Plant Viruses

Bradley HillmanDept. of Plant Biology and Pathology

339 Foran Hall, Cook932-9375 X 334

hillman@aesop.rutgers.edu

Comparative Virology course website: http://www.rci.rutgers.edu/~bhillman/comparative_virology/Index.htm

Plant Viruses

• Introduction and history– Why study plant viruses?– How do they relate to animal viruses?– How has their study impacted virology?

• Symptoms• Composition and structure• Taxonomy and nomenclature

– Four families contain both plant and animal viruses– Seven families contain only plant viruses– Many plant viruses belong to genera without family affiliation

Plant Viruses (cont’d)• Survey of some major plant viruses

– Positive strand RNA • RdRp supergroup 3 (Sindbis-related)

– Tobacco mosaic virus (Tobamovirus)– Brome mosaic virus (Bromoviridae)

• RdRp supergroup 2 (Flavivirus-related)– Turnip crinkle virus (Tombusviridae)– Red clover necrotic mosaic virus (Dianthovirus)

• RdRp supergroup 1(Picornavirus related)– Tobacco etch virus (Potyviridae)– Cowpea mosaic virus (Comovirus)

– Negative strand RNA• Sonchus yellow net virus (Rhabdoviridae)

– Ambisense RNA• Tomato spotted wilt virus (Bunyaviridae)

Plant Viruses (cont’d)• Survey of some major plant viruses (cont’d)

– Double-stranded RNA • Wound tumor virus (Reoviridae)

– Single-stranded DNA• Bean golden mosaic virus (Geminiviridae)

– Double-stranded DNA (pararetroviruses)• Cauliflower mosaic virus (Caulimoviridae)

• Expression strategies of + strand RNA viruses• Plant virus infection cycle• Cell-to-cell movement and movement within plants• Plant-to-plant transmission• Brome mosaic virus, a well-studied plant virus• Satellites, defective-interfering RNAs, viroids• Plant defense response to virus infection• Plant viruses and biotechnology

Host Systems: Plants 1• Eukaryotic, but fundamentally different from

animals• Plants don’t move, so vectors are very important

for moving viruses from one plant to another• Plants are autotrophic and easy to grow in

quantity– great bioreactors• Plants have rigid cell walls and very small cell-to-

cell connections (plasmodesmata)• Synchronous infection of many cells can be

achieved using plant protoplasts (primary cell cultures with cell walls removed)

Host Systems: Plants 2

• Developed plant cells are totipotent• Virus in one part of a plant moves to another

slowly by cell-to-cell connections; more rapidly through vascular system, mostly phloem

• Plant defense response system exists, but is less specific than vertebrate or invertebrate systems

• Plants are developmentally complex; viruses may be excluded from some tissues

CHARACTERISTICS OF PLANT PATHOGENIC VIRUSES

• Loss due to plant viruses is often difficult to quantify, but they are often of great importance as plant pathogens (fungi are most economically important )

• Viruses do not usually kill plants, and symptoms on plants are often subtle

• Virus diseases of plants are not subject to chemical control – no effective cure in individual plants

• Symptoms, serological, electron microscopic, or molecular methods are used to identify plant viruses

• Plant virus disease cycles often are dependent on vectors or alternate hosts

Selected highlights of plant virology research

Tulipomania – late 16th century

Traded for 1 Viceroy tulip bulb:

4 tons of wheat8 tons of rye4 fat oxen8 fat pigs12 fat sheep2 hogsheads of wine4 barrels of beer2 barrels of butter1000 lbs of cheese1 bed with

accessories1 full dress suit1 silver goblet

Before it was known to be caused by a virus, tulips with color breaking symptoms were prized and traded for large sums of goods – this led to “tulipomania” in the late 1500’s

• Adolf Mayer –1886 – showed that Tobacco mosaic virus was transmissible, could not find bacteria or fungi associated with disease

TMV

• Dmitri Ivanowski - 1892– showed that Tobacco mosaic virus was not retained by filters that retained all bacteria known at that time

