Modulation of west nile virus capsid protein and viral RNA interaction through phosphorylation 2

2.0

MATERIALS AND METHODS

2.1

Cell culture techniques
All solutions and media for cell culture were made with sterile ultrapure water

(Milipore, USA). All cell culture and media preparation work was performed under
aseptic conditions in a Class II Type A2 Biosafety Cabinet (ESCO Pte Ltd). Ltd.
Singapore). The cells used in this study were grown in 25 cm2 or 75 cm2 plastic tissue
culture flasks (Iwaki Glass, Japan.), and 24-well tissue culture plates (Greiner Bio – One,
USA). Cells were either cultured in a humidified 37 °C incubator (Thermo Fischer
Scientific, USA.) with 5 % carbon dioxide or a dry incubator (Memmert GmbH,
Germany).

2.1.1

Cell lines
The cell lines and the type of media used for each cell line used in this study are

listed in Table 2-1. All cell lines used are adherent cell types.

Table 2-1 Cells and media
Cell Line
Cell Media
Baby
Hamster RPMI
Kidney Cells
21

(BHK),
Human Embryonic DMEM
Kidney cells FT
(293FT)
Mosquito
cells L-15
derived from Aedes
albopictus (C6/36)

| Materials and methods

Cell Passage level
100 – 190
10 – 75
80 – 120

Origin
American
Type
Culture Collection,
USA
Invitrogen. Cat no.
R700-07
Kind gift from
Emeritus Professor
Edwin Westaway,
Australia.

26

2.1.2

Media and solution for cell culture
All culture media were supplemented with either 10 % or 2 % foetal calf serum

[FCS (PAA Laboratories GmbH, Austria)] and their formulations can be found in
Appendix 1A to C for the respective cell lines. The media pH was adjusted to
approximately 7.3 with solutions of 1 M sodium hydroxide and 1 M hydrochloric acid
(Appendix 1D).

2.1.3

Cultivation and propagation of cell lines
Flasks of cells were sub-cultured from confluent 75cm2 flask monolayer at a ratio

of 1 : 10 (BHK 21 and 293 FT cells) or 1 : 4 (C6/36 cells). The growth medium
(Appendix 1A to C) was first discarded. The monolayer was then rinsed with 5 ml of
phosphate buffered saline (PBS - Appendix 1E). This was followed by incubation with 2
ml of trypsin (Appendix 1F). The flask was left to incubate at 37 °C for 2 min and the
cells were dislodged by gentle tapping. Appropriate amounts of growth media were
added to the cell suspension to deactivate the activity of trypsin. Cell aggregates were
resuspended by pippetting up and down. The suspended cells were then aliquoted into
new tissue culture flasks. The cells were then incubated at 28 °C for C6/36 cells or 37 °C
for all other cell types.

2.1.4

Cultivation of cells in 6-well and 24-well tissue culture plate
A confluent cell monolayer of BHK or 293FT cells in a 75 cm2 tissue culture flask

was used to seed four 24-well or 6-well plate (Greiner Bio-One, St. Louis, USA). The
cell monolayer was treated as previously described (Section 2.1.3) to produce a single

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27


cell suspension. The cell suspension was then made up to a final volume of 10 ml using
appropriate cell culture growth medium. Aliquots of 1 ml of cell suspension were then
diluted in 24 ml or 30 ml of cell culture growth medium. Apporximately 2 x 105 cells or 1
x 106 cells was then transferred into each of the 24 wells or 6 wells, respectively. The
plates were then incubated at 37 °C in a humidified incubator (Thermo Fisher Scientific,
Massachusetts, USA) with 5 % carbon dioxide. The monolayers were confluent in 48 hr
and ready for use.

2.1.4.1 Cultivation of cells on cover slips
Individual glass cover slips were aseptically placed in 24-well tissue culture plate.
A confluent monolayer of cells from a 75 cm2 tissue culture flask was used to seed the
wells as described in Section 2.1.4. The plates were then incubated at 37 °C in a
humidified incubator supplemented with 5 % carbon dioxide until they were about 80 %
to 90 % confluent.

