CLONING AND EXPRESSION OF El AND E2 GENES OF ...

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CLONING AND EXPRESSION OF El AND E2 GENES OF CHIKUNGUNYA VIRUS IN Escherichia coli AND BACULOVIRUS SYSTEMS Anna Andrew . Master of Science (Medical Virology) 2013

Transcript of CLONING AND EXPRESSION OF El AND E2 GENES OF ...

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CLONING AND EXPRESSION OF El AND E2 GENES OF CHIKUNGUNYA VIRUS IN Escherichia coli AND

BACULOVIRUS SYSTEMS

Anna Andrew

. Master of Science (Medical Virology)

2013

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Pusat Khidmat Maklumat Akademik UNIVERSm MALAYSIA SARAWAK

CLONING AND EXPRESSION OF El AND E2 GENES OF CHIKUNGUNYA VIRUS IN Escherichia coli AND BACULOVIRUS SYSTEMS

P.KHIDMAT MAKLUMAT AKADEMIK

1111111I1111'~rllllllllll 1000246960

ANNA ANDREW

A dissertation submitted in fulfillment of the requirements for the degree of Master of Science (Medical Virology)

Institute of Health and Community Medicine UNIVERSITI MALAYSIA SARA W AK

2013

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ACKNOWLEDGEMENTS

First and foremost, I would like to express my deepest gratitude to my

supervisor, Dr Magdline Sia Henry Sum for intellectual guidance and words of

encouragement on numerous occasions, particularly at the beginning of this project, and

the freedom she has provided me for the last two years of my studies. Thanks also to

Director of Institute of Health and Community Medicine (lHCM), Associate Professor

Dr. David Perera for teaching me the countless number of methods and techniques in the

field of molecular biology. This dissertation project was made possible with the fund by

Universiti Malaysia Sarawak (UNIMAS), project number FPI(102)/lOO/2012(60).

I would especially like to acknowledge the staffs at IHCM, Fauzziah who is in

charge of the sequencer, Norkasinah who taught me her knowledge, Gan Li Kiun who I

always refer regarding tissue culture, all the lab assistants Hamidah, Cecelia, Siti and

Zila who help in and out throughout the study and also to all the postgraduate students

who was not only of tremendous help in my experiments in the lab, but also has become

my closest friends.

Above all, my deepest thanks and gratitude go out to my family, parents,

husband and my daughter. I could not have accomplished this without your support,

love, and guidance.

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Pusat Khidmat Maklumat Akademik ll~'VERSITI MAlAVSIA SARA AI(

ACKNOWLEDGEMrnNTS 11

TABLE OF CONTENTS iii

LIST OF FIGURES viii

LIST OF TABLES XI

ABBREVIATIONS xm

ABSTRACT XVI

ABSTRAK xvn

CHAPTER 1 LITERATURE REVIEW

1.1 Introduction to Alphaviruses

1.1.1 Genome organization and virion structure of alphavirus

1.1.1.1 Functions of non-structural and structural proteins 3

1.1.1.2 Eland E2 glycoproteins ofAlphavirus 5

1.1.2 Alphavirus life cycle 7

1.2 Chikungunya virus (CHIKV) 11

1.2.1 Epidemiology of CHIKV 12

1.2.1.1 History and geographic distribution 12

1.2.1.2 2004 - 2007 CHIKV outbreak 13

1.2.1.3 Malaysia CHIKV outbreak 16

1.2.2 Vectors 18

1.2.3 Clinical presentations of CHIKV infections 21

1.2.4 Laboratory investigations of CHIKV 24

1.3 Protein expression systems 27

1.3.1 E. coli expression system 29

1.3.2 Baculovirus expression system 31

1.4 Problem statements and objectives of the study 33

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CHAPTER 2 MATERIALS AND METHODS