• Martinus Beijerinck - 1898– repeated demonstration that Tobacco mosaic virus was not retained by filters that retained all bacteria known at that timeBelieved resultsDid extensive dilution experimentsShowed diffusion of infectious agent

through agarNamed “contagium vivum fluidum”, later

virus

Wendell Stanley – 1935

• At Rockefeller Foundation in Princeton• Crystallized TMV, thought it was only

protein

Stanley Hall, U.C. Berkeley

TMV

Bawden and Pirie - 1936

• Crystallized Tomato bushy stunt virus (TBSV); find that it and TMV contain phosphorous – conclude that it is not a protein, but is a nucleoprotein

TMV

TBSV

Markham and Smith - 1949

• Two classes of particles in purified Turnip yellow mosaic virus preparations: – light ones containing only

protein, which were not infectious

– heavy ones containing protein+nucleic acid, which were infectious

empty full

Myron Brakke - 1951

• Development of density gradient centrifugation– Isopycnic: particles reach

position of equal density in gradient

– Rate-zonal: Particles sediment differentially through medium as a function of size, shape, and density

– Equilibrium zonal: Combination of the above

Fraenkel-Conrat 1955-56

• Complete, infectious TMV particles can be reconstituted in vitro from the RNA and protein components

• RNA alone is infectious• RNA can be

“transcapsidated” in protein from closely related virus; resulting virus has properties of RNA strain

reconstitute in vitro

Virus Aa:RNA A

capsid a

RNA

protein

symptoms (A)

extract virus

virus Aa

inoculate plants

Fraenkel-Conrat – 1955-56 Transcapsidation

inoculate plants

Virus Aa:RNA A

capsid a

RNA

protein

symptoms (A)no

symptoms

extract virus

no virusvirus Aa

inoculate plants

Virus Bb:RNA B

capsid b

RNA

protein

symptoms (B)no

symptoms

extract virus

no virusvirus Bb

inoculate plants

Virus Ab:RNA A

capsid a

RNA

protein

symptoms (A)no

symptoms

extract virus

no virusvirus Aa

Crick and Watson – 1956

• TMV virions are composed of one nucleic acid and many identical protein subunits: RNA does not have the coding capacity to make many different subunits

Casper and Klug – 1962• Structure of Tomato bushy stunt

virus solved by X-ray crystallography, the first icosahedral virus so determined

Heinz Sanger – 1978

• Complete sequence of Potato spindle tuber viroid– First pathogen

sequence to be determined

– Yielded relatively little information that was immediately useful

1 cggaactaaa ctcgtggttc ctgtggttca cacctgacct cctgagcaaa aaagaaaaaa gataggcggc tcggaggagc gcttcaggga tccccgggga aacctggagc gaactggcaa aaaaggacgg tggggagtgc ccagcggccg acaggagtaa ttcccgccga aacagggttt tcacccttcc tttcttcggg tgtccttcct cgcgcccgca ggaccacccc tcgccccctt tgcgctgtcg cttcggctac tacccggtgg aaacaactga agctcccgag aaccgctttt tctctatctt cttgcttccg gggcgagggt gtttagccct tggaaccgca gttggttcct 359

Paul Ahlquist – 1984• Infectious viral RNA

transcribed in vitro from cDNA clones– Done with Brome mosaic

virus – with 3 RNAs– Brought reverse genetics

to RNA viruses

RNA

cDNA

RNA Inoculate plants

Roger Beachy – 1986

• Transgenic plants expressing TMV coat protein are resistant to virus infection

• First example of “pathogen-mediated resistance”

Bill Dougherty – 1991

• RNA was critical component in resistance in pathogen-mediated resistance

• All of the hallmarks that later came to be associated with PTGS and RNAi were first observed with Tobacco etch virus (TEV) (1993 Lindbo et al., Plant Cell 5:1749-1759)

