apoptosis and inhibition of cellular DNA syntiiesis: role of the SI and M2 genes. J Virol 70: 7984-91

134. Ulfrich CK, Groopman JE, Ganju RK (2000) fflV-1 gpl20- and gpl60-induced apoptosis in cultured endothehal cells is mediated by caspases. Blood 96: 1438-42

135. Vucic D, Kaiser WJ, Miller LK (1998) Inhibtor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM. Mol Cell Biol 18: 3300-9

136. Wang Y, Detiick B, Yu ZX, Zhang J, Chesky L, Hooks JJ (2000) The role of apoptosis within the retina of coronavims-infected mice. Invest Ophthalmol Vis Sci 41:3011-8

137. Ward VK, Kahnakoff J (1991) Invertebrate Iridoviridae. In: Kurstak E (ed) Vimses of Invertebrates. Marcel Dekker, New York

138. Wilkie NM, Ustacelabi S, Williams JF (1972) Characterization of temperature-sensitive mutants of adenovirus type 5: nucleic acid synthesis. Virology 51: 499-503

139. Williams MR (1998) Cotton Lisect Losses -1997. In: Beltwide Cotton Conference, San Diego, CA. National Cotton Council of America, pp 904-925

140. Williams RC, Smitii KM (1957) A crystallizable insect virus. Natiire 179: 119-20

141. WiUiams T (1996) The Iridovimses. Advances in Virus Research 467: 345-411

142. WiUiams T (1998) Invertebrate fridescent Vfruses. In: Miller LK and Ball LA (eds) The Insect Viruses. Plenum Press, New York The Vimses, pp 31-68

143. Willis DB, Goorha R, Granoff A (1984) DNA metiiyl ti^nsferase induced by frog virus 3. J. Virol. 49: 86-91

144. Willis DB, Granoff A (1978) Macromolecular synthesis in cells infected by frog virus 3. IX: Two temporal classes of early viral RNA. Virology 86: 443-453

145. Woodard DB, Chapman HC (1968) Laboratory studies with the mosquito iridescent virus (MIV). J Invertebr Patiiol 11: 296-301

30

146. Wright JE, Chandler LD (1991) Development of an attracticide for tiie boU weevil. In: Proceeding ofthe Beltwide Cotton Conferences, pp 299-303

147. Xeros N (1954) A second virus disease ofthe leatherjacket, Tipula paludosa. Nature 174: 562-565

148. Yao H, D'Souza S, Lodhi S, Bilimoria SL (1996) Gene expression of a virus causing arrest and mortality in the cotton boll weevil. In: 72nd Annual Meeting, SWARM, AAAS, Flagstaff, AZ, pp 35

149. Yu L, Henderson C, Houck M, Bilimoria SL (1996) Viral induced mortality and metamorphic arrest in the cotton boll weevil. In: 72nd Annual Meeting, AAAS, SWARM, Flagstaff, AZ, pp 35

150. Zhang Y, Feigenblum D, Schneider RJ (1994) A late adenovirus factor induces elF-4E dephosphorylation and inhibition of cell protein synthesis. J Virol 68: 7040-50

151. Zylber-Katz E, Weisman P (1975) Effects on host ceU polyribosomes following infection with frog virus 3 at a non-permissive temperature. Arch Virol 47: 181-5

31

CHAPTER II

REPLICATION OF CHILO IRIDESCENT VIRUS IN THE COTTON

BOLL WEEVIL, ANTHONOMUS GRANDIS AND THE

DEVELOPMENT OF AN DJFECTIVITY ASSAY.

Introduction

The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton in

the Western Hemisphere. In 1997, insect damage reduced profitability for the American

cotton farmer by $785 million; Texas producers lost $387 million [1,3]. As expected,

worldwide estimates are much higher. A recent task force has predicted that the cotton

boll weevil will have an economic inqiact exceeding $500 million per year in west Texas

alone. More than 9,200 jobs will be lost and at least 60 cotton gins will close if no new

technology is developed [3]. Clearly, safe and effective contiol measures are necessary.

Chemical pesticides are plagued with problems of resistance, targeting, and environmental

hazards. In addition, eradication by chemical methods is costly and inpractical for many

cotton-producing areas [2]. Microbial alternatives have the potential of limiting such

problems because of their narrow host range. Chilo iridescent vims (CIV) [6] is the only

vims shown to induce deformity and mortality in the cotton boll weevil. CIV belongs to

the fanaiy Iridoviridae, which are large, icosahedral cytoplasmic vimses vvo th a double-

stranded DNA genome [15]. The Iridoviridae axe divided into several genera, and CIV is

a member ofthe genus Iridovirus (small insect iridescent viruses, 130-140 nm in

diameter).

32

McLaughlin et al. [12] demonstrated infection ofthe boU weevil with CIV, using

extracts from iridescent insects and recovery of an iridescent peUet as the criterion for

infection. However, efficiency and production of viable progeny were not clearly

established. Given the increasing economic inqiortance ofthe cotton boll weevil, our

laboratory has undertaken a series of comprehensive, vertical studies on the biology of

CIV in this host. These include mortality, replication m. cell culture, and gene expression

studies [5,7, 16, 17]. Work in our laboratory has shown that CIV infection causes up to

70% mortality in the cotton boll weevil [17].

The present study is the first to investigate replication of a member ofthe genus

Iridovirus in the boll weevil. We studied CIV repUcation in boU weevil pupae using DNA

dot blotting, electron microscopy, and an infectivity assay developed in our laboratory.

Our data demonstrate that CIV undergoes a productive infection cycle in the cotton boU

weevil and suggest that the vims or its conponents have greater potential for boll weevil

control relative to earUer assessments.

Materials and Methods

Vims Rearing

Virus was raised in larvae ofthe greater wax moth, Galleria mellonella

(Lepidoptera, Pyralidae). Larvae (Sunfish Bait, Webster, WI). Infection was initiated in

larvae by nicking with bent forceps dipped in a vims suspension (500 ng/ml) in Buffer A

(150 mM NaCl, 50 mM Tris-HCl, pH 7.4). Larvae were incubated for two weeks at

33

21 °C in a bed of sawdust. They were observed on.altemate days and dead larvae were

discarded. After the incubation period, aU remaining larvae were stored at -80 °C.

Virus Purification

Virus purification was based on the method of KeUy and Tinsley [8]. Frozen

larvae were macerated in Buffer A, then passed through double-layered cheesecloth. The

extract was differentially centrifuged through two cycles (4,600 X g and 39,000 X g,

respectively) at 4 °C. Resuspended virus pellets were layered on 10-60% (w/v) sucrose

gradients in Buffer A and centrifuged (72,000 g) at 4 °C for 2 h. The vims was harvested

and then filtered through a series of 0.45 and 0.22 mrtMiUipore GV membranes. Viral

concentration was determined by spectrophotometry, where one optical density unit at

260 nm was equivalent to 55 |ig/ml vims [9].

Preparation nf Infected Samples

BoU weevUs (obtained from the GAST Insect Rearing Research Laboratory,

StarkviUe, MS) were infected in the first pupal stage. A tubercuUn syringe was dipped in a

vims suspension (500 jig/ml in buffer A), and pupae were then nicked immediately dorsal

to the base ofthe left elytra. Analysis ofthe nicking procedure using radioactive solutions

indicated that this technique deUvered an average of approximately 0.05 |al of the

suspension (or 25 ng vims) per nick. Infected weevils were incubated at 21 °C in Petri

dishes containing moist filter paper. Mock infection (performed with buffer A only) and

infection with heated virus (65 °C for 30 min in buffer A) were used as controls.

34

Viral DNA RepUcation

Individual boU weevils were macerated in 500 |al buffer B (0.4 M NaOH, 10 mM

EDTA) at 0, 1, 3, 5, and 7 days post infection. Fifty-microUter aUquots ofthe above

lysates were appUed to nitroceUulose membranes (S&S) and dot blot analyses were carried

out as described below.

Electron Microscopy

BoU weevils were harvested at 7 days post infection and placed in Kamovsky's

fixative (4% formaldehyde, 5% gluteraldehyde, 1% Tween 20 in PBS, pH 6.8). Pupae

were injected with fixative, placed under vacuum for 30 min, and then stored at 4 °C.

San^les were washed three times for 1 h each in PBS (pH 6.8), post-fixed in OSO4,

washed again, and dehydrated in a graded series of ethanol foUowed by soaking in

acetone. Infiltration was accon^Ushed via a graded series of Spurr's resin in acetone, and

embedding in Spurr's resin. Thin sections (approx. 70 nm) were cut on a Sorvell

Ultramicrotome, stained with uranyl acetate, and examined with a Hitachi HS-9

transmission electron microscope.

Infectivity Assay

Twenty-five boU weevil pupae (9-days old; weighing 12 mg ± 4.0 std. dev.) were

infected with CIV as described above. An excess number of pupae were set up to

con:q)ensate for high virus-induced mortaUty rate (70 percent) [17]. The pupae were

35

incubated at 21 °C. At 7 days p.i., three pupae from the survivors were pooled and

macerated in 500 |il Buffer A (150 mM NaCl, 50 mM Tris-HCl, pH 7.4). These lysates

were then seriaUy dUuted 10-fold and an excess number of 10 healthy boU weevU pupae

were infected with each dUution. These pupae were in tum incubated at 21°C for 7 days.

RepUcation of viral DNA in surviving indicator pupae was used to determine the titer.

The highest dilution of lysate from indicator pupae resulting in viral DNA repUcation (as

detected by dot blot analysis) was defined as the titer. Three pupae selected immediately

after inoculation ofthe 25 pupae in the primary infection served as day 0 san^les and

were processed immediately after selection exactly as described for the day 7 samples.

Primary mock-infected control pupae were inoculated with buffer without vims, incubated

at 21°C for 7 days, and processed as described above.

Dot Blot Analysis

A BioRad dot blot apparatus was used to ensure deUvery of equal amounts of

DNA per blot. The remaining material was stored at -20 °C and used for repUcate assays.

After blotting, membranes were hybridized and developed using standard procedures [4].

Briefly, membranes were air-dried and baked in a vacuum oven. Blots were prehybridized

for 4 h. Viral DNA was extracted using the method of Summers and Smith [14] and

prepared for use as a ^^P-labeled probe using the Boehringer Mannheim Random Priming

kit. Membranes were then hybridized overnight with 5 \iCi of ^P-CIV DNA, washed in

2x SSC (300 mM sodium chloride, 50 mM sodium citiate, pH 7.0) containing 0.1% SDS,

and exposed against Hyperfilm MP (Amersham).

36

Results

Evidence of DNA RepUcation in Pupae

In order to determine the relative levels of CIV DNA repUcation in the boU weevil,

lysates from individual CIV-infected pupae were analyzed by the dot blot procedure using

genomic DNA from purified virions as probe. Lysates were prepared at 0, 1, 3, 5, and 7

days post infectioa Figure 2.1 shows that labeled viral DNA probes did not hybridize

with DNA from mock-infected pupae or from pupae infected with heat-inactivated virus

(65° C; 30 min). Further, the viral DNA probe did not yield any signal with DNA

harvested from infected pupae at 0 and 1 day post infection, indicating that signal from

parental DNA was not detectable at the doses utilized to initiate infection. On the other

hand, strong signals were detected at 3, 5, and 7 days post infection, suggesting significant

increase in progeny viral DNA over the first three days of infectioa The above data were

highly reproducible.

Formation of Conylete Vims Particles

To determine whether conqilete virus particles were formed in boU weevU pupae,

infected boU weevils were prepared for electron microscopy 7 days post infection.

Electron micrographs of macerated tissue from the thorax of infected insects showed large

amounts of virogenic stroma (Figure 2.2A). Vims particles were located exclusively in

the cytoplasm (Figure 2.2B) and the vast majority of them were conqilete. Scanning

electron microscopy showed a distinct difference in eye morphology between infected and

37

uninfected insects (data not shown). Additional transmission electron microscopic studies

of eye tissue showed localization and particle arrangement similar that in thoracic tissue.

Moreover, highly stmctured patterns of virus particles were observed, predominantly in

eye tissue (Figure 2.2C). Large numbers of viral particles were consistently observed, and

there were very few eir^ty particles, indicating that the infection was very efficient. Close

inspection of virus particles revealed the intemal membrane, characteristic ofthe

Iridoviridae (Figure 2.2D). Negative staining of tissue fragments from other parts ofthe

pupae suggested that virus was present throughout infected insects. However, the latter

evidence does not necessarily represent virus repUcation in associated tissue.