2.2

Infection of cells and purification of virus

2.2.1

Viruses
The virus used in this study is the Sarafend strain of West Nile virus. It was a kind

gift from Emeritus Professor Edwin Westaway. The virus was propagated in either BHK
or C6/36 cells.

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28


2.2

Infection of cell monolayers for virus propagation
A confluent monolayer of cells was used for infection. The cell culture

supernatant was discarded and the monolayer was rinsed with 5 ml of PBS (Appendix
1E). Either 1 ml or 2 ml of virus suspension were used to infect cells in 75 cm2 or 175
cm2 tissue culture flask, respectively. The flask was incubated at 37 °C for 1 hr and
rocked every 15 min to ensure even infection. After 1 hr, unabsorbed virus was washed
off with 5 ml of appropriate maintenance medium (Appendix 2A and 2B) and an
appropriate volume of the same medium was added to the flask. Subsequently, the
infected BHK or C6/36 cell culture flask was either incubated at 37 °C in a humidified
incubator supplemented with 5 % carbon dioxide or at 28 °C in a dry incubator,
respectively.

2.2.3

Preparation of virus pool
Infection was carried out as described in Section 2.2.2. The virus was harvested

when cytopathic effects were pronounced, usually 24 hr post infection (p.i.). To obtain
extracellular virus, the infected cell culture supernatant was removed from the flask and
spun at 3000 x g for 10 min to remove cellular debris. Intracellular virus was obtained by
harvesting the monolayer of cell. The cells were detached from the flask with an
appropriate amount of trypsin (Appendix 1F). Trypsin was neutralize with an equal
maintenance medium. The resulting cell suspension was sedimented at 800 x g for 10
min and followed by 3 cycles of freeze-thawed action. The cell debris were removed by
spinning at 3000 x g for 15 min. Approximately 500 µl of infected supernatant
(extracellular virus) or clarified cell lysate (intracellular virus) was aliquoted into sterile

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29


cryovials (Nalgene, USA) and immediately snapped frozen in -80 °C liquid ethanol. The
frozen cryovials (Nalge Nunc International, Roskilde, Denmark) were then stored in a 80 °C freezer.

2.2.4

Concentration and purification of virus
Supernatant containing virus was prepared as described in Section 2.2.3.

Concentration of virus was achieved by placing the supernatant into a Vivaspin 100000
MW centricon filtration device (Satroius, Germany) and spinning at 3000 x g until the
desired volume is attained. The partially purified virus is either snap frozen in -80 °C
liquid ethanol or further purified by density gradient.
Density gradient is performed with the OptiPrep density gradient medium (AxisShield, Norway). The medium was diluted with 1M Tris-HCl buffer, pH7.4 (Appendix
2C) to obtain a range of concentrations (20 % - 50 %). A 5 ml continuous density

gradient was prepared by layering different concentrations (20 %, 30 %, 40 % and 50 %)
of 1 ml each of the OptiPrep medium on top of one another starting with the most
concentrated solution at the bottom. The virus supernatant was layered on top of the
gradient and spun at 35000 rpm (Beckman L8-ultracentrifuge, USA) for 16 hr at 4 °C in a
SW 55 rotor (slow acceleration, no brakes). After the spin, 1.5 ml of the medium was
removed from the bottom of the tube. The next 1 ml of medium was extracted from the
bottom and diluted 1:1. One ml of the diluted extract was layered over 4 ml of 15%
OptiPrep medium as a cushion. The layered cushion was spun at 35000 rpm in a SW 55
rotor for 3 hr at 4 °C. After the spin, the supernatant was removed and the virus pellet
was resuspended in Tris-HCl buffer, pH 7.4.