SYMPTOMS

Plant Virus Symptoms• Viruses rarely kill plants• Most severe disease usually in least well-adapted

host/pathogen systems• Levels of tissue specificity differ among plant viruses

– Some infect all or most tissues– Most cause symptoms only in aerial portions– Some accumulate only in roots– Symptoms in fruit or flowers may be most harmful

• Systemic symptoms only in developing tissue• Local necrotic lesions undetectable in natural

infections

Plant Virus Symptoms

• Stunting - common

• Mosaics & mottles - common

• Ringspots - common

• Abnormal growth/tumors/enations - rare

• Blights - rare

• Wilts – rare

• Systemic necrotic lesions – relatively rare

Blight

Stem pitting – usually results in loss of woody perennials

Ringspots, oakleaf

Deformities

Phyllody – tissue destined to develop flower parts instead develops leaves

Wilt

Necrotic roots

Mosaics – very common

Tomato mosaic

Alfalfa mosaic

Mosaics on monocots are streaks or stripes

Maize streak Maize mosaic

Necrotic lesionsSystemicLocal

“Local lesion” or “hypersensitive” response is an apoptotic response. Cells within a short distance of the initially inoculated cell begin to undergo programmed cell death in advance of virus invasion, preventing further virus spread.

Hypersensitive response – an apoptotic reaction to infection

May be viral coat protein or another viral gene product

TAXONOMY AND NOMENCLATURE

Virus taxonomy and nomenclature• Modified binomial is used• Taxonomy depends on particle properties, nucleic

acid properties and especially sequence• Family is the highest taxonomic level that is

commonly used; ends in viridae, e.g., Bromoviridae• Genus ends in suffix virus, e.g., Bromomovirus • Species is usually the commonly used virus name;

it is italicized in formal usage, e.g., Brome mosaic virus

• Small genome sizes, gene shuffling make broad taxonomic schemes difficult (above Family level)

COMPOSITION AND STRUCTURE

Virus properties: Plant viruses are often simpler than animal viruses

• Genome sizes 0.3 - 300 kb; plant viruses 0.3-30 kb• May have single-stranded or double-stranded RNA

or DNA genome; most plant viruses ssRNA• If RNA, may be + or – sense; most plant viruses +

sense ssRNA• May have one or many proteins in particles; most

plant viruses have 1-2• May or may not have lipid envelope; most plant

viruses do not

Types of plant virus genomes

• double-stranded (ds) DNA (rare)

• single-stranded (ss) DNA (rare)

• ssRNA, negative sense (rare)

• ssRNA, positive sense (common)

• dsRNA (rare)

Virus types, by nucleic acid

DNA RNA

ss ds ss ds

env naked env naked env naked env naked0 5 9 12 9 14 2 50 100 200 300 200 600 10 300

FamiliesSpecies

Host typeVertebrateInvertebratePlantFungusBacteria

- + ++ ++ ++ ++ - ++ - + ++ - ++ ++ - ++ - ++ - + + +++ - + - - - + + + + +++ - + + +++ - + + -

Plant viruses are diverse, but not as diverse as animal viruses – probably because of size constraints imposed by requirement to move cell-to-cell through plasmodesmata of host plants

Plant viruses often contain divided genomes spread among several particles

Basic Plant Virus Structures

Helix (rod) e.g., TMV

Icosahedron(sphere)e.g., BMV

Helical symmetry• Tobacco mosaic virus is typical,

well-studied example• Each particle contains only a

single molecule of RNA (6395 nucleotide residues) and 2130 copies of the coat protein subunit (158 amino acid residues; 17.3 kilodaltons)– 3 nt/subunit

– 16.33 subunits/turn

– 49 subunits/3 turns

• TMV protein subunits + nucleic acid will self-assemble in vitro in an energy-independent fashion

• Self-assembly also occurs in the absence of RNA

TMV rod is 18 nanometers (nm) X 300 nm

Cubic (icosahedral) symmetry

• Tomato bushy stunt virus is typical, well-studied example

• Each particle contains only a single molecule of RNA (4800 nt) and 180 copies of the coat protein subunit (387 aa; 41 kd)