Infectivity of Progeny Virus

To determine if infectious viral titer increased in boU weevil pupae, we developed

an endpoint dilution assay. A detailed experimental design is presented in Materials and

Methods. Viral DNA repUcation, as detected by dot blot analysis, was used as an

indicator. Twenty-five primary pupae were inoculated with virus. Three pupae from the

survivors (each weighing 12 mg ± 4.0 std. dev.) were pooled and macerated at 0 and 7

days post infection (as weU as mock-infected insects at 7 days post tieatment) and seriaUy

diluted 10-fold. Each dilution was then used to infect batches containing an excess of 10

healthy pupae. Individual, surviving pupae from the newly-infected insects were analyzed

for DNA repUcation after incubation for 7 days. The highest dilution inducing detectable

levels of viral DNA in the newly infected pupae was defined as the titer. TripUcate assays

(one insect per dUution and per repUcate) showed a titer of at least 10 over 7 days. Three

38

separate experiments were performed, each yielding results within one order of magnitude

those shown ki Figure 2.3. Mock-infected pupae incubated for 7 days and pupae infected

with day 0 san^les (macerated within 1 h p.i.) did not yield any signal The results

suggest significant production of viable CIV progeny in boU weevil pupae.

Discussion

EarUer work by McLaughlin et al. [12] showed that the boU weevil is susceptible

to infection by CIV. These workers used blue coloration of insects and the abiUty to

obtain iridescent viral peUets as indicators of infection. However, details ofthe infection

process such as efficiency, the extent of infection, and viabiUty of progeny virus were not

studied. In the present study, we demonstrate that upon CIV deUvery via nicking ofthe

integument, there is repUcation of viral DNA and copious particle formation. We also

describe an assay that demonstrates increase in infectious virus titer in this systeia

Although our study focused on thorax and eye tissue, negative staining of tissue fragments

from other parts ofthe organism suggested that virus was present throughout infected

pupae.

Typical paracrystaUine arrays were frequently observed, but other, highly

organized arrays were also seen, particularly in eye tissue. These consisted of repeating

columns four to five vims particles in width. These particles appeared to be arranged in a

three-dimensional (and possibly heUcal) partem because individual particles in these

columns faded above and below the plane of focus in a consistent pattern. It is possible

that CIV particles in this host are associated with cytoskeletal filaments such as actin, as

39

described for Autographa califomica MNPV (AcMNPV; Baculoviridae:

Nucleopolyhedrovirus) in lepidopteran ceUs [13].

In order to demonstrate the infectivity of progeny virus, we designed an endpoint

dilution assay that detected at least a 10^-fold increase in infectious virus titer over a 7-day

period. This is the first study showing such an effect at the organismal level for a member

ofthe genus Iridovirus. Previous work with mosquito iridescent virus {Chloriridovirus)

showed infectivity, but not in an easily quantifiable manner [10, 11]. Our assay can be

easily adapted to measure infectious titer for any virus and should be useflil for viral

systems that lack assays based on cytopathic effect, such as TCIDso or plaque assays.

In summary, we show that Chilo iridescent vims imdergoes complete and productive

infection in boU weevil pupae upon deUvery via nicking ofthe integument. The cotton boU

weevil is an economicaUy important pest in the Western Hemisphere, and the present study

establishes it as an efficient host for CIV. Our results provide inportant baseUne data for

future studies on the transmission of this vims in the boU weevil and other hosts. These

results wiU also faciUtate future studies on pathology, tissue tropism, and metamorphic stage-

related susceptibiUty ofthe boU weevil to CIV. Although iridescent vimses are not highly

transmissible in the field [15], our data indicate that in a laboratory context, CIV undergoes a

productive infection cycle in the cotton boU weevil. Genetic manipulation of CIV or use of

viral genes responsible for pathogenesis could yield potentiaUy inportant tools toward a boU

weevil control strategy.

40

0 1 3 5 7 DPI M A V

Figure 2.1: Replication of Chilo iridescent vims (CIV) in boll weevil pupae. Alkaline lysates of individual boll weevil pupae were made at the indicated days post infection. The resulting lysates were blotted onto nitrocellulose and hybridized with " P-labeled total viral DNA as described in Materials and Methods. Signal was detected in vims-infected pupae (V), but not in mock-infected pupae (M) or in pupae treated with vims heated at 65 °C for 30 min (A).

41

Figure 2.2: CIV particle formation in boll weevil pupae. Insects were infected with CIV as described in Materials and Methods, incubated for 7 days at 21 °C, sectioned, stained with uranyl acetate, and observed by electron microscopy. A) Large numbers of complete vims particles and extensive virogenic stroma were evident (X 10,000). B) Cytoplasmic localization (nucleus (n) is shown) of virions was clear (X 10,000). C) Highly stmctured arrays of vims were observed, particularly in eye tissue (X 50,000). D) A higher magnification exhibited the characteristic intemal membrane (m) of CIV (X 138,000).

42

LOG DILUTION -1 -2 -3 -4 -5 -6 -7

0

7

7

7

M

• • • • • t

• • • • • #

• • • • t •

Figure 2.3: End-point dilution assay and increase in infectious CIV titer in boll weevil pupae. See Materials and Methods for details. Primary vims-infected pupae were incubated at 21 °C for 0 and 7 days (mock-infected pupae were incubated for 7 days). To measure increases in infectious viral titer, serial 10-fold dilutions of were prepared from pools of three pupae, and aliquots of each dilution were used to infect healthy batches of boll weevil pupae. These pupae were also incubated for 7 days at 21 °C. The presence of vims in the dilutions was then determined by titrating for viral DNA by dot blot hybridization. The highest dilution inducing detectable replication of viral DNA was defined as the titer. The results of three replicates showed an average increase in infectious viral titer of at least 10 -fold over the 7-day incubation period. Mock-infected pupae incubated for 7 days, or pupae infected with lysates of primary insects retrieved at day 0 post infection-did not yield any signal.

43

References

1. Beltwide Cotton Conferences, San Diego, CA, January 1998

2. BiUmoria SL (1991) The biology of nuclear poljiiedrosis vimses. In: Kurstak E (ed) Viruses of Invertebrates. Marcel Dekker, New York, pp 1-72

3. BoU Weevil Task Force Special Repori 97-101 1997

4. Brent R, Moore DD, Kingston RE, Ausubel F (1993) Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York

5. D'Costa SM, Yao H, Zhang R, BiUmoria SL (1999) Tenporal classification and mapping of Chilo iridescent virus transcripts. In: 99th General Meeting, ASM, Chicago, IL, pp 624

6. Fukaya M, Nasu S (1966) A Chilo Iridescent Vims (CIV) from the rice stem borer, Chilo suppresalis Walker (Lepidoptera, PyraUdae). AppUed Entomology and Zoology 1: 69-72

7. Henderson C, Johnson C, Lodhi S, BiUmoria S (1999) RepUcation of an insect virus in the cotton boU weevil, Anthonomus grandis. In: 99th General Meeting, ASM, Chicago, IL, pp 624

8. KeUy DC, Tinsley TW (1972) Proteins of iridescent virus type 2 and 6. Microbios Letters 9: 75-93

9. KeUy DC, Tinsley TW (1974) Iridescent vims repUcation: a microscopic study of Aedes aegypti and Antharaea eucalypti ceUs in culture infected with iridescent virus types 2 and 6. Microbios 9: 75-93

10. Linley JR, Nielsen HT (1968) Transmission of a Mosquito iridescent vims in Aedes taeniorhynchus. I. Laboratory experiments. J Invertebr Pathol 12: 7-16

11. Matta JF, Lowe RE (1970) The characterization of a Mosquito iridescent virus (MTV). I. Biological characteristics, infectivity, and pathology. J Invertebr Pathol 16: 38-41

12. McLaughUn RE, Scott HA, BeU MR (1972) Infection ofthe boU weevil by Chilo iridescent virus. J Invertebr Pathol 19: 285-290

44

13. Ohkawa T, VoDonan LE (1999) Nuclear F-actin is required for AcMNPV nucleocapsid morphogenesis. Virology 264: 1-4

14. Summers MD, Smith GE (1987) A manual of methods for baculovirus vectors and insect ceU culture procedures. Texas Agricultural Ejqierimental Station BuUetin

15. WiUiams T (1998) Invertebrate Iridescent Vimses. In: MiUer LK and BaU LA (eds) The Insect Viruses. Plenum Press, New York, pp 31-68

16. Yao H, D'Souza S, Lodhi S, BiUmoria SL (1996) Gene expression of a vims causing arrest and mortaUty in the cotton boU weevil. In: 72nd Annual Meeting, SWARM, AAAS, Flagstaff, AZ, pp 35

17. Yu L, Henderson C, Houck M, Bilimoria SL (1996) Viral induced mortaUty and metamorphic arrest in the cotton boU weevil. In: 72nd Annual Meeting, AAAS, SWARM, Flagstaff, AZ, pp 35

45

CHAPTER III

INDUCTION OF APOPTOSIS AND INHEBITION OF PROTEIN

SYNTHESIS BY A VIRION PROTEIN EXTRACT

FROM CHILO IRIDESCENT VIRUS

Introduction

Virus infection frequently results in ceU death by triggering a specific ceU death

response, or apoptosis [38]. Organisms are often able to respond to viral infection by

quickly destroying infected ceUs before the invading virus has completed an infection

cycle. Apoptosis is characterized by the fragmentation of ceUular DNA and packaging of

ceUular contents into smaUer vesicles (a process commonly referred to as blebbing) to

prevent leakage of ceUular contents. In mammaUan cells, the interferon system responds

to viral infection by activating 2'-5'-oUgoadenylate synthetase (2'-5' A synthetase) and a

protein kinase (PKR) [32, 35]. This synthetase activates RNase L, which destroys single-

stranded RNA. PKR phosphorylates the tianslation initiation factor eIF-2a, resulting in

the inhibition of protein synthesis. Studies with vaccinia virus (a cytoplasmic DNA virus

and thus, related to the Iridovimses in gene expression strategy) show that PKR induces

inhibition of protein synthesis and Ukely triggers the apoptosis response [2, 27, 40, 44].

Apoptosis has been demonstrated in many vimses including the family

Baculoviridae among insect viruses [15, 25]. In addition, two baculovirus genes, p35 and

iap, have been inqiUcated in the inhibition of apoptosis [7, 15, 17, 25]. Sequencing ofthe

CIV genome has revealed several genes with high similarity to the baculovims iap present

in the genome [1, 34]. However, no activity has been demonstrated for any of these

46

genes. In this study, we show that a virion extract from CIV induces apoptosis in cotton

boU weevil (AG3A) [39] and spmce budworm (CF124T) [6] ceU Unes. This phenomenon

is indicated by characteristic blebbing morphology as weU as DNA fi^gmentation.

Apoptotic fragmentation was shown by separating purified DNA on agarose gels.

This research shows that a virion extract not only induces apoptosis in treated budworm

(CF) and boU weevil (AG) ceUs, but also inhibits protein synthesis in the same ceU Unes.

The inhibition of protein synthesis is a key event in the induction of apoptosis by the

interferon system, and has also been demonstrated in another Iridovirus, Frog Vims 3. In

addition, earUer reports have shown that CIV infection and treatment with soluble extracts

from virions inhibit DNA, RNA, and protein synthesis in mosquito ceU Unes [12].

However, no flirther reports on this Une of research have emerged. We continue this

research using pulse-labeUng experiments to show that a protein conqionent ofthe CIV

extract inhibits protein synthesis. This inhibition is intense and rapid, with greater than

80% inhibition (using 10 |ag/ml) at three hrs post treatment. It is not clear at this point

whether the induction and inhibition of apoptosis are caused by the same polypeptide(s)

and therefore, should be studied further. In this study, we show that a soluble virion

extract from CIV induces apoptosis and inhibits protein synthesis in treated cells. The

blebbing response of apoptosis to this extract was quantified using an endpoint dilution

assay and DNA degradation was indicated by the electiophoretic separation of DNA

extracted from treated cells. Inhibition of protein synthesis was demonstrated by SDS-

PAGE analysis of radioactively pulsed protein sanples from treated cells. These results

47

suggest that coir^onents or genes from CIV may be useflil as a control measure for the

corton boU weevU.

Materials And Methods

CeU Line Maintenance

IPRI-CFl24T cells (CF) from the spmce budworm, Choristoneura fumiferana [6]

and BRL-AG-3A ceUs (AG) from the boU weevil, Anthonomus grandis [39] were grown in

Hink's TNM-FH medium [23] supplemented with 10% Fetal Bovine Serum (HyClone

laboratories) in Coming 25 cm^ flasks (Fisher) at 28 °C. CF and AG cells were typicaUy

subcultured every 6 days at a 1:10 ratio.

Virus Rearing

Chilo iridescent vims [20] was reared in larvae ofthe greater waxmoth, Galleria

mellonella. Waxmoth larvae (Sunfish Bait Company, Webster, WI) were nicked with

sharpened forceps dipped in a virus suspension (0.5 mg/ml). Larvae were checked every

three days, and dead or pupated insects were discarded. Survivors were frozen at -20°C

after a two-week incubation.

48

Vims Purification and Quantitation

Vims was purified from waxmoth larvae using a modification ofthe method of

KeUy and Tinsley [26]. The virions were differentiaUy centrifiiged and the sucrose-

gradient centrifiigation step was omitted to obtain higher virus yields. Quantitation of

vims was performed by spectrophotometric analysis. One unit of absorbance at 260 nm

(A260) represented 55 |ig/ml of virus [26].