2.1 Propagation of Chikungunya virus 35

2.1.1 Source of Chikungunya virus 35

2.1.2 Maintenance of Vero Cell Culture ' 35

2.1.3 Inoculation of Chikungunya Virus in Vero cells 36

2.2 Cloning and expression in E. coli expression system 37

2.2.1 Gene amplification 37

2.2.1.1 Primers design 37

2.2.1.2 RNA extraction 40

2.2.1.3 RT-PCR 43

2.2.2 Preparation of inserts 47

2.2.3 Ligation of insert into pET SUMO vector 48

2.2.4 Transformation into competent Mach 11M -T 1 R E. coli 49

2.2.5 Screening for positive clones 50

2.2.6 Preparation of plasmid DNA 50

2.2.7 Sequencing PCR 53

2.2.8 Transfonnation into BL21 (DE3) 54

2.2.9 Small scale gene expression 55

2.2.10 SDS-PAGE and Western blot 55

2.2.11 Solubility of the expressed product 58

2.2.12 Cell extracts preparation 58

2.2.13 Nickel chelate column chromatography 60

2.2.14 Concentration of purified protein 61

2.3 Cloning and expression in baculovirus expression system 61

2.3.1 Gene amplification 62

2.3.1.1 Primers design 62

2.3.1.2 RNA extraction 62

2.3.1.3 RT-PCR 62

2.3.2 Preparation of inserts 65

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2.3.3 Digestion of vector and inserts with RE 66

2.3.4 Ligation 67

2.3.5 Transformation into competent Machl™_TIR E. coli 67

2.3.6 Screening for positive clones 67

2.3.7 Preparation of plasmid DNA 68

2.3 .8 Sequencing PCR 68

2.3.9 Transform into MAX Efficiency® DHlOBac™ 68

2.3.10 Bacmid preparation 70

2.3.11 Transfection 72

2.3.11.1 Maintenance of Insect cells (Sf9 cells) 72

2.3.11.2 Transfecting Sf9 cells 72

2.3.12 Amplifying baculovirus stock 74

2.3.13 Titration ofbaculovirus stock 74

2.3 .14 Expressing recombinant protein 76

2.3.15 Determining time points for optimal protein expression 77

2.3.16 SDS-PAGE and Western blot 77

2.3.17 Concentrating recombinant proteins 77

2.3.18 Nickel chelate column chromatography 79

2.4 Analysis of purified recombinant proteins 79

2.4.1 Virus titration assay 79

2.4.2 Virus neutralization assay 81

2.4.3 Evaluation of the recombinant proteins 82

2.5 Development of serological test based on recombinant antigens 83

2.5.1 Immunoblot assay 83

2.5.2 Dot blot assay 84

2.5.3 Evaluation of the serological assay 85

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CHAPTER 3 RESULTS

3.1 Virus propagation 87

3.2 Cloning and expression in E. coli system 87

3.2.1 Cloning and expression of complete CHIKV Eland

CHIKV E2 in E. coli expression system 87

3.2.2 Cloning and expression of partial CHIKE 1 and

CHIKE2 in E. coli expression system 90

3.2.2.1 Gene amplification 90

3.2.2.2 Screening for positive clones 90

3.2.2.3 Small scale expression of recombinant proteins 97

3.2.2.4 Expression of recombinant protein at different

conditions 99

3.2.2.5 Protein solubility test 104

3.2.2.6 Purification of recombinant proteins 107

3.3 Cloning and expression of complete CHIKV El and

CHIKV E2 in baculovirus expression system 109

3.3.1 Gene amplification. 109

3.3.2 Digestion profile of vector and inserts 109

3.3.3 Screening for positive clones 112

3.3.4 Transfecting Insect cells (Sf9 cells) 117

3.3.5 Titration ofbaculovirus stock 117

3.3.6 Determining time points for optimal protein expression 120

3.3.7 Concentrating culture supernatants 124

3.3.8 Purification of recombinant protein 125

3.4 Analysis of the purified recombinant proteins 130

3.4.1 Virus titration assay 130

3.4.2 Virus neutralization assay 130

3.4.3 Preliminary evaluation of the recombinant proteins 134

3.4.3.1 E. coli recombinant proteins 134

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3.4.3.2 Baculovirus recombinant proteins 137