• Viruses similar to TBSV will self-assemble in vitro from protein subunits + nucleic acid in an energy-independent fashion

TBSV icosahedron is 35.4 nm in diameter

Protein Subunits Capsomeres

T= 3 Lattice

C

N

GENOME ORGANIZATIONS

Plant virus genome organizations

• Very compact• Most are +sense RNA viruses, so translation

regulation very important• Use various strategies for genome expression• Only a few genes absolutely required:

– Replicase– Coat protein– Cell-to-cell movement protein

• Other genes present in some viruses

Plant viruses have members in all 3 supergroups of + strand RNA viruses

From Principles of Virology, Academic Press 1999

Genome expression of + strand RNA viruses

• Most use more than one strategy– Polyprotein processing– Subgenomic RNA– Segmented genome– Translational readthrough– Frameshift– Internal initiation of translation (without scanning)– Scanning to alternative start site (truncated product)– Alternative reading frame (gene-within-a-gene)

Polyprotein processing

• Post-translational cleavage of viral proteins may occur in cis or in trans

• Some viruses use polyprotein processing exclusively to regulate gene expression

• Many viruses use polyprotein processing as one of several regulation mechanisms

• Examples:– Potyviruses*– Comoviruses*– Closteroviruses– Carlaviruses

Subgenomic RNA

• Similar to traditional mRNA, but synthesized from an RNA template

• Many viruses use polyprotein processing as one of several regulation mechanisms

• Examples:– Tobamoviruses (TMV)*– Bromoviruses (BMV)*– Tombusviruses (TBSV)– Potexviruses (PVX)

Segmented genome

• Positive sense RNA genomes are usually encapsidated in separate particles

• Segmented genomes lend themselves to recombination

• Examples:– Bromoviruses (Brome mosaic virus, BMV)*– Dianthoviruses (Red clover necrotic mosaic virus,

RCNMV)*– Hordeiviruses (Barley stripe mosaic virus, BSMV)

Translational readthrough

• Usually UAG codon is read through using suppressor tyrosine tRNA

• Common mechanism in plant viruses• Examples:

– Tobamoviruses (Tobacco mosaic virus, TMV)*– Dianthoviruses (Red clover necrotic mosaic virus,

RCNMV)*– Hordeiviruses (Barley stripe mosaic virus, BSMV)

Translational frameshift

• Typically +1 or -1• Common mechanism in plant viruses• Examples:

– Luteoviruses (Barley yellow dwarf virus, BYBV)*– Dianthoviruses (Red clover necrotic mosaic virus,

RCNMV)*– Closteroviruses (Beet yellow vein virus, BYVV)

Internal initiation

• Cap-free translation

• Less complex in plant viruses than in animal viruses

• Examples:– Potyviruses (Tobacco etch virus, TEV)*– Sobemoviruses (Southern bean mosaic

virus, (SBMV)*

Tobacco mosaic virus is a typical positive-sense RNA plant virus with a 6.4 kilobase genome

INFECTION CYCLE

Plant Virus Life Cycle• Virus entry into host

– no attachment step with plant viruses– by vector, mechanical, etc. – must be forced– requires healable wound – delivery into cell

• Uncoating of viral nucleic acid– may be co-translational for + sense RNA viruses– poorly understood for many

• Replication– replication is a complex, multistep process– viruses encode their own replication enzymes

Plant Virus Life Cycle 2• Cell-to-cell movement

– cell-to-cell movement through plasmodesmata– move as whole particles or as protein/nucleic

acid complex (no coat protein required)

• Long distance movement in plant– through phloem– as particles or protein/nucleic acid complex (coat

protein required)

• Transmission from plant to plant– requires whole particles

Typical RNA-containing plant virus replication cycle

From Shaw, 1996 Ch. 12 in Fundamental Virology (Academic Press)

2. RNA is released; translates usinghost machinery

3. Replication in cytoplasm

4b. New virus particlesare assembled

1. Virus particle enters first cell through healable wound

4a. Infectious TMV RNA is shuttled to adjacent cell through plasmodesmata, by virus-codedmovement protein