Preparation of Virion Extiact

CHAPS (3-(3-Cholamidopropyl) dimethylammonio-1-propanesulfonate), a non-

ionic detergent (Sigma), was used to prepare the virion extract from differentiaUy

centrifuged virions in a method similar to that of Cemtti and DevaucheUe [13]. This

procedure differed mainly in the use of detergents (CHAPS, rather than the

octylglucoside, used by the previously mentioned researchers). We found that CHAPS

treatment yielded fewer polypeptides in the extract than octylglucoside treatment whUe

maintaining the same, or higher, activity. Virions were centrifiiged, and the resulting

peUet was suspended in the foUowing buffer: 10 mM Tris-HCl, IM KCl, pH 7.4, and 10

mM CHAPS. The vims suspension was incubated at 30 °C for 15 min to extract soluble

proteia The suspension was layered on 10% sucrose cushions and centrifuged at 36,000

rpm for 2 hrs in a Beckman SW41-Ti rotor. The clear supernatant was extensively

dialyzed using an Amicon stirred ceU apparatus (model no. 12) and MSI, Ultrasep discs

(25 mm) at 4 °C, 50 psi for approximately 4 hrs using virus buffer (50 mM Tris-HCl pH

7.4, 150 mM NaCl) to remove detergent. The final product was filtered using 0.22

49

MiUipore GV disposable filter and stored in microtubes at -70 °C. SDS-PAGE analysis

revealed no significant difference in polypeptide conqiosition between virion extracts

prepared from virus that was differentiaUy centrifiiged and extracts from virus purified by

sucrose gradient.

Bradford Assay for Determining Protein Concentration

Protein concentration was determined by the Bradford method [8] using bovine

serum albumin as a standard. The standard curve was then plotted, and the concentration

ofthe unknown determined.

Apoptosis Assay

Twenty microUters of virion extract dilutions were combined with 20 ^1 of ceU

suspension (7.5 x 10 ceUs/ml for both CF and AG ceUs). Fifteen microUters ofthe

resulting mixture were then added to Nunc Terasaki (60-weU) plates (Fisher). Two plates

were used in order to perform a dupUcate experiment. Various dilutions ofthe virion

extract to be tested were added to microtubes in a volume of 20 nl/repUcate (assays were

performed in dupUcate). The same volume of ceU suspension was then added to each

microtube. Concentrations used were 7.5 x 10 ceUs/ml for both CF and AG ceUs. Fifteen

microUters of each dilution was then added to each Terasaki plate. The plates were then

placed in a plastic bag containing a moistened paper towel (to maintain high humidity) and

incubated at 28 °C. CeUs were examined at 24 and 48 hrs post treatment for blebbing.

50

Endpoint Dilution Assay (Tissue Culture Toxicity Dose. TCTDsn) of Virion Extract

Once the phenomenon of apoptosis was estabUshed in each ceU Une, an end point

dilution assay was developed to conpare the sensitivities ofthe different ceU Unes to the

virion extract. The TCTDso assay was based on a viral end-point titiation assay [10].

Serial ten-fold dUutions ofthe virion extract were made in microtubes (10"' through 10"*)

in a final volume of 160 1 con^lete TNM-FH medium. An equal volume of ceU

suspension (3.5 x 10 ceUs/ml for CF or AG cells) was added to each dilution. Ten

microUters of each dilution was then added to a row (six weUs) in each of three Terasaki

plates. The first and last rows were set up as controls, without the protein preparation.

The plates were then placed in a plastic bag containing moist paper towel and incubated at

28 °C overnight. Wells showing greater than 50% blebbing ofthe total ceU population

were scored positive. The endpoint titer was calculated using the method of Reed and

Muench [33].

DNA Fragmentation Assay

CF124T or AG3A ceUs (3x10^ ceUs/weU) were added to 6-weU dishes in a total

volume of 3 ml. The trays were incubated at 28 °C overnight to aUow attachment. The

ceU monolayers were washed once with unsupplemented medium (TNM-FH) and 1 ml of

vims (larval derived; 10 |ig/ml), virion extract (CHAPS extract; 3 ng/ml), or actinomycin

D (positive control; 4 |ig/ml) was added into the respective weUs. Three negative controls

were included in the experiment. Mock-infected (virus mock) and mock-treated (extract

51

mock) cells were treated under the same conditions as virus-infected and virus extract-

treated, respectively, except that they did not contain virus or vims extract. Untreated

ceUs were not treated or washed in any way. The treatments were adsorbed for 1 hr at

21°C on a Bellco rocker platform at 2.5 rpia After adsorption, the volume in each weU

was made up to 3 ml with conplete TNM-FH medium (containing 10% fetal bovine serum

and 0.5 |ig/ml gentamicin), and the trays were incubated for 24 hrs at 21 °C for vims-

infected and 28 °C for extract-treated sanqiles. DNA was harvested 24 hrs after infection

or treatinent using 0.4M Tris-HCl (pH 7.5), 0.1 EDTA, 0.1% SDS and 200 ng/ml

Proteinase K solution (Sigma). After incubation at room tenqjerature for 12-16 hrs,

samples were extracted with phenol and ethanol precipitated [9]. Subsequently, the

samples were treated with 20 |ig/ml of RNase A (Sigma) for 30 min at 37 °C to digest any

residual RNA. Twenty micrograms per milUUter of each sarqile were then analyzed by

electrophoresis in 1.2% agarose gels. The DNA gels were stained with ethidium bromide

and observed under UV Ught.

Inhibition of Protein Synthesis

CF124T or AG3A ceUs were seeded at a concentration of 6.25 x 10 ceUs/ml ki

Coming 24-weU trays (Fisher) in 400 ^l of complete TNM-FH. Medium was removed

from the weUs and cells were washed with unsupplemented medium. CeUs were then

treated with 150 nl ofthe desired protein solution for a one-hour absorption. The medium

volume was then returned to 400 il for two hrs of incubatioa The ceUs were then starved

of methionine for one hr in 100 ^1 ExceU 401 methionine-free medium (JRH Biosciences)

52

and then labeled for two hrs with 150 1 of 80 i cyml 35s-methionine. CeUs were

removed from weU surfeces using a mbber poUceman. Samples were then centrifuged for

30 seconds and then resuspended in SDS-PAGE sample buffer. The san^les were boUed

for 5 min before loading and then fractionated on a 10% SDS-polyacrylamide gel. The

gels were fixed in 50% methanol, stained in Coomassie blue overnight, and destained in

50% methanol. Gels were treated in En^Hance (DuPont NEN) fluorography solution for

15 min, precipitated in ice-cold water for 15 min, and then dried at 60 °C for 4 hrs. The

dried gels were then exposed to X-ray film.

Results

Detection of Apoptotic Morphology

In order to obtain morphological evidence for apoptosis, budworm (CF) and boU

weevU (AG) ceUs were observed by phase-contrast microscopy. Both AG and CF ceU

lines were treated with the virion extract as described in Materials and Methods.

Observation ofthe ceUs after overnight incubation showed characteristic ceU blebbing

(Figure 3.1). In AG cells, some blebs remained attached to the cell, forming a "corona"

effect. As expected, mock-treated control ceUs did not show any blebbing.

Endpoint DUution Assay (Tissue Culture Toxicity Dose. TCTDsn) of Virion Extract

In order to develop an accurate assay for toxicity and to determine the exact

amoimt of virion extract required to induce blebbing, an assay similar to the TCIDso assay

53

used for baculovimses [10] was designed as described in Materials and Methods. The

results of this assay are summarized in Table 3.1. It was foimd that CF cells were more

sensitive requiring a mean of 6 ng/ml to give an endpoint titer, whereas AG ceUs required

a mean of 47 ng/ml. The amount of virion extiact required/7er cell was approximately

100 fg/ceU for AG ceUs and 10 fg/ceU for CF ceUs.

DNA Fragmentation in CF and AG CeUs

Experiments described above indicate that the virion extract induces blebbing

characteristic of apoptosis. Since DNA degradation is another haUmark of apoptosis,

assays designed to detect this effect were performed as described in Materials and

Methods. Figures 3.2 and 3.3 show that the virion extract induces DNA degradation

resulting in the fragmentation or smearing of DNA (lanes labeled E for both CF and AG

cells). Cells treated with infectious virus and the negative controls (mock-treated for

vims, mock-treated for extract) did not show any DNA degradation. The positive control,

actinomycin D, resulted in the characteristic degradation. A 1 kb DNA ladder (Gibco

BRL) was used as a marker.

Tnhihifinn of Protein Synthesis

Previous work with CIV showed that viral infection inhibited the synthesis of

DNA, RNA, and protein in mosquito ceU Unes [12]. The inhibition was even more

pronounced when ceUs were treated with a soluble extract from the vims. These assays

were performed as radiation uptake assays quantified by Uquid scintiUation counting. We

54

performed pulse-labeUng experiments to directly observe the rate of protein synthesis in

the CF124T and AG3A ceU Unes. CeUs treated with the extiact showed inhibition of

protein synthesis of over 85% (as determined by scanning densitometry) in both ceU Unes

at a concentration of 10 ^ig/ml (10 fg/ceU) as conqjared to imtieated cells and cells treated

with heated (65 °C, 30 min) extract (Figure 3.4).

Discussion

Chilo iridescent virus (CIV) has been shown to infect the corton boU weevU [30],

and earUer work in our laboratory has shown that CIV infection causes death and severe

deformity in infected boU weevils by an unknown mechanism [45]. In addition, CIV

infection and treatment with a CIV extract inhibit macromolecular synthesis in mosquito

ceU Unes [12].

The present study clearly shows that a soluble extract from CIV induces apoptosis

in two distinct ceU Unes (AG and CF), as observed by ceU blebbing and DNA

fragmentatioa Accurate and methodical testing ofthe virion extract for toxicity by the

TCTDso (based on the Reed and Muench method [33]) assay indicated that apoptosis

occurs in both ceU Unes. To yield a 50% titer, CF cells required only 6 ng/ml, whereas AG

cells required at least 47 ng/ml. CeUs treated with virion extract prepared from uninfected

larvae using the CHAPS extraction method did not yield any blebbing, indicating that the

blebbing effect is virion-specific. DNA fragmentation, the other iirportant haUmark of

apoptosis, was demonstrated in both ceU Unes by fractionating DNA from treated ceUs in

agarose gels. This fiagmentation was conqiarable to that generated by the positive

55

control, actinomycin D, which is a known chemical inducer of apoptosis [24]. As

expected, the negative controls (protein mock, mock-treated and imtreated) did not

exhibit this phenomenon, indicating that the active factor for apoptosis is one or more of

the virion proteins.

Having demonstrated the occurrence of DNA fragmentation, the apoptotic

sensitivity of each ceU Une to the extract was determined. CF and AG ceUs treated with

serial dUutions of virion extract were tested for DNA fragmentatioa It was found that a

minimum of 2 |ag/ml was required to generate the DNA degradation in both ceU Unes.

Lower concentrations ofthe virion extract (0.2 ng/ml in AG and 0.02 ig/ml in CF ceUs)

were able to induce blebbing but not DNA degradation as detected by agarose gel

electrophoresis. Although, it is known that DNA fragmentation is not always associated

with apoptosis [4, 11], a possible explanation for this effect in our system could be that

lower concentrations of virion extract induced blebbing, but the DNA degradation was not

extensive enough to be detected by our method.

We have shown that infection with heat-treated vims (30 min at 65 °C) and

tieatment of cells with heated virion extiact did not induce apoptosis, whereas infection

with whole virus resulted in negligible levels of DNA degradation [19]. The lack of

apoptosis seen with viral infection may be explained by the presence of putative

homologs ofthe baculovims inhibitor of apoptosis (iap) gene in the CIV genome [1, 34].

This suggests that CIV has evolved to prevent apoptosis that would normaUy occvir upon

infection by ejqiressing anti-apoptotic genes.

56

It is weU-estabUshed that some vimses are able to block the host apoptotic

response by expressing proteins that interfere with the ceU's abiUty to transduce a

particular ceU death signal or that interfere with the function of components of a ceU death

pathway [21]. Although apoptosis has been most extensively studied in mammalian

viruses, significant research has also been performed in baculovimses. Mutants ofthe

baculovirus anti-apoptotic genes (both iap an.dp35) induced apoptosis in infected cells

rather than a conplete, productive infection [16, 22, 29,41,42] indicating that these

genes prevented the apoptosis defense mechanism.

Although apoptosis is clearly demonstrated with both ceU Unes, the induction

process in not understood. One possibUity is the inhibition of protein synthesis induced by

the extract. Existing Uterature [2, 27,40, 44] has connected inhibition of protein synthesis

in systems involving vaccinia virus to the interferon-induced protein kinase that

phosphorylates and inactivates the translation initiation factor eIF-2a. Also, infection with

Frog virus 3 (another iridovirus) has been shown to cause phosphorylation and

inactivation ofthe eIF-2a factor [14, 43]. This suggests that a protein kinase might be

involved in similar processes in the CIV system. Our laboratory has shown that kinase

activity does exist in the virion extract (see Chapter IV/ unpublished data), and isolation

and characterization ofthe kinase gene is ongoing. SDS-PAGE analysis and kinase assays

of FPLC fractions have shown association of kinase activity with 17- and 44-kDa

polypeptides. It is possible that these polypeptides are also responsible for apoptotic

activity.