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2.2.5 Plaque assay
BHK cells were subcultured and grown in 24-well plates as described in Section
2.1.4. Ten-fold serial dilutions of the virus sample were prepared in virus diluent down to
10-8 (Appendix 2D). Aliquots of 100 µl from each dilution were transferred in triplicates
onto confluent cell monolayers in the wells. The plate was rocked every 15 min for 1 hr
at 37 °C with 5 % carbon dioxide to ensure even distribution of virus inocula. The diluted
virus inocula were removed, and the cell monolayers were washed once with PBS
(Appendix 1E). One ml of overlay medium (Appendix 2E) was pipetted into each well.
The trays were incubated at 37 °C in a humidified 5 % carbon dioxide incubator. Plates
were incubated for two days before the overlay medium was removed for staining. The
plaques were visualized by staining the monolayer with 0.5 % crystal violet in a 25 %
formaldehyde solution (Appendix 2F) for at least two hr at room temperature on orbital
shaker. The crystal violet solution was removed for proper hazardous chemical disposal
and the plate was washed under a running tap to remove residual dye. Plaques were

counted and the titre was calculated as PFU per ml of supernatant (PFU/ml).

2.2.6 Extraction of virus RNA
Viral RNA extraction was carried out using QIAmp® Viral RNA extraction kit
(Qiagen, USA). Virus pool for RNA extraction was obtained at described in Section
2.2.3. Extraction of viral RNA was performed by adding the kit’s lysis buffer to the virus
and subsequently loading the lysate into a spin column. RNA was isolated from the
supernatant by centrifugation. RNA bound to the column’s membrane was then eluted
with 35 µl of DEPC-treated water (Appendix 3A).

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31


2.3

Molecular techniques

2.3.1

Cloning vectors, expression vectors and infectious clones constructs.
The following Table (Table 2-2) shows the list of vectors and constructs used in

this study.

Table 2-2 List of genes and regions cloned.
Name
P28-His-C
pCMVmyc-C

Gene or regions cloned
Amino-terminal
Histidine-tagged mature
capsid protein
Amino-terminal cMyctagged mature capsid
protein

p1.3-transfer

The first 1.3kb of the
WNV genome

pWTIC

WNV infectious clone

Vector backbone
Pet28 A (Novagen)
pCMV-myc
(Clontech)
PBR322 (Promega)
PBR322 (Promega)

Purpose
Expression of C
protein in bacteria
cells.
Expression of C
protein in bacteria

cells.
A carrier vector
for the
mutagenesis of
the C protein.
Production of
WNV virus

All the above clones were constructed previously in the laboratory except for P28-His-C
which was constructed specifically for this project. Myc-C was constructed by
Bhuvanakantham Raghavan while p1.3-transfer and pWTIC was constructed previously
(Li et al., 2005).

2.3.2

List of primers
Table 2-3 shows a list of the names of the primers used and its corresponding

purposes. The sequences, of these primers can be found in Appendix 3B

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Table 2-3. List of the names of primers used and its purpose
No.
1
2
3

4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33

34
35
36
37
38
39
40
41

Primer Name
His-C F
His-C- R
Sense RNA 3 UTR F
Sense RNA 3 UTR R
Sense RNA 5 UTR F
Sense RNA 5 UTR R
Sense RNA 5 UTR+C F
Sense RNA 5 UTR+C R
A-sense RNA 3 UTR F
A-sense RNA 3 UTR R
A-sense RNA 5 UTR F
A-sense RNA 5 UTR R
A-sense RNA 5 UTR+C F
A-sense RNA 5 UTR+C R
RNA 1F
RNA 1R
RNA 2F
RNA 2R
RNA 3F
RNA 3R

RNA 4F
RNA 4R
RNA 5F
RNA 5R
RNA 6F
RNA 6R
RNA 7F
RNA 7R
RNA 8F
RNA 8R
RNA 9F
RNA 9R
RNA 10F
RNA 10R
RNA 11F
RNA 11R
RNA 12F
RNA 12R
A-sense RNA 1F
A-sense RNA 1R
A-sense RNA 12R

| Materials and methods

Purpose
Cloning of C protein
Synthesize sense 3’ UTR viral RNA
Synthesize sense 5’ UTR viral RNA
Synthesize sense 5’ UTR+C viral RNA
Synthesize anti-sense 3’ UTR viral RNA