3.5 Development of serological assay 143

3.5.1 Immunoblot assays 143

3.5.2 Dot blot assay 147

CHAPTER 4 DISCUSSION AND CONCLUSION

4.1 Recombinant proteins expressed by E. coli system 154

4.2 Recombinant proteins expressed by baculovirus system 158

4.3 E. coli vs Baculovirus expression system 159

4.4 Limitation of the study 161

4.5 Recommendation and future research 162

4.6 Conclusion 162

REFERENCES 165

APPENDICES 182

Publication and Presentation 210

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LIST OF FIGURES

Figure 1.1 Cross Section of virus particle 2

Figure 1.2 Organization of the Alphavirus genome 4

Figure 1.3 The Semliki Forest virus (SFV) E 1 confonnation 6

Figure 1.4 The Chikungunya virus (CHIKV) E2 confonnation 8

Figure 1.5 Summary of the CHIKV replication cycle 10

Figure 1.6 Global distribution and the three major genotypes of CHIKV 14

Figure 1.7 Chikungunya outbreaks in Indian Ocean Island (2005 - 2006) 15

Figure 1.8 Transmission cycles of CHIKV 20

Figure 1.9 Biological diagnosis of Chikungunya 25

Figure 1.10 T A Cloning diagram 30

Figure 1.11 Bac-to-Bac® Baculovirus Expression System procedures 32

Figure 2.1 Flowchart of cloning and expression strategies in E. coli

expression system 38

Figure 2.2 The amino acid sequence of E 1 of CHIKV analyzed

with PROTEAN, LaserGene DNAStar software 41

Figure 2.3 The amino acid sequence of E2 of CHIKV analyzed

with PROTEAN, LaserGene DNA Star software 42

Figure 2.4 Flowchart of cloning and expression strategies used

in baculovirus expression system 63

Figure 3.1 The cytopathic effect (CPE) of CHIKV inoculated in

Vero cell line 88

Figure 3.2 Agarose gel electrophoresis analysis ofRT-PCR products

of complete CHIKV Eland E2 for cloning into E. coli system 89

Figure 3.3 Agarose gel electrophoresis profile ofRT-PCR products of

partial CHIKV Eland E2 for cloning into E. coli system 91

Figure 3.4 Colonies screening of recombinant partial CHIKV E 1 in

pET SUMO vector/Mach 1 ™ -T 1 R E. coli via colony-PCR 92

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Figure 3.5 The partial CHIKV E 1 clone 10 recombinant plasmid

sequence in pET SUMO vector 94

Figure 3.6 Colonies screening of recombinant partial CHIKV E2

in pET SUMO vectorlMach1™-T1 R E. coli via colony-PCR 95

Figure 3.7 The partial CHIKV E2 clone 7 recombinant plasmid sequence

in pET SUMO vector 96

Figure 3.8 Analysis of small scale expression of partial CHIKV E 1

recombinant protein 98

Figure 3.9 Analysis of small scale expression of partial CHIKV E2

recombinant protein 100

Figure 3.10 Analysis of partial CHIKV E1 recombinant protein at

different conditions 102

Figure 3.11 Analysis ofpartial CHIKV E2 recombinant protein at

different conditions 103

Figure 3.12 Solubility test for recombinant partial CHIKV E 1 protein 105

Figure 3.13 Solubility test for recombinant partial CHIKV E2 protein 106

Figure 3.14 Protein purification gel profile 108

Figure 3.15 Agarose gel electrophoresis profile ofRT-PCR products for

cloning into baculovirus expression system 110

Figure 3.16 Agarose gel electrophoresis profile of digested products 111

Figure 3.17 Colonies screening of recombinant CHIKV E 1 in

pFastBacDual vector and transfonned into MachI ™ -T1 RE. coli 113

Figure 3.18 The sequence of the complete CHIKV E 1 clone 16

recombinant plasmid in pFastBacDual vector 114

Figure 3.19 Colonies screening of recombinant CHIKV E2 in

pFastBacDual vector and transfonned into Mach1™_T1R E. coli 115

Figure 3.20 The sequence of the complete CHIKV E2 clone 10

recombinant plasmid in pFastBac Dual vector 116

Figure 3.21 Cytopathic effect (CPE) of Sf9 cells transfected with

recombinant bacmid 118

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Figure 3.22 Baculovirus stocks titration 119