57

Interferon, an antiviral and anticeUular cytokine produced by mammaUan cells in

response to viral infection and other stimuli, might be an inducer of apoptosis through

transcriptional activation ofthe RNA-dependent protein kinase (PKR) gene [18]. Activation of

PKR by dsRNA, a frequent by-product of virus infection, leads to inhibition of protein

synthesis and induction of apoptosis in the infected ceU. This has been shown to be a central

component ofthe mechanism by which interferon induces apoptosis in vaccinia and other viral

systems. It is possible that a similar mechanism exists in the insect cells studied. Perhaps a

similar eIF-2a kinase is activated, resulting in the inhibition of protein synthesis and

consequently, apoptosis. A second possibUity is also related to a vaccinia vims example. A

vaccinia vims gene product BIR has been shown to phosphorylate the conqionents ofthe 40s

ribosomal subunits (S2&Sa) [3, 5] and inhibit translation [28]. SimUarly, kinase activity has

been associated with CIV, but this activity has not been isolated [31]. The BIR gene product

has also been inqiUcated in the inhibition of protem synthesis observed with vaccinia vims

infection. We have also shown the existence of a BlR-like gene in CIV. Based on the above,

we postulate two possible mechanisms for induction of apoptosis by CIV. The first mechanism

may involve interaction of a capsid corrponent with a ceUular receptor and activation ofthe

eIF-2a mediated pathway of inhibition. The second mechanism may involve phosphorylation

of ribosomal proteins by a BIR-Uke protein expressed by CIV. The BIR ORF has also been

identified in cowpox [37] and variola [36] vimses. There is clearly a conservation of this gene

within the vaccinia-related vimses, and with the finding of this gene in CIV, possibly

cytoplasmic DNA viruses, in general

58

It has been shown in baculovimses that insects have developed an apoptotic response

in order to combat viral infection by killing cells and preventing the completion of the viral

replication cycle. The virus, on the other hand, has apparently been able to counteract this

effect with anti-apoptosis genes. These genes prevent the cell death cycle, thus allowing a

complete replication cycle to occur. The present report is the first suggesting that this

partem is also observed in other insect viruses. The CIV system should be further developed

for a berter understanding of virus-host interactions, noting similarities and differences

between CIV and the well-studied baculovims systems. The resulting data from CIV may

eventually lead to identification and cloning of these genes for the production of transgenic

plants.

59

TREATED UNTREATED

CF

AG

Figure 3.1: Photomicrography of apoptotic morphology (X 900) induced by a virion extract of CIV. Cells were mock treated and treated with virion extract at a concen­tration of 2 (J.g/ml in Terasaki plates, then incubated at 28 °C overnight.

60

MW E EM AD

Figure 3.2: DNA fragmentation, characteristic of apoptosis, observed in CF cells. MW, molecular weight marker; E, virion extract (2 pig/ml); EM, mock for virion extract; AD, actinomycin D (4 |ag/ml); V, vims (10 ng/ml); VM, mock for vims.

61

MW E EM AD VM

Figure 3.3: DNA fragmentation, characteristic of apoptosis, observed in AG cells. MW, molecular weight marker; E, virion extract (2 ng/ml); EM, mock for virion extract; AD, actinomycin D (4 ng/ml); V, vims (10 ng/nnOi VM, mock for vims.

62

M A 10 25 50 M A 10 25 50

&

AG CELLS CF CELLS

Figure 3.4: Inhibition of protein synthesis in CF and AG cells induced by the virion extract. M, mock; A, heated virion extract (30 min and 65 °C); 50, 25, and 10 refer concentration of virion extract in ng/ml.

63

Table 3.1: TCTDso assay of CIV extt-act in CF and AG ceUs.

VIRION EXTRACT

(ng/ml)

20

2.0

0.2

0.02

0.002

0.0002

50% ENDPOINT

TITER (ng/ml) MEAN (ng/ml)

NUMBER OF WELLS WITH > 50% BLEBBING

BOLL WEEVIL CELLS (AG)

REPLICATE

1 2 3

++++++

++++++

++++++

58

++++++

++++++

++++++

++

37

++++++

+++-I-++

++++++

+

~ ~ ~

47

47

BUDWORM CELLS (CF)

REPLICATE

1 2 3

++++++

++++++

++++++

++++++

" • " " " •

• " " " " •

6

+++++-I-

++++++

++++++

++++++

" " " " " •

" * • " " " " "

6

++++++

++++++

+++++-

++++++

. . .

7

6

CeUs were treated with CHAPS extract from virions and assayed as described in Material and Methods. WeUs with greater than or equal to 50% blebbing were scored positive (+) and those with fewer than 50% blebbing were scored as negative (-). Endpoint titer was the highest dUution in which 50% ofthe wells were postitive, and was coir juted by the method of Reed and Muench [33]. The CHAPS virion extract induces apoptotic morphology in both ceU Unes, even at very low concentrations. The endpoint titer for CF ceUs is approximately 8 times lower than the titer for the AG ceUs.

64

References

1. Bahr U, Tidona CA, Darai G (1997) The DNA sequence of Chilo iridescent vims between the genome coordinates 0.101 and .391; similarities in coding strategy between insect and vertebrate iridovimses. Virus Genes 15: 235-245

2. Balachandran S, Roberts PC, Kipperman T, BhaUa KN, Compans RW, Archer DR, Barber GN (2000) Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaUng pathway. J Virol 74: 1513-23

3. Banham AH, Leader DP, Smith GL (1993) Phosphorylation of ribosomal proteins by the vaccinia vims BIR protein kinase. FEBS Lert. 321: 27-31

4. Barres BA, Hart IK, Coles HS, Bume JF, Voyvodic JT, Richardson WD, Raff MC (1992) CeU death and control of ceU survival in the oUgodendrocyte Uneage. CeU 70: 31-46

5. Beaud G, Sharif A, Topa-Masse A, Leader DP (1994) RiTxisomal protein S2/Sa kinase purified from HeLa ceUs infected with vaccinia virus corresponds to the BIR protein kinase and phosphorylates in vitro the viral ssDNA-binding proteia J Gen Virol 75: 283-93

6. BUimoria SL, Sohi SS (1977) Development of an artached strain from a continuous insect ceU line. In Vitro 13: 461-466

7. Bimbaum MJ, Clem RJ, MUler LK (1994) An apoptosis-inhibiting gene from a nuclear poljdiedrosis vims encoding a polypeptide with Cys/His sequence motifs. J Virol 68: 2521-8

8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utUizing the principle of protein-dye binding. Anal. Biochem. 72: 248-54

9. Brent R, Moore DD, Kingston RE, Ausubel F (1993) Current Protocols in Molecular Biology. Greene Publishing Associates and WUey-Interscience, New York

10. Brown M, Faitikner P (1975) Factors affecting the yield of virus in a cloned ceU Une of Trichoplusia ni infected with a nuclear polj^edrosis virus. J. Invertebr. Pathol 26: 251-257

65

11. Catchpoole DR, Stewart B (1993) Etoposide-induced cytotoxicity in two human T-ceU leukemic Unes: delayed loss of membrane penneabiUty rather than DNA fragmentation as an indicator of programmed ceU death. Cancer Res. 53: 4287-96

12. Cemrti M, DevaucheUe G (1980) Inhibition of host macromolecular synthesis in cells infected with an invertebrate virus. Archives of Virology 63: 297-

13. Cemrti M, DevaucheUe G (1990) Protein con^osition of Chilo iridescent vims. In: Darai G (ed) Molecular Biology of Iridovimses. Kluwer Academic Press, Boston, pp 81-112

14. Chinchar VG, Dholakia. JN (1989) Frog vims 3- induced translational shutoff: activation of an eIF2 kinase in vims infected ceUs. Virus Res. 14: 207-224

15. Clem RJ, Fechheimer M, MUler LK (1991) Prevention of apoptosis by a baculovirus gene during infection of insect cells. Science 254: 1388-90

16. Clem RJ, MUler LK (1994) Control of Programmed CeU Deatii by the Baculovims Genes p35 and iap. Molecular and CeUular Biology 14: 5212-5222

17. Crook NE, Clem RJ, MUler LK (1993) An apoptosis-inhibiting baculovirus gene with a zinc finger-Uke motif J Virol 67: 2168-74

18. Diaz-Guerra M, Rivas C, Esteban M (1997) Activation of the IFN-inducible enzyme Rnase L causes apoptosis of animal ceUs. Virology 236: 354-363

19. D'Souza S, Henderson C, Lodhi S, Glass A, Yao H, BUimoria S (1997) CeU Culture Models for RepUcation of an Insect Vims Pathogenic to the Corton BoU WeevU. In: 73rd Annual Meeting, SWARM, AAAS, CoUege Station, TX, pp 4

20. Fukaya M, Nasu S (1966) A ChUo Iridescent Virus (CIV) from the rice stem borer, Chilo suppresalis Walker (Lepidoptera, PyraUdae). AppUed Entomology and Zoology 1:69-72

21. Gooding LR (1992) Virus proteins that coimteract host immune defenses. CeU 71: 5-7

22. Hawkins CJ, Uren AG, Hacker G, Medcalf RL, Vaux DL (1996) Inhibition of interleukin 1 beta-converting enzyme-mediated apoptosis of mammalian ceUs by baculovirus IAP. Proc Natl Acad Sci U S A 93: 13786-90

23. Hink WF (1970) EstabUshed insect ceU Une from the cabbage looper, Trichoplusia ni. Natiire 226: 466-467

66

24. Ijiri K, Potten CS (1983) Response of intestinal ceUs of differing topographical and hierarchical status to ten cytotoxic dmgs and five sources of radiation. Br J Cancer 47: 175-85

25. Kamita SG, Majima K, Maeda S (1993) Identification and characterization ofthe p35 gene of Bombyx mori nuclear pol>iiedrosis virus that prevents virus-induced apoptosis. J Virol 67: 455-63

26. KeUy DC, Tinsley TW (1972) Proteins of iridescent virus type 2 and 6. Microbios Lerters 9: 75-93

27. Kftiler KV, Shors T, Perkins KB, Zeman CC, Banaszak MP, Biesterfeldt J, Langfield JO, Jacons BL (1997) Double stianded RNA is a trigger for apoptosis in vaccinia virus-infected cells. J. Virol 71: 1992-2003

28. Kovacs GR, Moss B (1998) Regulation of gene expression by the vaccinia vims protein kinase-1. In: Twelfth Intemational Poxvirus Symposium, St.Thomas, United States Virgin Islands, pp 66

29. Manji GA, Hozak RR, LaCount DJ, Friesen PD (1997) Baculovirus inhibitor of apoptosis functions at or upstream of the apoptotic suppressor P35 to prevent programmed ceU death. J. Virol 71: 4509-16

30. McLaughlin RE, Scort HA, BeU MR (1972) Infection of tiie boU weevU by ChUo iridescent virus. J Invertebr Pathol 19: 285-290

31. Monnier C, DevaucheUe G (1976) Enzyme activities associated with an invertebrate iridovims: nucleotide phosphohydrolase activity associated with iridescent virus type 6 (CIV). J Virol 19: 180-6

32. Pestka S, Langer JA, Zoon DC, Samuel CE (1987) Interferons and their actions. Annu. Rev. Biocheia 56: 727-77

33. Reed L, Muench H (1938) A sinqile method for estimating fifty percent endpoints. American Journal of Hygiene 27: 493-497

34. Schnitzler P, Hug M, Handermann M, Janssen W, Koonin EV, DeUus H, Darai G (1994) Identification of genes encoding zinc finger proteins, non-histone chromosomal HMG protein homologue, and a putative GTP phoshohydrolase in the genome of ChUo iridescent virus. Nucleic Acids Research 22: 158-164

35. Sen GC, Lengyel P (1992) The interferon system; A bird's eye view of its biochemistry. J. Biol Chem. 267: 5017-20

67

36. ShcheUamov SN, BUnov VM, Sandakhchiev LS (1993) Genes of variola and vaccinia vimses necessary to overcome the host protective mechanisms. FEBS Lett 319: 80-3

37. Shchelkunov SN, Safronov PF, Totinenin AV, Petiov NA, Ryazankina 01, Gutorov W , Kotwal GJ (1998) The genomic sequence analysis of the left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins. Virology 243: 432-60

38. Shen Y, Shenk TE (1995) Viruses and apoptosis. Curr. Opia Gea Develop. 5

39. StUes B, McDonald IC, Gerst JW, Adams TS, M. NS (1992) Initiation and characterization of five embryonic ceU Unes from the corton boU weevil, Anthonomus grandis, in a commercial serum-free medium. In Vitro CeU Dev. Biol. 28A: 355-363

