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The biological role of Asn-linked glycosylations of

DENV-2 E protein

Sook Kwan Leang

April, 2008

A thesis submitted for the degree of

Master of Philosophy at

The Australian National University

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Table of Contents

v State~ent

vi Acknowledge~ents

vii Abbreviations

ix Abstract

-Chapter 1-

Background

1.1 Introduction ........................................................................ , .. 2 1.2 DENV disease ........................................................................ 3 1.3 DENV and other flaviviruses ....................................................... 5 1.4 DENV geno~e and polyprotein processing ....................................... 6 1.5 DENV life cycle ...................................................................... 8 1.6 DENV structural proteins and their function ..................................... 9

1.6.1 Capsid protein ............................................................... 9 1.6.2 prMIMprotein .............................................................. 10 1.6.3 E protein ... ................................................................... 11

1.7 Asn-linked glycosylation of the flavivirus E protein ............................ 19 1.8 Biological role of flavivirus E protein Asn-linked glycosylation .............. 20

1.8.1 Role ofE protein glycosylation in attachment to the host cellular receptor ..................... .. , ..................................... 20

1.8.2 Role ofE protein glycosylation in replication ............ ............... 22 1.8.3 Role of E protein glycosylation in entry ... ............................... 24 1.8.4 Role ofE protein glycosylation in virulence ...... ....................... 26

1.9 Objectives ............................................................................. 28

--- Chapter 2 --­

Materials and Methods

2.1 Site-directed mutagenesis ........................................................... 30 2.1.1 DENV-2 genome-length parental plasmids ...... ........................... 30 2.1.2 Fusion PCRfor site-directed mutagenesis .............................. 31 2.1.3 In vitro transcription of infectious RNA transcripts ... ................ 35

2.2 Electroporation of infectious RNA transcripts into BHK-21 cells ............ 37 2.2.1 Electroporation efficiency of infectious RNA transcripts in

Vero cells ............... ...................................................... 37 2.3 Sequence analysis .................................................................... 38

2.3.1 Shuttle-plasmid derivatives ................................................ 38 2.3.2 Loss-of-Asn-linked-glycan DENV-2 variants ...... ...................... 39

2.4 Cell lines and viruses ................................................................ 40 2.4.1 Plaque purification ......................................................... 41

2.5 Viral growth properties ............................ , ................................. 41 2.5.1 Viral growth in Vero cells .......... ....................................... .41 2.5.2 Viral growth in C6/36 cells ........... .................................... .42 2.5.3 Viral growth in BHK-21 cells ............ ................................. 42

2.6 F ACS analysis ........................................................................ 42 2.6.1 Percentage of virus infection in Vero cells ..... ........................ .42 2.6.2 Percentage of virus infection in C6/36 cells ............................ .43

2.7 Determination of virus titre ......................................................... 43 2.7.1 Plaque assay ................................................................. 43 2.7.2 TCIDso .................................................................... .... 44 2.7.3 qRT-PCR ............................................. ........................ 45

2.8 Virus characterisation ............................................................... 47 2.8.1 Virus specific infectivity in Vero and C6/36 cells ... .................... 47 2.8.2 C6/36 cell membranefosion assay .. ...................................... 47 2.8.3 Acid stability assay .................. ....................................... 48 2.8.4 Temperature sensitivity assay ............................................. .48

2.9 Biochemical analysis of E protein ................................................. 48 2.9.1 Metabolic labelling and radio-immuno-precipitation .......... ........ 48 2.9.2 Determination of intracellular and secreted E protein ................ .49 2.9.3 Endo-H and PNGase-F digestion ......................................... 50 2.9.4 SDS-PAGE ................................................................... 50

2.10 [5,6-3H]-uridine labeling of intracellular viral RNA ........................... 51 2.11 DENV-2 virulence in (IFN-a/y-R-/-) mice ....................................... 51

ii

- Chapter 3 ---

The generation of loss-of-Asn-linked-glycan DENV-2 E protein

from infectious cDNA clones

3.1 Introduction ........................................................................... 53 3.2 Results ................................................................................. 54

3.2.1 Electroporation efficiency of loss-0f-Asn-153-linked-glycan DENV-2 variants ............................................................. 54

3.2.2 Electroporation efficiency of loss-0f-Asn-67-linked-glycan DENV-2 variants ............................................................. 55