Figure 3.23 Different days harvest of CHIKV E 1 baculovirus stock 122

Figure 3.24 Different days harvest of CHIKV E2 baculovirus stock 123

Figure 3.25 Protein expression of concentrated culture supernatant of

CHIKV E1 in baculovirus system 126

Figure 3.26 Protein expression of concentrated culture supernatant of

CHIKV E2 in baculovirus system 127

Figure 3.27 Complete CHIKV E1 recombinant protein purification gel profile 128

Figure 3.28 Complete CHIKV E2 recombinant protein purification gel profile 129

Figure 3.29 Purified E. coli recombinant proteins 135

Figure 3.30 Evaluation of the purified E. coli recombinant proteins with

patient sera 136

Figure 3.31 Purified complete CHIKV E 1 recombinant proteins 139

Figure 3.32 Evaluation of the purified CHIKV E1 recombinant proteins

generated by baculovirus system 140

Figure 3.33 Evaluation of the unpurified CHIKV E2 recombinant proteins

generated by baculovirus system 141

Figure 3.34 The reactivity ofthe recombinant proteins with PRNT50 positive

and negative sera in immunoblot assay 145

Figure 3.35 The reactivity of the recombinant proteins with PRNT 50 positive

and negative sera in IgM dot blot assay 150

Figure 3.36 The reactivity of the recombinant proteins with PRNTso positive

and negative sera in IgG dot blot assay 151

Figure 4.1 The position of partial CHIKV E 1 genome cloned and expressed

by (x) Yathi et aI., 2011 and (y) in this study 155

Figure 4.2 The alignment of partial CHIKV E2 amino acids generated by

E. coli system in this study and E2EP3 protein 157

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LIST OF TABLES

Table 1.1 Summary of differences of sign and symptoms between

CHIKV fever and dengue fever 23

Table 2.1 Oligonucleotide primers used for PCR amplification for

the purpose of cloning into E. coli system 39

Table 2.2 PCR cycling conditions used to amplify the gene of interest 45

Table 2.3 The annealing temperature used in each set of primers 46

Table 2.4 Primers used for screening of E. coli recombinant clones and

the expected PCR product size 51

Table 2.5 Oligonucleotide primers used for PCR amplification for

the purpose of cloning into baculovirus system 64

Table 2.6 Primers used for screening of baculovirus recombinant

clones and expected size 68

Table 2.7 Matrix table used to calculate the screening test

characteristics of sensitivity and specificity 86

Table 3.1 Sera and neutralization end titer of the sera used

for preliminary evaluation of the recombinant proteins 131

Table 3.2 Neutralization end titer and other laboratory information of

each sera used in the development of serological assay 132

Table 3.3 Summary of preliminary evaluation of the

recombinant proteins expressed by E.coli and

baculovirus system 142

Table 3.4 Recombinant proteins with their respective size tested in

immunoblot assay 144

Table 3.5 Relative sensitivities and specificities of anti-CHIKV

IgG immunoblot assay using recombinant proteins 146

Table 3.6 Relative sensitivity and specificity of anti-CHIKV IgM and

IgG Dot Blot assay using recombinant CHIKV E 1 protein 152

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Table 3.7 Sensitivity and specificity of anti-CHIKV IgM Dot Blot assay