40. Tan SL, Katze MG (1999) The emerging role of the interferon-induced PKR protein kinase as an apoptotic effector: A new face of death? J. Interferon. Cytokine Res. 19: 543-54

41. Vucic D, Kaiser WJ, Harvey AJ, MUler LK (1997) Inhibition of reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (lAPs). Proc Natl AcadSciUSA94: 10183-8

42. Vucic D, Kaiser WJ, MiUer LK (1998) A mutational analysis ofthe baculovims inhibitor of apoptosis Op-L\P. J. Biol Chem. 273: 33915-21

43. WilUs DB, Goorha R, Chinchar VG (1985) Macromolecular synthesis in ceUs infected by frog vims-3. Curr. Topics Microbiol Immunol. 116: 77-106

44. Yeung MC, Chang DL, Camantigue RE, Lau AS (1999) Inhibitory role ofthe host apoptogenic gene PKR in the establishment of persistent infection by encephalomyocarditis vims in U937 ceUs. Proc Nati Acad Sci U S A 96: 11860-5

45. Yu L, Henderson C, Houck M, BUimoria SL (1996) Viral induced mortaUty and metamorphic arrest in the corton boU weevU. In: 72nd Annual Meeting, AAAS, SWARM, Flagstaff, AZ, pp 35

68

CHAPTER IV

IDENTIFICATION AND CHARACTERIZATION OF A CW

OPEN READING FRAME WITH HIGH SIMILARITY TO

THE VACCINIA VIRUS BIR GENE

Introduction

Several members ofthe FamUy Iridoviridae are known to drasticaUy inhibit gene

expression in their hosts. This phenomenon has been weU studied in another cytoplasmic

DNA virus, vaccinia vims, which serves as an exceUent model for the study of host gene

ejqiression in the Iridoviridae. One mechanism of inlubition in vaccinia virus is through

induction ofthe interferon host defense system [6], which phosphorylates the translation

factor eIF-2a. Another mechanism of inhibition also exists involving the BIR protein

kinase [7]. The vaccinia virus BIR protein kinase has been shown to phosphorylate

ribosomal proteins [1,2] and was iiiqjUcated in the inhibition of protein synthesis in a

separate study [7]. In this study, we describe the characterization of a BlR-Uke gene in

the Chilo iridescent virus (CIV) genome. Sequencing and analysis of CIV DNA in our

laboratory revealed regions in adjacent EcoR I fragments (B and U) with similarity to the

BIR protein of vaccinia vims. PCR primers were therefore designed to ampUfy the DNA

between the two regions. The PCR product was sequenced and then analyzed for

similarity to the vaccinia BIR gene and for the presence of kinase-related signature

sequences. Northern blotting analysis using PCR-generated internal, gene-specific probes

confirmed that the BIR-Uke gene is expressed in CIV-infected boU weevU ceUs (BRL-

69

AG3A [13]). With this work, we show that a putative homolog ofthe vaccinia BIR

proteui kinase exists in the CIV genome and is expressed upon infection. The potential

role of this protein in the inhibition of macromolecular synthesis and selective ceU death as

weU as in^iUcations for contiol ofthe corton boU weevU are discussed.

Materials and Methods

DNA Sequencing and Analysis

Sequencing was performed using a Perkin-Elmer/AppUed Bipsystems Model 310

DNA sequencer. As part of a sequencing project in our laboratory, several EcoR I

fragments of our restriction fragment library were sequenced. After BLAST analysis of

DNA sequenced from fragments B and U showed open reading frames with similarity to

the vaccinia BIR protein, the region between tiiese two sequenced portions was ampUfied

by PCR. The resulting anqiUcon was then sequenced by primer waUdng. AUgnment of the

putative CIV ORF protein sequence with the vaccinia BIR protein sequences was

performed using the CLUSTALW program. Both sequences were analyzed for motife

using the PROSITE program.

PCR AnyUfication for DNA Sequencing

Two sets of PCR primers were designed from the above CIV sequence to ampUfy

the CrV BIR-Uke sequence. The first set of primers was designed to an^Ufy an intemal

sequence (1.2 kb) ofthe BIR-Uke gene that would be used for Northern blorting. The

second set of primers was designed to ampUfy a longer region (2.0 kb) that would be

70

suitable for DNA sequencing. PCR anpUfication ofthe BIR-Uke region was performed

using a Perkin-Ehner GeneAn^ PCR System 2400 thermal cycler. Forward and reverse

primer sequences synthesized for ampUfication ofthe intemal fragment were: 5'-ATGGA

TCTTAAAGACGAATTTATTC-3' and 5'-ATTTTGATGATGGTTTCAAAATACG-3',

respectively. Forward and reverse primer sequences synthesized for an iUfication ofthe

larger fragment were: 5'-TTTGGTAGTTGGGAACGGCTCATCT-3' and 5'-AAGTTGA

AAAAACCAATGTAATGAC-3', respectively (Figure 4.1). PCR reactions were

performed (in a final volume of 50 nO using 5 \i\ of lOX Fisher Biotech reaction buffer,

1.5 mM MgCl2, 200 nmoles of each NTP, 66 ng/ml of template DNA, 1 nmole of forward

and reverse primer, 2.5 units of Takara Taq polymerase (Fisher Biotech), and 32.5 nl H2O.

After an initial denaturation step at 94 °C for 5 minutes, 30 cycles of denaturation,

anneaUng, and elongation were performed successively at 94 °C, 55 °C, and 72 °C for 1

minute each. A final elongation was performed at 72 °C for 7 minutes. Products for each

PCR reaction are seen in Figure 4.2.

Vims Infections and Passaging

BRL-AG-3A [13] and IPRI-CF-124T [3] ceUs were grown to 80% confluency in

Coming 25 cm^ flasks at 28 °C and infected with ceU culture-derived CIV at an MOI of

20 lU/ml. Adsorption was for 1 hour at 21 °C and infected ceUs were incubated at 21 °C.

Viral infections were carried out according to the method of D'Costa [5]. Briefly, ceUs

were first infected with larval-derived vims imder one-step growth conditions. CeU

culture medium was harvested after four days and centrifuged. Supernatant was harvested

71

and saved as passage #1 (PI) vims. This was then used as a stock for subsequent ceU

culture-derived virus infections.

RNA Isolation and Northem Blot Analysis

BRL-AG3 A ceUs were infected with virus as described above and total RNA was

extracted by differential phenol extraction [10] at 24 h p.i. RNA was solubilized in 100%

formamide and fractionated on 1% agarose gels containing formaldehyde. Northem blots

(Sambrook, et al [10]) were performed using primers intemal to the CIV open reading

frame. The forward and reverse primers for the intemal fragment were 5'-ATGGATCTT

AAAGACGAATTTATTC-3' and 5 '-ATTTTGATGATGGTTTCAAAATACG-3',

respectively as described earUer. Blorting was performed on a nitroceUulose membrane

(MSI, Westborough, MA) and probed under conditions of high stringency (50%

formamide, 5X SSC, 0.1% SDS at 42 °C). The 1.2 kb probe was random-prime labeled

according to the manufecturer's instmctions (Boehringer Mannheim). Molecular weights

ofthe transcripts were determined using a standard RNA ladder (0.24-9.5 kb; Gibco-

BRL).

Results

Amplification and Sequencing ofthe BIR-Uke ORF

Sequencing ofthe CIV EcoR I fragments B and U revealed regions of DNA with

similarity to the BIR protein kinase of vaccinia virus. In order to estabUsh if these were

separate portions ofthe same open reading frame, PCR was performed using primers from

72

each restriction fragment using regions outside the open reading frame. The PCR

anqiUfied product was separated on a 1% agarose gel and a product of approximately 2.0

kb was extracted and purified (using the QIAquick DNA extraction kit (Qiagen, Valencia,

CA). Sequencing of this PCR product using primer waUcing showed a single open reading

frame containing relevant sequences observed in the EcoR I fragments B and U regions.

The conplete open reading frame is 1.2 kb, with the conq)lete sequence shown in Figure

4.3.

Similarity and Analysis ofthe CIV Open Reading Frame

BLAST analysis performed on the CIV open reading frame showed a high degree

of similarity with many protein kinases. High similarity was observed with the vaccinia

virus BIR protein kinase, and strong similarity was also seen with open reading frames

from other poxvimses [11, 12], as weU as the human genes, VRKl and VRK2 [8], and a

protein isolated fromMus musculus [16]. However, the vaccinia BIR is the only highly

related kinase with a known function. The similarity ofthe predicted amino acid sequence

ofthe ORF vvdth the vaccinia BIR is 56% over a 308 amino acid region, with 38% identity

over the same region. Similarity and identity ofthe predicted amino acid sequence ofthe

CIV ORF with other protein sequences is shown in Table 4.1. AUgnment ofthe predicted

amino acid sequence ofthe CIV open reading frame and the vaccinia BIR was performed

using the CLUSTALW program [14] and the results are seen in Figure 4.4. The vaccinia

BIR and putative CIV homolog amino acid sequences were scanned for motifs using the

PROSITE database. Analysis revealed the presence of a serine/threonine protein kinase

73

signature sequence in both proteins. In addition, an ATP binding motif characteristic of

protein kinases was found on the CIV open reading frame, but not in the vaccinia BIR.

Northem Analysis ofthe BIR-Like Gene

In order to confirm if the CIV BIR is expressed in infected BRL-AG3A cells,

Northem blot analysis was performed. A probe was constmcted by PCR using primers

intemal to the CIV ORF and used to detect the presence ofthe transcript in lysates taken

from virus infected and mock infected ceUs 24 hr p. i. The results show the presence of a

1.8 kb transcript in virus infected ceUs and no transcript in mock infected cells (Figure

4.5).

Discussion

Chilo iridescent virus (CIV) is the only viral agent known that has been shown to

infect the corton boU weevU, a highly important pest of corton. Work in our laboratory

has shown that CIV causes death and metamorphic deformity at a rate of nearly 70% over

controls [15]. However, the mechanism of this activity has not yet been estabUshed.

EarUer studies have demonstrated that CIV infection, or treatment with a virion extiact of

CIV resulted in inhibition of macromolecular synthesis [4], but this activity has not been

isolated. Sequence analysis of CIV EcoR I restriction fragments B and U performed in

our laboratory revealed a single ORF with simUarity to the vaccinia virus protein kinase,

BIR. The BIR protein kinase has been shown to phosphorylate the Sa and S2 ribosomal

74

proteins [1,2] and has been impUcated in the inhibition of protein synthesis [7]. These

studies suggest a potentiaUy in^ortant role for this enzyme in the CIV repUcation cycle.

The polymerase chain reaction was used to an^Ufy approximately 2 kb of CIV

DNA overlapping the ends ofthe EcoR IB and U restriction fragments. This DNA was

then sequenced by primer waUdng and analyzed for similarity to other known genes and

for the presence of known protein motifs. The results show that the CIV open reading

frame has extensive similarity with the BIR protein kinase and simUar kinases in other

members ofthe Poxvirus famUy. Motif analysis using PROSITE showed that the open

reading frame has two motife characteristic of protein kinases. The first of these is a

serine/threonine protein kinase signature, and the second is a protein kinase ATP binding

signature. This strongly suggests that the open reading frame encodes a protein kinase.

Northem analysis performed using an internal PCR-an^Ufied probe confirmed the

expression of this open reading frame.

Although the precise fimction ofthe BIR protein kinase in vaccinia infections has

not been determined, this enzyme appears to be essential to the viral Ufe cycle. Early

studies used tenperature sensitive mutants ofthe BIR gene which were found defective in

viral DNA repUcation [9]. Other studies have shown that mutations in the BIR kinase

significantly delay the normaUy pronpt inhibition of protein synthesis [7]. Other work in

our laboratory has shown that CIV also rapidly inhibits host protein synthesis, suggesting

that these effects may be related. Additional reports have shown that the BIR protein

kinase phosphorylates the Sa and S2 nTx)somal proteins [2], which creates a tantaUzing

link between phosphorylation activity and the inhibition of protein synthesis. It is quite

75

possible that the BIR-Uke gene of CIV is involved in the phosphorylation of ribosomal

proteins in infected cells or in host shutdown by other mechanisms. The feet that

homologs ofthe BIR kinase have been found in other viruses [11, 12] suggests the BIR-

Uke CIV gene may have a key fimction in CIV repUcatioa Our study provides in^ortant

and essential data for the elucidation of that function in a virus, which has potentiaUy

important impUcations for cotton boU weevU control

76

- • 2,015 bp product (for sequencing) ^

Forward: 5 -TTTGGTAGTTGGGAACGGCTCATCT-3

Reverse: 3-CAGTAATGTAACCAAAAAAGTTGAA-5

1,236 bp CIV BlR-like gene

»^^183 bp product (for Northern

Forward: 5-ATGGATCTTAAAGACGAATTTATTC-3

Reverse: 3 -GCATAAAACTTTGGTAGTAGTTTTA-5

Figure 4.1: Primers used for PCR amplification. Two sets of PCR reactions were performed. The first reaction amplified a 2,015 bp region surrounding the CIV BlR-like gene and was used for DNA sequencing. The second reaction amplified a 1,183 bp region intemal to the open reading frame and was used for Northern blotting. The different regions are depicted and the primers used for the respective amplifications are shown. The 1,236 bp open reading frame is also shown.