Cell-to-Cell Movement of Plant Viruses

• Plant viruses move cell-to-cell slowly through plasmodesmata

• Most plant viruses move cell-to-cell as complexes of non-structural protein and genomic RNA

• The viral protein that facilitates movement is called the “movement protein” (MP)

• Coat protein is often dispensable for cell-to-cell movement

Cell-to-Cell Movement of Plant Viruses

• Several unrelated lineages of MP proteins have been described

• MPs act as host range determinants

• MP alone causes expansion of normally constricted plasmodesmata pores; MPs then traffic through rapidly

• MPs are homologs of proteins that naturally traffic mRNAs between cells

• MPs may act as suppressors of gene silencing

Plant cells are bound by rigid cell walls and are interconnected by plasmodesmata, which are too small to allow passage ofwhole virus particles.

Plasmodesma

http://www.ncbi.nlm.nih.gov/ICTVdb/WIntkey/Images/d7.gif

Understanding virus infection and movement through plants requires understanding architecture of dicotyledonous plants and the connections between different cell types.

This has been studied extensively with GFP-labeled virus

Some plant viruses radically modify plasmodesmata, allowing for cell-to-cell movement as whole particles

Systemic spread of plant viruses is primarily through vascular tissue, especially phloem

Plant Virus Transmission• Generally, viruses must enter plant through

healable wounds - they do not enter through natural openings (no receptors)

• Insect vectors are most important means of natural spread

• Type of transmission or vector relationship determines epidemiology

• Seed transmission is relatively common, but specific for virus and plant

Plant Virus Transmission

• Mechanical transmission– Deliberate – rub-inoculation– Field – farm tools, etc.– Greenhouse – cutting tools, plant handling– Some viruses transmitted only by

mechanical means, others cannot be transmitted mechanically

• Transmission by vectors: general– Arthropods most important– Most by insects with sucking mouthparts

• Aphids most important, and most studied• Leafhoppers next most important

– Some by insects with biting mouthparts– Nematodes are important vectors– “Fungi” (protists) may transmit soilborne viruses– Life cycle of vector and virus/vector relationships

determine virus epidemiology– A given virus species generally has only a single

type of vector

Plant Virus Transmission by Vectors

• Insect transmission (vectors)– Aphids most important– Leafhoppers– Whiteflies– Thrips– Mealybugs– Beetles– Mites (Arachnidae)– Ants, grasshoppers, etc. – mechanical– Bees, other pollinators – pollen transmission

Types of vector relationshipsTerms apply mainly, but not exclusively, to

aphid transmission

• Non-persistent transmission– virus acquired quickly, retained short

period (hours), transmitted quickly– “stylet-borne” transmission– virus acquired and transmitted during

exploratory probes to epidermis

Types of vector relationships

• Persistent transmission– virus acquired slowly, retained long period

(weeks), transmitted slowly– circulative or propagative transmission– virus acquired and transmitted during

feeding probes to phloem

Types of vector relationships

• Semi-persistent transmission– virus acquired fairly quickly, retained

moderate period (days), transmitted fairly quickly

– virus acquired and transmitted during exploratory probes

Brome mosaic virus

• Relatively little studied prior to 1980• Relatively narrow host range• Causes no important disease• Mechanically transmitted, probably not

vectored• Similar to Alfalfa mosaic virus and

Cucumber mosaic virus, two important plant pathogens

• Now most thoroughly understood plant virus at RNA level

BMV structure• Rigid isometric particles 27 nm• RNA1 (3.2 kb) and RNA2 (2.9 kb) packaged alone;

RNA3 (2.1 kb) and RNA4 (1.2 kb) packaged together

• Particle is held together primarily by protein/RNA interactions

• With RNA, 180-subunit, T=3 particles predominate; without RNA, 120-subunit, T=1, particles