using the complete CHIKV E 1 recombinant protein

with anti-CHIKV IgM Immunofluorescent assay (IgM) 153

Table 4.1 Summary of cloning and expression ofEland E2 genes

of chikungunya virus using two cloning systems 164

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A

Ae

BF

bp

C

C

CBB

cDNA

CHIKV

CMC

CO2

CPE

DMEM

DNA

E. coli

El

E2

ECSA

EEE

ELISA

ER

ABBREVIATIONS

Alanine

Aedes

Barmah forest

base pair

Capsid

Celsius

Coomassie brilliant blue

complementary Deoxyribose nucleic acid

Chikungunya Virus

carboxymethyl-cellulose

Carbon dioxide

Cytopathic effect

Dulbecco's Modified Eagle's Medium

Deoxyribose nucleic acid

Escherichia coli

Envelope 1

Envelope 2

East Central South African

Eastern equine encephalitis

Enzyme linked immunosorbent assay

Endoplasmic reticulum

Xlll

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FBS

HI

HIS

HRP

IFA

IgG

IgM

ICT

IIF

IMR

IPTG

kb

kDa

LB

MID

mM

MOl

NaOH

NC

NCR

NDV

Nickel-HRP

NPHL

Foetal Bovine Serum

Haemagglutination Inhibition

Histidine

horseradish peroxidase

Immunofluorescent assay

Immunoglobulin G

Immunoglobulin M

Immunochromatographic test

Indirect immunofluorescent

Institute for Medical Research

isopropyl-(3-D-thiogalactosidase

kilobases

kilo daltons

Luria Bertani

Middleburg

mili molar

Multiplicity of infection

Sodium hydroxide

Nucleocapsid

Non-coding region

Ndumu

Nickel with horseradish peroxidase

National Public Health Laboratory

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ns

ONN

ORF

PBS

PCR

pfu

pi

PRNT

RNA

rpm

RT-PCR

SOS

SDS-PAGE

SF

Sf9

sgRNA

S.O.C

TBE

UHQ

UV

V

VEE

WEE

Non-structural

O'Nyong-nyong

Open Reading Frame

Phosphate Buffered Saline

Polymerase Chain Reaction

Plaque forming unit

post-infection

Plaque Reduction Neutralization Test

Ribonucleic acid

revolution per minute

Reverse transcription polymerase chain reaction

Sodium dodecyl sulphate

sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Semliki forest

Spodoptera Jrugiperda cells

subgenomic RNA

Super Optimal Broth with catabolite repression

Tris-borate-EDTA

Ultrapure high quality

Ultraviolet

Valine

Venezuelan equine encephalitis

Western equine encephalitis

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Cloning and Expression of El and E2 Genes of Chikungunya virus in

Escherichia coli and Baculovirus Systems

Abstract

(Chikungunya fever caused by Chikungunya virus (CHIKV) has re-emerged with

large outbreaks in various parts of the world and grabbed the attention of researchers

worldwide. Due to lack of simple and rapid diagnostic method for the identification of

the virus infection, assessing the epidemic potential and implementing appropriate

control measures are often delaye~ In this study, Envelope 1 (E 1) and Envelope 2 (E2)

genes of CHIKV from a local isolate were cloned into both E. coli and baculovirus

systems as CHIKV -specific diagnostic reagents. The antigenicity of the expressed

products was assessed with CHIKV positive and negative patient's sera. Partial CHIKV

El and E2 were cloned into E. coli system while complete CHIKV El and E2 were

cloned into baculovirus system. All the recombinant proteins were found to be antigenic

except for the partial CHIKV E 1 recombinant protein generated by E. coli system. None

of the recombinant antigens showed cross reactivity with anti-dengue virus serum

sample. These results suggest that the recombinant proteins generated in this study might

be useful in the development of diagnostic tools such as ELISA, for the detection of

CHlK.V infections. Even though the direct comparison of the two systems is not

possible, baculovirus system was seems to be better as they are more compatible with

eukaryotic proteins because of similar codon usage rules, providing better expression

levels and fewer truncated proteins than in bacteria.