3.2.3 Electroporation efficiency of loss-0f-Asn-67-plus-Asn-153-linked-glycan DENV-2 variants ....... .57

3.2.4 Plaque morphology ofloss-of-Asn-linked-glycan DENV-2 variants ....... ..................................................... 57

3.2.5 Sequence analysis of loss-of-Asn-linked-glycan plaque isolates of DENV-2 ....................................................................... 59

3.2.6 Biochemical analysis ofloss-of-N-linked-glycan E proteins .......... 63 3.3 Discussion ............................................................................. 72

--- Chapter 4 ---

Biological role of the Asn-153-linked glycan in the E protein of DENV-2 strains, NGC and PUQ-US

4.1 Introduction ........................................................................... 76 4.2 Result. ................................................................................. 77

4.2.1 Growth properties ofloss-of-Asn-153-linked-glycan variants in Vero cells ......... ......................................................... 77

4.2.2 Effect of loss-0f-Asn-153-linked-glycan on virus specific infectivity in Vero cells ...... ................................................ 79

4.2.3 Growth properties ofloss-of-Asn-153-linked-glycan variants in C6136 cells ............ .................................................... 81

4.2.4 Loss-of-Asn-153-linked-glycan increases virus specific infectivity in C6136 cells ... ................................................. 84

4.2.5 Effect of loss-0f-Asn-153-linked-glycan mutation on acid stability and temperature sensitivity ...... .......................... 85

4.2.6 Effect ofloss-of-Asn-153-linked-glycan mutation on cell-cell membrane fusion of mosquito cells ............................. 88

III

IV

4.2.7 Loss of the Asn-153-linked glycan reduces virus virulence in IFN-a/y-K/- mice ..................... '" ..................... 90

4.2.8 Effect of loss-0f-Asn-153-linked-glycan mutation on E protein secretion and virus particle release ........................... 91

4.3 Discussion ................................................................. '" ......... 95

--- Chapter 5 ---

Requirement for the Asn-67-1inked glycan in the E protein ofDENV-2 strain, PUO-

218 during virus production in mammalian cells

5.1 Introduction ........................................................................... 98 5.2 Results ................................................................................. 99

5.2.1 Growth properties ofloss-of-Asn-67-linked-glycan virus variants in BHK-21 cells ........ ...................................................... 99

5.2.2 Effect ofN124S mutation in the Eprotein gene ofPUO-218 on the growth properties ofloss-of-Asn-67-linked-glycan PUO-218 variant and loss-0f-Asn-67-Asn-153-linked-glycan PUO-218 variant...... ..... ......... ................ ............. ......... 101

5.3 Discussion ............................................................................. 103

- Chapter 6 --­

Concluding Remarks

Concluding Remarks .......................................................................... 106

xii References

xxx Appendix

v

Statenlent

I certify that to the best of my knowledge, this thesis contained no materials previously

published or written by another person, except where due reference is made in the text of

the thesis. I also acknowledge that plasmid pDVWS601 was provided by Dr. Peter

Wright, and plasmids pPUO.FL, pPUO.T69L, pPUO.N67Q and pPUO.N67D were

constructed and provided by Dr. Eva Lee. The technical work involved in the animal

study was performed by Megan Pavy. All other experiments were performed by me,

under the supervisions of Dr. Eva Lee and Dr. Mario Lobigs at the Molecular Virology

Laboratory, JCMSR, ANU.

Sook K wan Leang

(18/04/08)

Vi

Acknowledgements

The completion of this research project would not have been possible without the support

and guidance from the following people. To them, I express my sincere appreciations and

best wishes.

To Dr. Mario Lobigs, my project supervisor, for giving me this opportunity to

undertake this project and also for his supervision, guidance, patience and

encouragement;

To Dr Eva Lee, my project co-supervisor, for her supervision, motivation,

guidance and most importantly for her patience and encouragement throughout the

project;

To Professor Amo Miillbacher, my project advisor, for his guidance and great

motivations;

To all the staff at the Molecular Virology Laboratory, Megan Pavy, Paivi Lobigs,

Siti Addis, Jo Shuang, Saila Ismail, Max Larena and Dr. Jay Bettadapura, for their

friendships, assistance and technical support;

To Cameron McCrae, for his help with the sequences and Hapreet Vohra and

Sarah Dawson for their assistance in the F ACS lab;

To Esmee Weil, Karen Edwards and Anna Cowan for their friendship and

assistance;

To Charis Teh, for proof-reading my thesis;

Most of all to my family, especially my beloved dad, Mr. Leang Fok Lam, for his

unfailing love and support;

Last but not least to all my friends, especially to Florence, Fei-Chuin, Soo-Huey,

Chatwin, Tricia, Christine, Ju Lynn, Dom & Leslie, Timothy and Isaac, for their constant

prayers, company, comfort, support and words of encouragement.