Brome mosaic virus genome organization

• 3 genomic RNAs, one subgenomic RNA

• Only RNAs 1 and 2 required for replication in protoplasts

• 3’-terminal 200 bases of segments nearly identical

• All three genomic and subgenomic RNA are capped

• Non-templated C and A at 3’-ends

Capping Helicase

Polymerase

Movement Capsid

Why is BMV such a powerful system• 3 RNAs

– only 1 and 2 required for replication– RNAs can be studied independently

• All three promoter types (+, -, sg) found on RNA 3, which is not required for infection

• Replication is fast in plant protoplasts• Infects monocot and dicot host plants• Replicates in yeast, best eukaryotic genetic

system• Efficient transcriptase/replicase complex has

been isolated for in vitro studies• Structurally stable particles allow for

encapsidation studies

Brome mosaic virus contributions• 1980

– studies of 3’-terminal pseudoknot (Ahlquist/Kaesburg)

• 1984 – complete BMV sequence (Ahlquist/Kaesburg)– 3’-terminal replicase recognition site (- strand

promoter) identified (Ahlquist/Hall)– Nonstructural proteins of BMV, TMV, and alfaviruses

are similar (Haseloff; also Ahlquist)– First infectious transcripts from a cloned RNA virus

genome (Ahlquist)

• 1985– Continued dissection of 3’-end functions

Brome mosaic virus contributions

• 1986– CAT gene substituted for CP (French/Ahlquist)

• 1987 – Requirement of intercistronic region of RNA 3 for

replication (French/Ahlquist)

• 1988– Identification of subgenomic RNA promoter

(French/Ahlquist)

• 1990– Rescue of CCMV deletion mutants by recombination

(Allison/Ahlquist)

Brome mosaic virus contributions• 1992

– Determination of 5’-terminal sequences involved in transcription and replication (+ strand promoter (Pogue/Hall)

– TMV movement protein can substitute for BMV MP (DeJong/Ahlquist)

• 1993 – BMV replication in yeast (Janda/Ahlquist)– BMV transcription and replication requires compatibility between

polymerase and helicase proteins (Dinant/Ahlquist)

• 1994– Recombination between viral RNA and transgenic plant

transcripts (Green/Allison)

• 1995– Formation of RdRp in yeast requires coexpression of RNA 1 and

2 proteins (Quadt/Ahlquist)

Brome mosaic virus contributions• 1997

– Inducible expression of active RNA 3 replicons in yeast from DNA plasmids (Ishjkawa/Ahlquist)

– Yeast mutations in multiple complementation groups inhibit BMV RNA replication and transcription (Ishjkawa/Ahlquist)

• 1998– Specific residues critical for recognition of the 33 nt subgenomic

RNA promoter by the BMV RdRp identified, demonstrating functional homology of RNA and DNA promoters (Siegel/Kao)

• 1999– BMV 1a protein functions in in vitro, in yeast, and in plants in

methylation of GTP and cap analogs (Ahola/Ahlquist; Kong/Kao)– BMV RdRp can use RNA, DNA, or hybrid to initiate RNA

synthesis, suggesting that transition from RNA to DNA world may have been relatively easy (Siegel/Kao)

Brome mosaic virus contributions• 2000

– Host protein associated with efficient RNA template selection identified (Diez/Ahlquist)

– BMV protein 2a (RdRp) is directed to ER by capping/helicase-like 1a protein (Chen/Ahlquist)

• 2001– Factors regulating template switching during RNA

synthesis by viral RdRps identified using in vitro assays (Kiml/Kao)

• 2002– 3’-terminal tRNA-like structure of BMV RNAs mediate

particle assembly (Choi/Dreher/Rao)– Crystallographic structure of BMV solved

(Lucas/McPherson)

Brome mosaic virus contributions• 2003

– The BMV 3’-terminal core promoter element, stem-loop C (SLC), functions at different positions on the template, can initiate RNA synthesis internally, and can potentiate RNA synthesis from a cellular tRNA template (Ranjith-Kumarl/Kao)