Synthesize anti-sense 5’ UTR viral RNA
Synthesize anti-sense 5’ UTR+C viral RNA
Synthesize fragment 1- 1017 of viral RNA
Synthesize fragment 960-1974 of viral RNA
Synthesize fragment 1920-2934 of viral RNA
Synthesize fragment 2880-3894 of viral RNA
Synthesize fragment 3830-4839 of viral RNA
Synthesize fragment 4790-5804 of viral RNA
Synthesize fragment 5758-6772 of viral RNA
Synthesize fragment 6728-7742 of viral RNA
Synthesize fragment 7698-8702 of viral RNA
Synthesize fragment 8655-9669 of viral RNA
Synthesize fragment 9613-10617 of viral RNA
Synthesize fragment 10053-11057 of viral RNA
Synthesize ant-sense fragment 1- 1017 of viral
RNA
Synthesize anti-sense fragment 10053-11057 of

33


42
43
44
45
46
47
48
49
50

51
52
53
54
55
56
57

A-sense RNA 12R
RNA RT 1 Rev
RNA RT 2 Rev
RNA RT 3 Rev
RNA RT 4 Rev
RNA RT 5 Rev
RNA RT 6 Rev
RNA RT 7 Rev
RNA RT 8 Rev
RNA RT 9 Rev
RNA RT 10 Rev
RNA RT 11 Rev
A-sense RNA RT 2 Rev
A-sense RNA RT 12 Rev
Seq C F
Seq C R

viral RNA

Reverse primers for real-time polymerase chain
reaction for the above generated RNA fragments

Forward and reverse sequencing primers for capsid
protein

All primers were synthesized either by Proligo Singapore Pte Ltd or Sigma-Aldrich. The
primers are partially purified through desalting procedures performed by the
manufacturers.

2.3.3

Bacteria strains
The One Shot DH5α Escherichia Coli (E.Coli) competent cells (Invitrogen, USA)

were used for the transformation and propagation of cloning vectors. BL2-CodonPlus
competent E.coli cells (Stratagene, USA) were used for the expression of proteins. All the
bacteria cells were grown in either Luria-Bertani (LB) broth (Appendix 4A) or LB agar
supplemented with the appropriate antibiotic (Appendix 4B).

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2.3.4
2.3.4.1

Agarose electrophoresis
RNA agarose gel electrophoresis
To cast a 1.5 % RNA agarose gel, 1.5 g of agarose (1st Base, Singapore) was

mixed with 74 ml of deionised water and brought to a boil in a microwave. When the

molten agarose cooled, 10 ml of 10 x MOPS buffer (Appendix 5A) and 8.75 ml of 37 %
formaldehyde was added to give final concentration of 1 x MOPS and 2.2 M of
formaldehyde, respectively. The molten agarose was poured into a gel casting tray and
the combs inserted. When the gel had solidified, it was transferred into the gel
electrophoresis tank filled with 1 x MOPS buffer and 2.2 M formaldehyde. RNA samples
were prepared by adding an appropriate amount of RNA loading buffer (Ambion, USA)
and 1 µl of 10 mg/ml ethidium bromide was added into the sample to aid visualization
after electrophoresis. The sample was heated to 65 °C for 10 min immediately prior to
loading. Approximately 6 µl of RNA marker (Ambion, USA) was added in another well
to indicate the relative size of the RNA. The sample was electrophorized at 120 volts for
40 mins. The DNA bands were then visualize under UV light (UV transilluminator,
Vilber Lourmat, UK) and captured with ChemiGenius (Syngene, UK).

2.3.4.2

DNA agarose gel electrophoresis
To cast a 1.2 % DNA agarose gel, 2.4 g of agarose (1st Base, Singapore) was

mixed with 200 ml 1 x TBE buffer (Appendix 5B) in a 500 ml conical flask and heated to
a boil in a microwave. The flask of molten agarose was cooled and 7 µl of 10 mg/ml of
ethidium bromide solution (Sigma, USA) was added and mixed. The molten agarose was
then poured into a gel casting tray and the gel combs were inserted. The solidified gel

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35


was then submerged in the gel electrophoresis tank (Bio-Rad, USA) filled with 1 x TBE
buffer (Appendix 5B). An appropriate amount of DNA sample was mixed with 6 x gel

loading buffer (Promega, USA) before loading into the well. Approximately 6 µl of 1000
bp or 100 bp DNA markers (Promega, USA) was added in another well to indicate the
relative size of the DNA sample. The sample was electrophorized at 120 volts for 40
mins. The DNA bands were then visualize under UV light (UV transilluminator, Vilber
Lourmat, UK) and captured with ChemiGenius (Syngene, UK).