Vll

Abbreviations

fi-OG ~g

~Ci ~L ABTS

ADE APS Asn ATP BHK-21 BSA eDNA CRD CTP dNTPs DI DII Dill DC-SIGN

DHF DENV-I DENV-2 DENV-3 DENV-4 DENV DSS endo-H E ELISA EM EMEM EDTA ER Fe FACS FITC GTP h HBSS HEPES

n-octyl-fi-D-glucoside microgram micro-Curie micro-litre assay peroxidise substrate 2.2' -asino-di-[3-methyl-benzthiazoline sulfonate] antibody-dependent enhancement of disease ammonium persulphate Asparagine adenosine 5'-triphosphate Baby hamster kidney cells bovine serum albumin complementary nucleic acid carbohydrate-recognition domain cystidine 5' -triphosphate deoxyribonucleotide triphosphate domain I domain II domain III dendritic cell-specific intercellular adhesion molecule 3-grabbing non­integrin dengue haemorrhagic fever Dengue virus serotype 1 Dengue virus serotype 2 Dengue virus serotype 3 Dengue virus serotype 4 Dengue virus dengue shock syndrome Endoglycosidase H Envelope protein enzyme-linked immunosorbent assay electron-micrograph Eagle's minimal essential medium Ethylenedinitrilotetraacetic acid endoplasmic reticulum Fragment, crystallisable Fluorescence-activated cell sorting fluorescein isothiocyanate guanine 5' -triphosphate hour Hanks' balanced salt solution 4-(2-hydroxyethyl )-l-piperazineethanesulfonic acid

HS HSP IFNy IL-2 IL-8 l.p.

l.V.

JEV LLC-MK LPS mAb mIll MES Met MST NGC OD p.l prM PBS PNGase-F PFU qRT-PCR RT-PCR PCR rpm RNA sec sE SAP SDS SLEV TBEV TCID50

Thr TNF-n U UTP V WNV YFV

Heparan sulphate heat shock protein interferon-gamma interleukin-2 interleukin-8 intraperitoneal intravenous Japanese encephalitis virus LLC-monkey kidney cells lipopolysaccharide monoclonal antibody minute 2-(N-morpholino )ethanesulfonic acid methionine mean survival time New GuineaC optical density post-infection precursor membrane protein phosphate-buffer saline Peptide-N4-( acetyl-B-glucosaminyl)- asparagine amidase plaque forming unit quantitative reverse transcriptase- polymerase chain reaction reverse transcriptase-polymerase chain reaction polymerase chain reaction revolution per minute ribonucleic acid second soluble E protein shrimp alkaline phosphatase sodium dodecyl sulfate St Louis encephalitis virus Tick-borne encephalitis virus Tissue culture infectious dose at 50% Threonine tumour necrosis factor-alpha unit uridine 5' -triphosphate voltage West Nile virus Yellow fever virus

Vlll

IX

Abstract

The envelope proteins of many viruses are glycosylated, where the glycans on the virion

surface have been shown to be important, in addition to their strictly structural role, in

various aspects of the virus life cycle, such as receptor-binding and escape from antibody

recognition. Dengue virus (DENV) is in terms of human disease frequency the most

important member of the flaviviruses, a group of small, enveloped, plus-strand RNA

viruses. The four DENV serotypes differ from all other flaviviruses by the presence of

two glycans on the envelope (E) protein, one linked to Asn-153, a glycosylation which is

well conserved among the flaviviruses, and a second linked to Asn-67, which is unique to

DENY. While the presence of glycan at Asn-153 has been shown by others to influence

fusion of viral with cellular membranes, infectivity, assembly and/or release and

virulence, the glycan at Asn-67 has been suggested to be critical in virus attachment to

the cell surface molecule, DC-SIGN, a putative cellular receptor for DENV. In this study,

I have used a reverse genetics approach to prevent glycosylation at Asn-67, Asn-153, or

both carbohydrate acceptor sites, in order to investigate their physiological function(s).