– Systematic, genome-wide identification of host genes affecting replication of BMV (Kushner/Ahlquist)

• 2004– Two distinct types of homologous RNA recombination in

BMV replication (Bujarski)

• 2005– Gold nanoparticles encapsidated in BMV coat protein

form normal, solid particles (Rao)

Some major BMV contributors

• Ahlquist – most prolific, many aspects• Hall – Cis-acting RNA elements• Dreher – 3’-terminal structure• Kao – recent dissection of cis-acting RNA

elements• Bujarski – Intra-strand and inter-strand RNA

recombination • Rao – RNA packaging

BMV promoters for + strand RNA synthesis

• Promoter sequence for genome-length plus strand synthesis is on 3’-end of minus strand; corresponding sequences on plus strand are important for efficient transcription

• 5’-terminal regions are less highly conserved than 3’-terminal regions

• Internal poly(A) tract of variable length precedes subgenomic promoter and is required for efficient sgRNA transcription

• Core promoter elements

Three classes of 3’ termini among + sense RNA viruses

TLS = tRNA-like structureHet = Heteropolymeric, non-tRNA- like sequenceAn = poly(A) tail

A. Full phylogeny, Supergroups 1-3

B. Supergroup 3From Dreher 1999, Annu. Rev. Phytopathol. 37: 151-174

Type of 3’-terminal structure cannot be predicted based on polymerase phylogeny alone, supporting the role of interviral recombination in selection of 3’-end.

Brome mosaic virus 3’-terminal tRNA-like structure is a multifunctional domain

• Promoter for – strand RNA synthesis

• RNA protection against nuclease

• Can be charged with tyrosine in vitro and in vivo

• (ATP, CTP) tRNA nucleotidyl transferase adds terminal C and A residues

• Involved in RNA packaging into virions – intact tRNA-like fold is required

• Relatively little involvement of 3-end of BMV RNA in translation regulation

Infection of yeast by Brome mosaic virus constructs

Brome mosaic virus replication

• 1a protein (capping & helicase) localizes to ER

• 1a protein recruits 2a protein (RdRp) during active translation of 2a

• Viral RNA templates are recruited to nascent replication complex by 1a protein

• RNA replication occurs in membrane-bound, capsid-like spherules

Identification of host genes involved in BMV replication (Kushner/Ahlquist 2003, PNAS 100:15764)

• Used yeast – genetics easy, many mutants available

• Provides information about replication, not systemic infection

• Transform with inducible two plasmid system requiring replication for reporter gene expression

• Screen for replication-associated genes by monitoring luciferase expression

• 4500 yeast mutants examined (~80% or yeast genes)

• ~100 yeast genes whose deletion altered BMV-directed expression of luciferase by > 3 fold

Identification of host genes involved in BMV replication (Kushner/Ahlquist 2003, PNAS 100:15764)

• Mutants that were positive in the luciferase screen were examined further by northern and western blot

Tomato bushy stunt virus genome

Comparative properties of BMV and TBSV

• TBSV– A T=3 icosahedral virus with a 40

kDa capsid subunit and monopartite RNA genome of 5 kb

– RdRp supergroup 2– No known helicase domain– 5’ end of RNA uncapped; 3’ end has

no poly(A) or tRNA-like structure– Expression of 5 gene products via

cap-independent translation (1) readthrough (1), subgenomic RNAs (2), and internal initiation (1)

– Defective-interfering (DI) RNAs replicate to high levels in permissive plant host; equally high in yeast

• BMV– A T=3 icosahedral virus with a 20

kDa capsid protein subunit and tripartite RNA genome of 8 kb

– RdRp supergroup 3

– No known helicase domain

– 5’ end of RNA capped; 3’ end has tRNA-like structure aminoacylated with tyrosine

– Expression of 4 gene products via normal cap-dependent translation (3) and subgenomic RNA (1)

– Defective-interfering (DI) RNAs present, not prevalent

TBSV replication constructs for yeast assay – based on trans replication of a defective interfering RNA