77

2.0

P M

1.2

Figure 4.2: PCR amplification ofthe putative CIV BIR homolog. Two separate sets of PCR primers (see Fig. 4.1) were used to amplify viral DNA external to the open reading frame (A) for DNA sequencing and intemal to the open reading frame (B) for Northem blotting. The PCR product (P) and molecular weight markers (M) are shown.

78

map nnlts 0.728 £

B 1 90 180 270 360 450 540 630 720 810 900 990 1080 1170 1260 1350 1440 1530 1620 1710 1800 1890 1980

TGTAGTTGGGAACGGCTa^TCTTCAATTAATTGAATTAAT<K»TTTTTATTrTCGAAAAGGTCGTCGTTT<nTAAATTTAATA;^ GTGATTCATTTAATTTAAAATTAGGAATATCGTTTTCGTCJU^TAACAATTTTTATAGTTTTACTATCTTTTACTTCT^ GTTATATATCCATTTTTATTTAAAATTTCTACAAAATTTCTTCCTAOVTCATCTGCTTTATCATCTGTATCACTTTCATATC TATAACTGCACaiCGACTCTTCTTCAATTTCTTTATATAATTTATGGATATCTGTATTGAATGTAAAAACATATCCTTTTTCTAATGGTT CATCAAAATACTTTAAAAATTCTTTTCTTCCTTTTAAAATATTAAGATCTGCGTCATCTGAATTTTTACSATAAGAATCTACATCAAGA AATAACAAAATACCTCTTTCATACAATTCTATAAATACGTTTTCAAGCATTTTATTAGGTAAAAAATTACTTATAACGTTAAGAAAATT GAAGTTTAAATTTTTAACTATAAACATTTTTAATGACACAAAATGGATCTTAAAGACGAATTTATTCAAATAATTAAAAAGTATTCTGA ACTTTCTCTTAAGGAATCTGAAAACGAAACAAAAAAATATATACAAGAAGTATTTAACGTATCGTTGTCTGAAGTATTTATAAAAGAAC CATCAAAAOVGGAAACaiTTAAAACAGGAACCAACTCAAGAAACCAATGGATGTATTTATATTTTTAAAAAAGGAAAAAATATAGGACAA AAATGTGGTTCTGGAAAATCTAAATTIT^STTATAAACATAAAAAGAATGATTTAAATATAAAAGTGAATCAAGAAAGAAATATTAAAAA ACCTATACCGAAACCTATACCAACAAACAAAATTGAAACCGGATCAGTTATCAATCAGAATTGGTTTATAGGAACTTCAATAGGTAAGG GAGGTTTTGGAGAAATATATTCGGCTGCTAAATTTAATGATCATGGTAACTACAAAGACGACGATTTTAGTTTTGCAATTAAAATCGAA CCCAAAAGTAATGGACCCTTATTTGTTGAAATGCATTTTTACAAACGCGTTATTGTAGAAAAAGAAATTGAAAAATTTAAACTTCAAAA AAATATTCAATATTTAGGATTACCTAAATATTATGGATCTGGATTGTACAATGATTATAGATATATTGTTATGGAAAAGTATGATTCAA ACATTGATAAGTTGTTTAGAAATGGTGACTTAAATTCTTCTACAATTTTACAAATAGGAATTOUU^TATOAAACATTGTGGAGTACaiTC CATTCTAAAGGATATATTCATGCTGACATTAAGGGCGAAAATATACTATTGAAAGAAAATGATACTTCTCATATTTATTTAGTTGATTT TGGATTATGTTCTCGTTATAGTAACATTTATCAACCAGATCCTAAAAAAGCACATAATGGTACATTAAAATATACAAGCAGAGATAGCC ATCAAGGTGTTGAATCJ^GAAGAGGAGATTTAGAAATTCTAGGCTATAATATGATAGAGTGGTCTGGTGGTATTTTACCATGGCAACAT TTATGTAAAAAGAGTGCTGGAAAAAAGATTTTAAACGAAGTTTCAGAAAGTAAAATAAAATATATQAATGATTTATCTTTGTTTTTTAA CAAAGTTCAATTTAACGACACCAATTTAAAAAATAAATTACAAACGTATTTrGAAACCATCATCUU^AATAACATTTGAAGAATTACCAC CTTATCAACTATTACATAACATTTTTAACTAACATATTTAACTAATTTTTATTTTAATGGACAATGTCCATTAAAATAAATAAGAGGAG CAGTACGAATTAATTAACAATTAGTAATTCTTGCCTAGCTTGTTTGATAACACTTTCTGGTATAGATTTAAATTCTTCATGAACGGATA CACCGCTTTTCAACCATTACTTGCCAAAAATAATCTGTCATTACATTGGTTTTTTCAA

Figure 4.3: Mapping and sequencing ofthe BlR-like gene in the CIV genome. A) The open reading frame was identified and mapped to a region (fiUed block) spanning the EcoR I site separating fragments B and U. EcoR I sites (marked "E") are indicated with Unes on the circular map and given a letter designation in the inset. B) The nucleotide sequence ofthe 2 kb sequenced region. Probable start and stop sites are bold and underlined.

79

CIV MDLKDEFIQIIKKYSELSLKESENETKKYIQEVFNVSLSEVFIKEPSKQETLKQEPTQET BIR -

CIV NGCIYIFKKGKNIGQKCGSGKSKFCYKHKKNDLNIKVNQERNIKKPIPKPIPTNKIETGS BIR MNFQGLVLTDN

: * ...

ATP binding CIV VINQNWFIGTSIGKGGFGEIYSAAKFNDHGNYKDDDFSFAIKIEPKSNGPLFVEMHFYKR BIR CKNQ-WWGPLIGKGGFGSIYTTN D-NNYWKIEPKANGSLFTEQAFYTR

CIV VIVEKEIEKFKLQKNIQYLGLPKYYGSGLYN DYRYIVMEKYDSNIDKLFRNGD - -L BIR VLKPSVIEEWKKSHNIKHVGLITCKAFGLYKSINVEYRFLVINRLGADLDAVIRANNNRL

s/t kinase CIV NSSTILQIGIQILNIVEYIHSKGYIHADIKGENILLKENDTSHIYLVDFGLCSRYSN-- -BIR PKRSVMLIGIEILNTIQFMHEQGYSHGDIKASNIVLDQIDKNKLYLVDYGLVSKFMSNGE

CIV - -lYQPDPKKAHNGTLKYTSRDSHQG-VESRRGDLEILGYNMIEWSGGILPWQHLCKKSA BIR HVPFIRNPNKMDNGTLEFTPIDSHKGYWSRRGDLETLGYCMIRWLGGILPWTKISET-

. : * : * . * * * * : : * . * * * ; * * * * * * * * * * * . * • * * _ * * * * * * * ; : . : .

CIV GKKILNEVSESKIKYMN-DLSLFFNKVQFNDTNLKNKLQTYFETIIKITFEELPPYQLLH BIR - - KNCALVSATKQKYVNNTATLLMTSLQYAPR ELLQYITMVNSLTYFEEPNYDEFR

* **.***.* :*::..:*: :* *: :.:*:* * * : : :

CIV NIFN BIR HILMQGVYY

. * .

Figure 4.4: AUgnment ofthe CIV BIR-Uke protein predicted amino acid sequence with that ofthe vaccinia BIR protein kinase. The CLUSTALW program was used to perform the aUgnment, and the PROSITE program was used to scan each amino acid sequence for motifs. Signature sequences characteristic of serine/threonine protein kinases were observed for both CIV and vaccinia sequences, whUe an additional ATP binding site characteristic of protein kinases was observed in the CIV sequence. These signatures are indicated above the sequence and by bold-feced type in the sequence. Levels of low simUarity (.), high simUarity (:), and identity (*) are indicated.

80

V M

1.8 kb

Figure 4.5: Expression of CIV BlR-like gene in infections of 5i?Z,-/iG-i^ cells. Cells were infected with CIV at a multiplicity of infection of 20. Infections were carried out under one-step conditions and incubated at 21 °C. Total cellular RNA was extracted at 24 h p.i. and fractionated on a 1.0 % denaturing agarose gel. RNA was blotied onto nitrocellulose membrane and northem analyses carried out using PCR-amplified intemal sequences of CIV BlR-like gene as probe.

81

Table 4.1: Comparison ofthe predicted CIV BlR-lUce polypeptide to other known proteins.

ORGANISM Yaba monkey tumor vims' Mus musculus

vaccinia virus Homo sapiens

Homo sapiens

fowlpox virus*

GENE Yb-C3R 51PK BIR VRKl VRK2

FPV 212

% IDENTITY 41

39 38 36 37 32

% SIMILARITY 60 59 56 58 54

55

BLAST conqiarison ofthe CIV open reading frame to identified genes shows a high amount of identity (exact amino acid match) and similarity (similar amino acids) to several putative protein kinases. The polypeptides marked ("*) aU belong to the famUy Poxviridae. To this point, only the function ofthe BIR gene in vaccinia virus has been studied.

82

References

1. Banham AH, Leader DP, Smith GL (1993) Phosphorylation of ribosomal proteins by the vaccinia vims BIR protein kinase. FEBS Lett. 321: 27-31

2. Beaud G, Sharif A, Topa-Masse A, Leader DP (1994) Ribosomal protein S2/Sa kinase purified from HeLa ceUs infected with vaccinia virus corresponds to the BIR protein kinase and phosphorylates in vitio the viral ssDNA-binding protein. J Gen Virol 75: 283-93

3. BUimoria SL, Sohi SS (1977) Development of an attached strain from a continuous insect ceU Une. In Vitro 13: 461-466

4. Cemrti M, DeauvecheUe G (1980) Inhibition of host macromolecular synthesis in ceUs infected with an invertebrate virus. Archives of Virology 63: 297-

5. D'Costa SM (1999) Regulation of gene expression and transcription mapping of an insect iridescent virus. Ph. D. Dissertation. Biology. Texas Tech University, Lubbock, Texas

6. Gale M, Katze MG (1998) Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol Ther. 78: 29-46

7. Kovacs GR, Moss B (1998) Regulationofgeneexpressionbythe vaccinia vims protein kinase-1. In: Twelfth Intemational Poxvims Synqiosium, St.Thomas, Uiuted States Virgin Islands, pp 66

8. Nezu J, Oku A, Jones MH, Shimane M (1997) Identification of two novel human putative serine/threonine kinases, VRKl and VRK2, with stmctural similarity to vaccinia virus BIR kinase. Genomics 45: 327-31

9. Renqiel RE, Traktman P (1992) Vaccinia vims Bl kinase: phenotypic analysis of ten:5)erature-sensitive mutants and enzymatic characterization of recombinant proteins. J Virol 66: 4413-26

10. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning, Second Edition edn. Cold Spring Harbor Laboratory Press, Cold Springs Harbor, NY

11. ShcheUcunov SN, BUnov VM, Sandakhchiev LS (1993) Genes of variola and vaccinia vimses necessary to overcome the host protective mechanisms. FEBS Lert 319: 80-3

83

12. ShcheUamov SN, Safronov PF, Totinenin AV, Petrov NA, Ryazankina 01, Gutorov W , Kotwal GJ (1998) The genomic sequence analysis ofthe left and right species-specific terminal region of a cowpox virus strain reveals unique sequences and a cluster of intact ORFs for immunomodulatory and host range proteins. Virology 243: 432-60

13. StUes B, McDonald IC, Gerst JW, Adams TS, M. NS (1992) Initiation and characterization of five embryonic ceU Unes from the corton boU weevil, Anthonomus grandis, in a commercial serum-free medium. In Vitro CeU Dev. Biol 28A: 355-363

14. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence aUgnment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nuc Acids Res 22: 4673-80

15. Yu L, Henderson C, Houck M, BUimoria SL (1996) Viral induced mortaUty and metamorphic arrest in the cotton boU weevU. In: 72nd Annual Meeting, AAAS, SWARM, Flagstaff, AZ, pp 35

16. ZeUco I, Kobayashi R, Honkakoski P, Negishi M (1998) Molecular cloning and characterization of a novel nuclear protein kinase in mice. Arch Biochem Biophys 352: 31-6

84

CHAPTER V

IDENTIFICATION OF KINASE ACTIVITY IN SOLUBLE

EXTRACTS OF CHILO IRIDESCENT VIRUS

Introduction

In Chapter HI, I describe the effects of a soluble extract from Chilo iridescent vims

(CIV). The CIV soluble virion extract inhibits protein synthesis in treated boU weevU

{Anthonomus grandis Boheman) and spmce budworm {Choristoneura fumiferana) ceU

Unes. The extract was also shown to induce apoptosis in these ceU Unes.