Single or multiple point mutation were introduced at the Asn-153 and Asn-67

glycosylation sites, respectively, on the E protein ofDENV-2 strains NGC and PUO-218,

using two infectious cDNA clones. DENV -2 strain, NGC, is a laboratory-adapted virus,

while strain PUO-218 is a low-passage natural isolate from Toxorhynchites splendens

mosquitoes.

In Chapter 3, I show that removal of the Asn-153-linked glycan in the context of the E

protein of both DENV-2 strains impaired virus growth in vertebrate cells. This was

reflected in reduced plaque size on Vero cell monolayers, although electroporation

efficiencies for the different variants were not reduced relative to that of parental

infectious clone-derived RNA. Removal of the Asn-67-linked glycan revealed a more

detrimental impact on growth phenotype in vertebrate cells, which was influenced by the

E protein background and the substitution chosen to prevent glycosylation. A T69A

mutation introduced into the PUO-218 E protein produced a viable virus, albeit one with

x

a very small plaque phenotype on Vero cells. Interestingly, when other substitutions

preventing Asn-67-linked glycan addition were introduced into the PUO-218 E protein,

virus viability was improved in the order: T69V > T69L, N67Q > N67D > T69A

(Chapter 5). Insertion of the T69A mutation into the E protein of DENV-2, NGC strain,

severely limited virus viability in vertebrate cells and following passage in Vero cells

only viruses with a pseudo-reversion (T69V) or compensatory mutation (K126T) could

be recovered. The latter change introduced a novel carbohydrate acceptor site into the

NGC E protein (AsnI24-MetJ25-ThrI26), which was utilised. Passage in Vero cells of the

loss-of-Asn-67-linked-glycan variant, rPUO.T69A, also produced a pseudo-revertant

with the compensatory mutation, N 124S, in the E protein. This mutation significantly

increased plaque size on Vero cells, but did not alter glycosylation status.

Growth properties of loss-of-Asn-153-linked-glycan variants in vertebrate (Vero) and

mosquito (C6/36) cells were further analysed in Chapter 4. Absence of the Asn-153-

linked glycan in the context of the NGC E protein reduced growth in Vero cells by -10-

fold in terms of virus particle production and release measured by qRT-PCR. In contrast,

virus particle production and release of the PUO-218 loss-of-Asn-153-linked-glycan

variant was not markedly different from that of the parent virus. Loss of the Asn-153-

linked glycan reduced the specific infectivity for Vero cells but increased that for C6/36

cells for both variants. I show that the latter may be due to an increase in the pH threshold

for fusion as a result of loss of the Asn-153-1inked glycan and discuss that this E protein

change may allow virus uptake into some cell types by cell surface fusion.

In Chapter 5, the requirement for the Asn-67-linked E protein glycan for virus production

in mammalian cells was investigated by introducing infectious RNA into Vero cells by

electroporation and measuring virion assembly and release by qRT -PCR. These

experiments were undertaken in view of the finding by two groups published during the

course of my study, Mondotte et al. (2007) and Bryant et aL (2007), that loss-of-Asn-67-

linked glycosylation on the DENV -2 E protein is lethal for virus growth in mammalian

cells as a result of a defect in virus assembly/release. My results clearly demonstrate that

Xl

this conclusion is not valid for the E protein of DENV-2 strain, PUO-218: I find that

loss-of-Asn-67-linked-glycan mutant, rPUO.T69V, is comparable to the parent virus in

terms of particle assembly and release and that the N67Q mutation used in the published

work reduces but does not abolish particle assembly and release, when in the context of

the PUO-218 E protein.

I also show that removal of both glycans from the DENV-2 E protein prevents the

recovery of viable virus with a stable mutant genotype (Chapters 3 and 5). Nevertheless,

given the importance of strain origin and type of substitution used for deletion of the

carbohydrate acceptor sites that I have demonstrated in this thesis, it is likely that the

engineering of a viable DENV variant lacking both E protein glycans is feasible.