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Pengklonan dan Ekspresi Gen "Envelope 1" dan "Envelope 2" Virus Chikungunya

Melalui Sistem Escherichia coli dan Baculovirus

Abstrak

Chikungunya adalah demam yang disebabkan oleh virus chikungunya (CHIKV)

dan wabak ini telah merebak ke kebanyakan negara dan menarik perhatian penyelidik di

seluruh dunia. Oleh kerana kekurangan kaedah diagnostik yang boleh men genal pasti

virus ini dengan cepat, mengenal pasti punca wabak dan tindakan untuk mengatasi

masalah tersebut biasanya tertangguh. Dalam projek ini, gen "Envelope 1" dan gen

"Envelope 2" virus chikungunya diklon melalui sistem E. coli dan baculovirus. Produk­

produk yang telah diekspreskan itu dikaji keantigenikannya dengan menggunakan serum

pesakit yang disahkan positif dan negatif terhadap virus chikungunya. Separa gen

"Envelope 1" dan gen "Envelope 2" telah diklon dan diekspreskan menggunakan sistem

E. coli manakala seluruh gen "Envelope 1" dan gen "Envelope 2" telah diklon dan

diekspreskan menggunakan sistem baculovirus. Semua protein rekombinan yang

dihasilkan telah disahkan keantigenikannya kecuali separa gen "Envelope 1" yang

dihasilkan melalui sistem E. coli. Semua rekombinan protein juga tidak menunjukkan

tindak balas apabila diuj i dengan serum dengue. Sehubungan itu, protein rekombinan

yang dihasilkan melalui kajian ini mempunyai potensi digunakan sebagai antigen

diagnostik seperti ujian ELISA bagi mengenal pasti penyakit chikungunya. Walaupun

perbandingan dua sistem yang digunakan tidak dapat dibandingkan secara menyeluruh,

sistem baculovirus didapati lebih sesuai menghasilkan protein eukariotik.

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CHAPTER 1

LITERATURE REVIEW

1.1 Introduction to Alphaviruses

The Alphavirnses genus of the family Togaviridae is an arthropod-borne

viruses (arboviruses) and consists of 30 species that has been classified into 7

antigenic complexes: Bannah Forest (BF), Eastern equine encephalitis (EEE),

Middleburg (MID), Ndumu (NDU), Semliki Forest (SF), Venezuelan equine

encephalitis (VEE), and Western equine encephalitis (WEE) (reviewed by Strauss

and Strauss, 1994). Depending on the geographic location from which they were

originally isolated, these viruses are classified as either New World alphaviruses or

an Old World alphaviruses. While New World alphavirnses typically cause

encephalitis in humans and other mammals (e.g. Venezuelan equine encephalitis

virus, Western equine encephalitis virus, Eastern equine encephalitis virus), infection

by Old World alphavirnses typically cause fever, rash, and arthralgia syndrome that

are rarely fatal (e.g. Semliki Forest virus, O'nyong-Nyong virus, Chikungunya

virus). Of the Old World alphaviruses, Chikungunya virus (CHIKV) is the most

important human pathogen, which has caused numerous outbreaks and become a

major global health problem (Enserink, 2007).

1.1.1 Genome organization and virion structure of alphavirus

The mature virion of alphavirns is a small, icosahedral-shaped, enveloped

and about 70 nm in diameter (Simuzu et aI., 1984). As shown in Figure 1.1, the

virion consists of three components: (i) an outer glycoprotein shell, (ii) a lipid bilayer

which derived from the host cell plasma membrane and (iii) an RNA-containing core 1

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Glycoprotein shell

Lipid bilayer

Virus RNA

Nucleocapsid

Figure 1.1 Cross Section of virus particle; red is the viral RNA, brown IS the nucleocapsid, green is the lipid bilayer, and yellow is the glycoprotein shell.