Exanqiles in the Uterature concerning another iridovirus (Frog virus 3) and a

cytoplasmic DNA virus (vaccinia vims) suggested that a protein kinase may be involved in

this cytocidal activity. Frog virus 3 (FV3) has also been shown to inhibit macromolecular

synthesis in infected ceUs [7], and Uke CIV, a soluble extract from FV3 also induces

inhibition of host synthesis [1]. In addition, infection with FV3 induces phosphorylation of

the initiation factor eIF-2a and prevents translation by interfering with binding ofthe

initiator tRNA molecule to the 40S ribosomal subunit [7]. It is unclear whether this

activity results from FV3 gene expression, as a direct result of phosphorylation by a viral

kinase, or by viral induction of a host kinase.

Evidence from multiple virus studies has indicated that viral kinases are inqjortant

in the abiUty ofthe virus to shut off host macromolecular synthesis. Previous studies with

CIV have indicated that kinase activity is associated with the virion, and our laboratory

has identified a putative homolog ofthe vaccinia BIR protein kinase in the CIV genome.

85

The BIR protein is a serine/threonine protein kinase that has been inpUcated in host

shutdown in vaccinia virus infections [11]. We hypothesize that a CIV kinase (possibly

the BIR-Uke protein) is responsible for triggering these inhibition and apoptotic responses

in budworm and boU weevU ceU lines.

It was therefore inyortant to confirm the presence of kinase activity in virion

extracts that induce inhibition and apoptosis and to artenqit isolation of relevant

polypeptides and genes. Our results suggest that soluble extiacts prepared by using two

different detergents show association of kinase activity with a single polypeptide in each

extract. The data indicate that a 17-kD and a 44-kD polypeptide are impUcated in the

CHAPS and OGE extracts, respectively. The inpUcations of these results, including the

48-kD predicted molecular weight ofthe BIR-Uke protein and arterr^its to isolate the

genes coding for these polypeptides, are discussed. An effort was made to obtain N-

terminal sequences from the candidate polypeptides in order to use this information for

locating the respective genes. This work did not achieve the desired results, but further

attempts to isolate this CIV protein kinase gene are ongoing in our laboratory.

Further analysis of these virion extracts and ofthe BIR-Uke gene should yield

usefiil genetic information about polypeptides responsible for kinase activity in CIV. This

approach could help identify the gene or genes inducing apoptosis and inhibition in CIV.

Isolation of a kinase gene and confirmation ofthe role ofthe BIR-Uke gene wiU have

important impUcations for the production of transgenic boU weevU-resistant cotton plants.

86

Material and Methods

Virus Rearing

Chilo iridescent virus [8] was reared in the larvae of the greater waxmoth,

Galleria mellonella. Larvae were nicked with sharpened forceps dipped in a vims

suspension (0.5 mg/ml). Larvae were checked every three days, and dead or pupated

insects were discarded. Survivors were frozen at -20°C after a two-week incubation.

Vims Extraction and Quantitation

Vims was extracted from waxworm larvae using a modification ofthe method of

Kelly and Tinsley [9], except that the sucrose-gradient centrifiigation steps were omitted.

The virions were harvested after differential centrifiigation to prevent loss of material in

the sucrose-gradient centrifiigation steps. Also, a Tris (150 mM NaCl, 50 mM Tris-base,

pH 7.4) buffer was used instead ofthe borate buffer used by Kelly and Tinsley.

Quantitation of virus was performed by spectiophotometric analysis. One unit of

absorbance at 260 nm (A^^J represented 55 \ig/ml of vims [9].

OGE Extract Preparation

The non-ionic detergent P-octylglucoside was used to prepare a virion extract

from differentiaUy centrifiiged virus using a modification ofthe method described by

Cemtti and DevaucheUe [6]. DifferentiaUy extiacted vims was centrifuged to a peUet and

resuspended overnight in detergent buffer (30 mM P-octylglucoside, 25 mM Tris-base, 1

M NaCl, pH 9). The resuspended vims was layered onto 10% sucrose (w/v) in SW-41

87

ultracentrifugation tubes (SorvaU) and centrifiiged for two hours at 36,000 rpm and 4 °C

in a Beckman SW-41-Ti rotor. The clear supernatant was extensively dialyzed using the

Amicon stirred ceU apparatus (model #12) with MSI, Ultrasep discs (10 kD MW cut-off)

at 4 °C and 50 psi nitrogen for approximately 4 hours using virus buffer (50niM Tris-HCl

pH 7.4, 150mM NaCl) to remove detergent. The final product was filtered using 0.22

MiUipore GV disposable filter and stored in microtubes at -70 °C.

CHAPS Extract Preparation

A second extract was prepared from differentiaUy centrifiiged virions using another

anionic detergent, CHAPS (3-(3-Cholamidopropyl) diinethylammonio-l-

propanesulfonate). Virions were centrifiiged, and the resulting peUet was rapidly

resuspended by pipetting in the foUowing buffer: 10 mM Tris-HCl IM KCl pH 7.4, and

lOmM CHAPS (instead of octylglucoside (OGE) used by Cemrti and DevaucheUe). The

virus suspension was incubated at 30 °C for 15 min to extiact soluble protein. The

suspension was then centrifiiged and dialyzed as described for the P-octylglucoside

extract. SDS-PAGE analysis with sUver staining revealed no difference in polypeptide

conposition and minimal differences in the amounts of some polypeptides between

CHAPS extracts prepared from vims that was differentiaUy centrifuged and extracts from

vims purified by sucrose gradient.

88

Fast Performance Liquid Chromatography

Fast performance Uquid chromatography (FPLC) of extract material was

performed for each extiact using a Superose-75 size-exclusion column at a flow rate of

0.5 ml/min {Pharmacia, Peapack, New Jersey). The buffer (containing detergent) for

each extract was present during the chromatography process in order to minimize

aggregation of polypeptides. The separation was performed at 4 °C to maximize

biological activity. Fractions were coUected in a volume of 250 nl and were analyzed for

kinase activity and for polypeptide composition. Molecular weight of polypeptides

contained in FPLC fractions was determined by conqiarison to standard proteins on sUver

stained SDS-PAGE gels.

KinRse Assay

The kinase assay was performed according to the method of Monnier and

DevaucheUe [12]. Briefly, sanples were incubated in microtubes containing a kinase

reaction mbrture (25 mM Tris, 8 mM MgC12, 200 ^M ATP, 0.02% NP-40, pH 9) with 1

mg/ml protamine {Sigma) as a substrate and 5 nCi [Y-^^P]-ATP (DuPont NEN) as label.

Sanqiles were incubated 30 min at 30 °C and 20 nl ofthe reaction mixture was spotted to

phosphoceUulose paper (Fisher). Unincorporated label was rinsed off the paper whUe

label boimd to protein remained attached. The paper disks were then analyzed in a

Beckman LC-2500 scintillation counter using ScintiSafe BD scintUlation fluid (Fisher).

Intact vims was used as a positive control and BSA was used as a negative control.

89

Amino-Terminal Protein Sequencing

A high concentration sanqile (1 mg/ml) ofthe CHAPS extract was separated on a

12% SDS-PAGE gel The polypeptides of this gel were transferred to an Immobilon P

membrane (MUUpore) using a Hoeffer TE 22 Mini Tank Transphor unit using CAPS

buffer (10 mM CAPS (3-[cyclohexylamino]-l-propanesulfonic acid), 10 % methanol pH

11). The blot was stained for 5 minutes in Coomassie Blue stain (0.1 % Coomassie Blue

R-250, 40% methanol, 1 % acetic acid) and destained for several minutes in 40%

methanol. After drying, the 17 kD polypeptide from the CHAPS extiact was removed

from the gel and sequenced in a Porion Instruments 2020 sequencer with on-Une Beckman

Gold HPLC system.

Southern Blorting with an OUgonucleotide Probe

Protein sequencing information was used to design an oUgonucleotide probe with

the least possible degeneracy. In an attempt to determine the approximate location ofthe

putative kinase gene, Southem blorting under low stringency (30 % formamide, 2 x SSC

(300 mM NaCl 50 mM sodium citrate), 0.1% SDS, 42 °C) conditions was performed

using the designed oUgonucleotide. The oUgonucleotide was labeled with [g-32P] ATP

using the Promega end-labeUng kit according to the manufecturer's instmctions.

Hybridization was aUowed for 48 hours before washing for 5 minutes at 21 °C (2 x SSC,

0.1% SDS) foUowed by 5 minutes washing at 42 °C in fresh solution ofthe same

composition.

90

DNA Sequencing ofthe Candidate Region

The CIV EcoR I fragment appearing to have been identified by the Southem

blorting was cloned into pBluescript (Stiatagene) and sequenced by primer waUdng using

a Perkin-Elmer ABI 310 Genetic Analyzer.

Results

Polypeptide Conyosition of Soluble Extracts

Extracts were prepared using the detergents and methodology described in

Materials and Methods. In order to determine their polypeptide conposition, these

extiacts were observed by SDS-PAGE analysis using 10% acrylamide gels and bands were

visuaUzed by sUver staining. The results for the octylglucoside extract (OGE) and CHAPS

extracts are shown in Figures 5.1 and 5.2, respectively. Twelve major bands were

observed in the OGE extract, whereas the CHAPS extract contained seven major bands.

The exact nuniber of polypeptides differed sUghtly with each extractioa TypicaUy for the

OGE extract, several bands were seen in 110-130 kD range, several approximately 50 kD,

a 44 kD polypeptide, a 36 kD polypeptide, and several more between 10 and 20 kD. The

typical CHAPS extract contained one or two bands in the 110 kD vicinity, and individual

bands of 69, 38, and 31 kD, as weU as 2-3 bands in the 15-18 kD range.

Kinase Activity of |3-octylgucoside and CHAPS Extracts

Kinase assays as described in Materials and Methods were performed on both the

P-octylglucoside (OGE) and CHAPS extracts. Both extiacts showed a high level of

91

kinase activity (determined by cpm of incorporated radioactivity) over boUed extract

controls (Tables 5.1 and 5.2, respectively) in tripUcate assays. Specific activity

(incorporated cpm per microgram protein) of these preparations was also calculated and

was substantiaUy higher than the activity of boUed extiact (5 min). The specific activity

for the CHAPS extract (3,993 cpm/ng) is approximately four-fold higher than that ofthe

OGE extract (1,068 cpm/ng), suggestmg that the CHAPS preparation contams the kinase

polypeptide as a much higher percent of total protein when conpared to the OGE extract.

Analysis of FPLC Fractions

Analysis ofthe FPLC firactions ofthe OGE and CHAPS was performed in order to

determine the polypeptide composition ofthe fractions and to determine which fractions

contained kinase activity. SDS-PAGE analysis ofthe resulting fractions as weU as the

void volume from chromatography showed that a large aggregate of polypeptides washed

through the column into the void volume, but several polypeptides were separated from a

large aggregate for each extract. The kinase activity ofthe fractions containing

polypeptides was then determined. SDS-PAGE analysis of OGE and CHAPS extract

fractions showed isolated bands of 44 kD and 17 kD, respectively, that corresponded to

kinase activity (Figures 5.3 and 5.4).

Polypeptide Sequencing and AppUcation to Gene Sequencing

Sequencing ofthe 17 kD polypeptide revealed the foUowing 23 amino acids ofthe

amino terminus: MNQNLIILSVGIAVLSAIFTSAY. A degenerate oUgonucleotide probe

(5' ATG AA(T/C) CA(A/G) AA(T/C) (T/C)T(A/T/G) AT(T/A) AT 3') was syntiiesized

92

using information from the first seven amino acids. Southem blorting using this probe

indicated that hybridization occurred with a portion ofthe CIV EcoR I fragment D.

However, sequencing information and analysis of this fragment did not show any open

reading frames or even any sequences matching the sequence ofthe oUgonucleotide probe.

In addition, no open reading frames were foimd vdth significant similarity to genes already

present in the GeriBank.

Discussion

Our laboratory has shown that protein extracts from Chilo iridescent virus (CIV)

inhibit protein synthesis and induce apoptosis in boU weevU and spmce budworm ceU Unes

(Henderson, Jayaraman, D'Costa, and BUimoria, in preparation). Research on other

cytoplasmic DNA viruses (vaccinia virus and Frog vims 3), has indicated that protein

kinases are involved in these processes during infection [3, 7, 10, 13, 14]. The BIR

protein kinase of vaccinia vims has been impUcated in the phosphorylation of ribosomal

proteins [4] and in the inhibition of host protein synthesis [11]. Frog vims 3 infection has

been shown to resuh in the phosphorylation of eIF-2a, thus preventing the formation of

the translation initiation complex [7, 14]. In addition, previous experiments with CIV

indicate the presence of a kinase activity associated v dth the virus particle [12]. However,

the polypeptide containing kinase activity was not identified.

