cloning and expression of el and e2 genes of
TRANSCRIPT
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
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
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.
ii
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
IV
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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
v
1 I
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
VI
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
VB
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
viii
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
IX
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
x
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
Xl
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
XlI
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
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
XIV
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.
XVI
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.
xvii
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
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)
2
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
3
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)
4
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).
5
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)
6