(Adapted from Sherman and Weaver, 2010)

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or nucleocapsid (Zuckennan et aI., 2004). The viral-encoded glycoproteins, E I and

E2 proteins fonned heterodimers that oligomerized into 80 prominent trimeric spikes

protruding perpendicularly to the virus surface. The nucleocapsid consists of

genomic positive sense RNA and is approximately 11,800 nucleotides long. The

genome resembles eukaryotic mRNAs in that it possesses 5' methyl-guanosine cap

structures and 3' poly (A) tail (Strauss and Strauss, 1994; Powers et aI., 2000;

Solignat et aI., 2009).

The coding sequence consists of two large open reading frames (ORFs); the

N-tenninal ORF encodes the non-structural polyprotein (nsP I, nsP2, nsP3 and nsP4)

and the C-tenninal ORF encodes the structural polyprotein (C, E3, E2, 6K and EI)

(Figure 1.2). The two ORFs are flanked by 5' and 3' non-coding regions (NCR) that

contain signals important for replication of the viral RNA (Zuckennan et aI., 2004).

1.1.1.1 Functions of non-structural and structural proteins

There are four non-structural proteins namely nsP I, nsP2, nsP3 and nsP4 in

alphavirus genome as previously mentioned. In general, the non-structural proteins

are required for viral replication and processing. Upon entry and uncoating, nsP I is

specifically required for synthesis of minus strand RNA (Strauss and Strauss, 1994).

This minus strand RNA in turn provides the template for synthesis of both new

genomic and subgenomic RNA. nsP2 of alphavirus has two structural and functional

domains. The N-tenninal domain serves as a RNA helicase to unwind the RNA-RNA

duplex during RNA replication and transcription while the C-tenninal part of the

protein has been associated with proteolytic processing of the alphavirus polyprotein

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Figure 1.2 Organization of the Alphavirus genome. The four non-structural proteins (nsPI-4) are translated as a single poyprotein directly from the positive-sense RNA genome. The structural proteins (C, E3, E2, 6K and E I) are translated from a subgenomic RNA (26S) transcribed from a separate promoter within the non-coding (NCR) junction region. 5' and 3' NCR regions flank the coding region.

(Reproduced from Tsetsarkin, 2009a)

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Pusat Khidmat Maklumat Akademlk UNIVERSm MALAYSIA ARAWAJ<

(non-structural proteinase) (Strauss and Strauss, 1994). The role of nsP3 is to

synthesize subgenomic 26S and negative strand RNA and nsP4 is thought to be the

RNA polymerase of the alphaviruses (Sherman and Weaver, 20 I0).

The structural polyprotein of alphavinls consists of capsid (C), two envelope

glycoproteins (El and E2) and two small proteins E3 and 6K. C forms a

nucleocapsid (NC) core structure beneath the viral membrane, encasing the viral

genome. C acts as an autoprotease, to recognize the genomic RNA, and to assemble

into an ordered protein shell (Warrier et aI., 2008). The two envelope glycoproteins

El and E2 that form spikes of the virus playa major role in viral encapsidation and

budding. The structure of El and E2 will be further discussed in section 1.1.1 .2. The

main function of the E2 glycoprotein during the course of the alphavirus life cycle is

interaction with specific cell surface receptor while El is responsible for triggering

fusion of the viral and target cell membranes during entry. The two small peptides

E3 and 6K protein act as a signal sequence where E3 signal for insertion of the

remaining polyprotein into the endoplasmic reticulum while 6K signal for the

downstream processing of the E I protein (Simuzu et aI., 1984).

1.1.1.2 El and E2 glycoproteins of Alphavirus

During alphavinls infection, the invasion of susceptible cells is mediated by

two viral glycoproteins, El and E2, which carry the main antigenic determinants and

form the glycoprotein shell at the virion surface. The Eland E2 proteins are similar

in shape with E2 proteins extend to the tips of the spikes (Mukhopadhyay et aI.,

2006).

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Figure 1.3 The Semliki Forest virus (SFV) EI confonnation; EI domain I, II and III are shown in red, yellow and blue, respectively and the fusion loop in orange. The N­ter is the N-tenninus and C-ter is the C-tenninus of the EI protein.

(Adapted from Voss et aI., 2010)

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