Yeast genome-wide screen reveals dissimilar sets ofhost genes affecting replication of RNA viruses

Tadas Panavas, Elena Serviene, Jeremy Brasher, and Peter D. Nagy* 2005 PNAS 102:7326-7331

• Replication of both BMV and TBSV is suppressed in about 100 out of 4,800 yeast knock-out mutants (YKOs)

• Of those 100 mutants, only 4 were common between the two viruses: three genes involved in protein metabolism (ubiquitin pathway), and the fourth a transcription regulator

• Another 10 mutants affecting replication of one or the other viruses represented genes with known functions in common. These were involved in: i) protein biosynthesis; ii) protein metabolism; iii) transcription/DNA remodeling

• Genes involved in protein targeting, membrane association, vesicle transport, and lipid metabolism affect TBSV, but none in BMV

Viroids

Viroids

• Very small, covalently closed, circular RNA molecules capable of autonomous replication and induction of disease

• Sizes range from 250-450 nucleotides• No coding capacity - do not program their own

polymerase• Use host-encoded polymerase for replication• Mechanically transmitted; often seed transmitted• More than 40 viroid species and many variants have

been characterized• “Classical” viroids have been found only in plants

Viroids are divided into two groups, based on site anddetails of replication

Viroid Diseases• Potato spindle tuber

viroid (PSTVd)– May be limiting to potato

growers– First viroid characterized– Many variants described– Control with detection in

mother stock, clean seed

PSTVd in tomato

PSTVd in potato

Viroid Diseases• Citrus exocortis viroid (CEVd)

– Causes stunting of plants, shelling of bark

– May result in little yield loss– May be useful to promote

dwarfing for agronomic advantage

– Transmitted though stock, graft– Control by removal of infected

plants, detection, clean stock

Citrus exocortis viroid

Apple crinkle fruit viroid Avocado sun blotch viroid

Citrus exocortis viroid

Healthy Infected

Potato spindle tuber viroid (PSTVd) is the most thoroughly characterized viroid disease

(From R. Owens, USDA, Beltsville)

Viroid structures-All are covalently closed circular RNAs fold to tightly base-paired structures-Two main groups of viroids: self-cleaving and non-self-cleaving-Non-self cleaving viroids replicate in nucleus and fold into “dog bone” or rod-like structure -Five domains identifiable in non-self-cleaving

-Left hand (LH) and right hand (RH) domains are non-base-paired loops-Single mutations to pathogenic domain often alter virulence-Mutations to conserved central domain are often lethal-Mutations to variable domain are often permitted

Minor variations in viroid sequence, and presumably attendantRNA structure changes, are associated with virulence differences

(From R. Owens, USDA, Beltsville)

Viroid replication• In nucleus or chloroplasts, depending on class of viroid• Chloroplast-associated viroids process into monomers by

ribozyme-mediated cleavage; nucleus-associated viroids process into monomers by using host-derived enzyme

• In both classes, host DNA-dependent RNA polymerase is the performs RNA polymerization on + and – strand RNA templates

Ribozyme-mediated Cleavage by host-factor

RZ RZ

RZ RZ HF HF

Viroid movement

• Traffic within cell through nuclear pores using VirP1, a nuclear localization protein that binds viroid RNA

• Traffic cell-to-cell through plasmodesmata

• Traffic long distance through phloem

• All of these processes are associated with host proteins

+ and – viroid strands are differentially localized within the nucleus

• Viroid strands of + polarity localized to nucleolus, as well as nucleoplasm

• Viroid strands of - polarity localized to only to nucleoplasm

Qi and Bing, 2003, Plant Cell 15:2566

Hepatitis delta• Hepatitis delta virus has many viroid-like properties, but

the RNA is larger (1.7 kb), is encapsidated, and encodes a virion-associated protein (hepatitis delta antigen)

• Intensifies HBV infection• HDV requires HBV as helper virus for encapsidation, so it

has satellite-like properties (like a “virusoid”)• Replicates in nucleus via cellular DNA-dependent RNA

polymerase II