In this study, we demonstrate kinase activity in soluble protein extracts prepared

using two different detergents (octylglucoside and CHAPS). Specific activity (cpm/ng

protein) based on kinase assays ofthe two extracts showed that the CHAPS extract

93

contains approximately four times more activity. We have also analyzed FPLC fractions

of these extracts by sUver-stained SDS-PAGE and kinase assay in an artempt to identify

single polypeptides that associate with kinase activity. The OGE analysis suggested that a

44-kD polypeptide was associated with kinase activity, whereas CHAPS extract analysis

suggested that a 17-kD polypeptide correlated with kinase activity. Further analysis is

needed to determme which of these polypeptides (or both) contains kinase activity. It is

possible that the 44-kD and 17-kD polypeptides are related and the 17-kD polypeptide is a

specific cleavage product of a larger proteia Interestingly, the molecular weight ofthe

44-kD polypeptide is within experimental error of that for the polypeptide predicted from

the BlR-hke open reading fimne.

We artempted to use N-terminal sequencing of these polypeptides to gain

information useful for the identification and isolation of their encoding genes. The 44

kD polypeptide was blocked at the N-terminus, but the 17 kD polypeptide yielded a

sequence 23 amino acids m length. Back tianslation ofthe amino acid sequence led us to

constmct a 20 nucleotide degenerate oligonucleotide to be used as a probe. Southem

blotting using this end-labeled oligonucleotide suggested that an approximately 5 kb

region in the EcoR I fragment D of CIV contained this gene. However, sequencing of

this DNA did not reveal the expected protein kinase gene, or any other open reading

frame with significant similarity to any known genes.

Recent sequencing information has revealed the presence of multiple protein kinase

genes in the CIV genome [2], suggesting that multiple polypeptides may be involved in the

cytocidal activities. In addition, our laboratory has discovered a CIV open reading fi-ame

94

with high similarity to the vaccinia BIR protein kinase gene (Henderson, Demirbag,

D'Costa, BUimoria, in preparation). The putative CIV homolog has a predicted

molecular weight of 48 kD based on the amino acid sequence, a reasonable approximation

to the 44 kD polypeptide observed with the OGE extract. We have considered the

possibUity that the 44 kD polypeptide may be cleaved under the conditions used for the

production the CHAPS extiact, thereby yielding the 17 kD polypeptide as a degradation

product. However, this possibUity has not yet been investigated.

Isolation of kinase polypeptides in CIV will lead to identification and cloning ofthe

kinase genes. We hypothesize that the kinase gene(s) may be useful for the constmction of

tiansgenic cotton plants with resistance to the boll weevil and possibly other insects.

95

kD

— 112

— 57

— 50 — 44

— 36

— 31

Figure 5.1: Polypeptide composition ofthe octylglucoside extract. Representative SDS-PAGE in 10% gel shows the polypeptide profile ofthe extract (E) and purified virion (V) as visualized by silver staining. Molecular weight markers (M) were also observed. The molecular weights of major polypeptides are indicated.

96

kD M V E

Figure 5.2: Polypeptide composition ofthe CHAPS extract. Representative SDS-PAGE in a 10% gel shows the polypeptide profile ofthe extract (E) and purified virion (V) as visualized by silver staining. Molecular weight markers (M) were also observed. The molecular weights of major polypeptides are indicated.

97

FRACTION # (FPLC)

KINASE ACTIVITY

11 13 14 15 16 17 19

120 802 1246 1129 376 402 96

'-$

54 51 48

- 44kD

36

— 32

20

k BUFFER ONLY OGE EXTRACT

Figure 5.3: Analysis of FPLC fractions from the octylglucoside extract. FPLC fractions were analyzed for both polypeptide content and kinase activity as described in Materials and Methods. The fractions showing the greatest kinase activity were observed using a 10% acrylamide gel and silver staining. Fraction number and kinase activity (cpm/ng protein) are shown above the lanes for their respective fractions. A profile ofthe CHAPS extract is also shown for comparison. An additional band (*) was observed at 38 kD, but this is likely an artifact since it was also seen in lanes containing buffer only.

98

FRACTION # (FPLC) 11

KINASE ACTIVITY

12 13 14 15

241 550 897 439 318

- 2 5 k D 24 kD

20 kD

14 kD - 1 2 k D

MW VIRUS CHAPS EXTRACT

Figure 5.4: Analysis of FPLC fractions from the CHAPS extract. FPLC fractions were analyzed for both polypeptide content and kinase activity as described in Materials and Methods. The fractions showing the greatest kinase activity (cpm/ng protein) were observed using a 10% acrylamide gel and silver staining. Fraction number and kinase activity are shown above the lanes for their respective fractions.

99

Table 5.1: Kinase activity ofthe OGE extract and contiols

Preparation

OGE Extt-act OGE Extract OGE Extract

Mean OGE Extract BoUed Extract

BoUed Extract

BoUed Extract

Mean Boiled Extract BSA

Virus

Replicate

1 2 3

1 2 3

ng Protein

20 20 20

20 20 20

20 20 30 30

CPM

24,801 20,457 19,462

21,573 178 172

296

215 173

9,415

Corrected for Boiled Extract

24,586 20,242 19,247

21,358 -37

-43 81

0 -42

9,200

Specific Activity

1,230 1,012 963

1,068 -1.9 -2.2 4.1

0 -1.4 307

Specific activity ofthe OGE extract in cpm of incorporated label per microgram protein is over three times that of whole virus.

100

Table 2: Kinase activity ofthe CHAPS extiact and controls

Preparation

CHAPS Extract CHAPS Extiract CHAPS Extract

Mean CHAPS Extract BoUed Extract BoUed Extract BoUed Extract

Mean Boiled Extract BSA (Mean)

Vims (Mean)

Mock Extract (Mean)

RepUcate

1 2 3

1 2 3

ng protein

10 10

10

10 10 10 10

10 10

20

20

CPM

40,874

41,740 38,422

40,345 480 405 363

416 472

10,085 1607

Corrected for Boiled Extract

40,458 41,324

38,006

39,929 64 -11

-53

0 56

9,669

1,191

Specific Activity

4,046 4,132

3,801

3,993 6.4 -1.1 -5.3

0 5.6 484

60

Specific activity ofthe CHAPS extract in cpm of incorporated label per microgram protein is over eight times that of whole virus. A mock CHAPS extract (performed using uninfected larvae) contained close to zero specific activity.

101

References

1. Aubertin AM, Hirth C, Travo C, Noimenmacher H, Kim A (1973) Preparation and properties of an inhibitory extract from frog virus 3 particles. J Virol 11: 694-701

2. Bahr U, Tidona CA, Darai G (1997) The DNA sequence of Chilo iridescent vims between the genome coordinates 0.101 and .391; similarities in coding strategy between insect and vertebrate iridoviruses. Virus Genes 15: 235-245

3. Balachandran S, Roberts PC, Kipperman T, BhaUa KN, Cortpans RW, Archer DR, Barber GN (2000) Alpha/beta interferons potentiate vims-induced apoptosis through activation ofthe FADD/Caspase-8 death signaUng pathway. J Virol 74: 1513-23

4. Beaud G, Sharif, A., Topa-Masse, A., and Leader, D. P. (1994). J. Gen. Virol. 75, 283-293. (1994) Ribosomal protein S2/Sa kinase purified from HeLa ceUs infected vvdth vaccinia vims corresponds to the BIR protein kinase and phosphorylates in vitro the viral ssDNA-binding proteia J Gen Virol 75: 283-93

5. Cemtti M, DeauvecheUe G (1980) Inhibition of host macromolecular synthesis in cells infected with an invertebrate virus. Archives of Virology 63: 297-

6. Cemtti M, DevaucheUe G (1990) Protein composition of Chilo iridescent vims. In: Darai G (ed) Molecular Biology of Iridovimses. Kluwer Academic Press, Boston, pp 81-112

7. Chinchar VG, Dholakia. JN (1989) Frog vims 3- induced translational shutoff: activation of an eIF2 kinase in virus infected ceUs. Virus Res. 14: 207-224

8. FiJcaya M, Nasu S (1966) A ChUo Iridescent Vims (CISO from the rice stem borer, Chilo suppresalis WaUcer (Lepidoptera, PyraUdae). AppUed Entomology and Zoology 1: 69-72

9. KeUy DC, Tinsley TW (1972) Proteins of iridescent vims type 2 and 6. Microbios Letters 9: 75-93

10. Kibler KV, Shors T, Perkins KB, Zeman CC, Banaszak MP, Biesterfeldt J, Langfield JO, Jacons BL (1997) Double stranded RNA is a trigger for apoptosis in vaccinia virus-infected ceUs. J. Virol 71: 1992-2003

11. Kovacs GR, Moss B (1998) Regulation of gene expression by the vaccinia virus protein kinase-1. In: Twelfth Intemational Poxvims Symposium, St.Thomas, United States Virgin Islands, pp 66

102

12. Monnier C, DevaucheUe G (1976) Enzyme activities associated viith an invertebrate iridovirus: nucleotide phosphohydrolase activity associated with iridescent virus type 6 (CIV). J Virol 19: 180-6

13. Tan SL, Katze MG (1999) The emerging role of tiie interferon-induced PKR protein kinase as an apoptotic effector: a new fiice of death? J. Interferon. Cytokine Res. 19: 543-54

14. WUlis DB, Goorha R, Chinchar VG (1985) Macromolecular synthesis in cells infected by frog vims-3. Curr. Topics Microbiol. Immunol. 116: 77-105

103

CHAPTER VI

CONCLUSIONS

The present study describes the biology and cytotoxicity of an insect vims {Chilo

iridescent virus) pathogenic to the cotton boU weevU, Anthonomus grandis. The boU

weevU is a devastating pest of cotton, causing over $300 mUUon in damages annuaUy in

the United States alone. Problems associated with chemical control of this pest require

alternate methods of control. Several potential biological control methods have been

studied, but Chilo iridescent virus (CIV) is the only virus known to infect the boU weevU.

Our laboratory has shown that CIV causes death and metamorphic deformity in up to 70%

of infected insects. It is cmcial to understand the cytotoxic mechanisms of this CIV in

order to more fiiUy utUize its biocontiol potential.

This study conclusively demonstrates that CIV undergoes a productive infection

cycle in the boU weevU. Dot blot analysis of infected insects showed that viral DNA is

repUcated in the boU weevil, and election microscopy indicates that con^lete vims

particles are formed. FinaUy, an infectivity assay developed in our laboratory showed that

high quantities of infectious vims were produced. This is the first study of a conplete

infection cycle in any host for the genus Iridovirus.

The remainder of this study focuses more on the molecular biology and

biochemical characteristics of CIV, including multiple cytopathic effects induced by a

soluble extract from CIV. The soluble extract drasticaUy inhibited protein synthesis of

treated spmce budworm and boU weevU cultured cells as early as three hours post

104

treatinent with only 10 ng/ml extrart. The extract also induces apoptosis m both ceU Unes,

demonstrated by both induction of apoptotic ceU morphology and fragmentation of host

DNA. Evidence with vaccinia virus (another cytoplasmic DNA virus) suggests that

inhibition of protein synthesis in infected ceUs leads directly to apoptosis. Even though

host defense mechanisms differ significantly between vertebrates and insects, it is possible

that a mechanism similar to that of vaccinia vims is present in CIV.

The BIR protein kinase of vaccinia vims has been inpUcated in the phosphoryl­

ation ofthe Sa and S2 ribosomal subunits. BIR has also been shown to effect the

shutdown of host protein synthesis. It is quite possible that these effects are directly

related. Sequence analysis ofthe CIV genome in our laboratory revealed the presence of

a BIR-Uke gene. Further analysis of this ORF shows the presence of two sequence motifs

characteristic of protein kinases. Further study into the role of this gene in CIV infection

and cytotoxicity should be intriguing.

Additional analysis ofthe soluble extract from CIV also revealed the presence of

protein kinase activity. Figure 6.1 shows hypothetical mechanisms of host inhibition by

CIV. Since these extracts are relatively simple, an attenqit was made to isolate the

polypeptide(s) responsible for the kinase activity. Analysis of this polypeptide(s) wiU lead

to the identification ofthe responsible gene, which may potentiaUy serve as an insecticidal

gene. We are also pursuing the possibUity that the factor or factors responsible for this

activity is not a kinase. Ongoing research in our laboratory includes amino acid sequence

analysis and isolation of genes coding for the major polypeptides in the CHAPS extract as

weU as testing gene products for apoptosis and other observed pathogenic effects.

105

Regardless of whether a cytotoxic gene codes for a kinase or another product, such a gene

could be invaluable in generating recombinant insect-resistant plants.

106

VACCINIA VIRUS LIR CIV

Figure 6.1: Working model for induction of apoptosis by CIV. We postulate that CIV may induce apoptosis by one of two mechanisms. First, CIV may induce an interferon-like system similar to vaccinia vims induction of interferon. This eventually results in the phosphorylation of initiation factors, thus inhibiting protein synthesis and inducing apoptosis. Second, CIV has been shown to contain a putative homolog ofthe vaccinia BIR protein kinase which has been shown to phosphorylate ribosomal proteins and inhibit host protein synthesis. It is quite possible that like vaccinia vims, CIV may have more than one cytotoxic mechanism.

107