2.3.5

Propagation and mutagenesis of the infectious clone of West Nile virus
The infectious clone of West Nile virus was previously constructed in the

laboratory (Li et al., 2005). The DNA plasmid encoding the infectious viral was
linearized with Xba I (Promega, USA) and the DNA was purified with QiaPrep miniprep
kit (Qiagen USA). The infectious West Nile virus RNA was in vitro transcribed with the
RiboMax kit (Promega) using purified and linearized DNA as a template. The transcribed
RNA was extracted using phenol:chloroform:isoamyl alcohol. 25:24:1, v/v (Invitrogen)
and precipitated with isopropanol on ice. The precipitated RNA was then pelleted at
16000 x g for 30 min and washed with 70 % ethanol in diethylpyrocarbonate (DEPC)treated water (Appendix 3A). The RNA was resuspended in DEPC-treated water and
quantitated with Nanodrop (Thermo Scientific, USA). Approximately 1 µg of the
infectious viral RNA was transfected into a 80 % - 90 % confluent monolayer of BHK
cells in a 75 cm2 tissue culture flask with 60 µl Lipofectamine 2000 (Invitrogen, USA).
The infectious cell culture supernatant was harvested when cytopathic effects were
apparent (usually 4 days post transfection). The virus was processed and propagated as

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36


described in Section 2.2.3 and 2.2.2, respectively.

Mutagenesis of WNV infectious clone was performed by site-directed
mutagenesis using p1.3-transfer vector as template (Section 2.3.7.2). After successful
mutagenesis, the vector was digested with 1 µl of BsiW I and Mlu I (New England
Biolabs, USA) each at 37 °C for 2 hr. The reaction was subjected to DNA electrophoresis
(Section 2.3.4.2) and the 1.3 kb fragment was excised from the gel. Thereafter the DNA
in the gel was eluted and purified (Section 2.3.8). Concurrently, the WNV infectious
clone was digested and subjected to electrophoresis in the same manner and the 11 kb
fragment was excised, eluted and purified from the gel. Approximately 7.5 µl of the 1.3
kb and 0.5 µl of 11 kb excised DNA fragments were assembled together with 1 µl each of
T4 DNA ligase and buffer in an eppendorf tube. The mixture was incubated at 4 °C
overnight. After incubation, 10 µl of the mixture was added into an eppendorf tube
containing 100 µl of frozen competent cells (Invitrogen, USA) that was thawed on ice.
The mixture of cells and DNA was incubated on ice for 20 min and heat-shocked at 42
°C for 45 sec. The cells were sedimented at 4000 x g for 1 min and the supernatant was
discarded. The cells were resuspended in 100 µl of LB broth and plated onto agar plate
containing 100 µg/ml of ampicilin. The plate was incubated at 37 °C overnight and
several colonies were picked for PCR colony screening (2.3.10.2). Positive clones were
sent for sequencing (Section 2.3.9) and propagated in bacteria.

2.3.6

Complementary DNA (cDNA) synthesis
Complementary DNA was synthesized from virus RNA sample isolated as

described in Section 2.2.6. A 10 µl viral RNA sample was mixed with 1 µl of 10 mM C

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37

protein specific primer or random hexamer and 1 µl of deoxynucleotide mix [(dNTP)
Promega, USA]. The mixture was then heated to 65 °C for 5 min and 4 °C for 1 min.
Subsequently, 4 µl of 5 x 1st strand synthesis buffer (Invitrogen, USA), 2 µl of
dithiothreitol (Invitrogen, USA), 1 µl of RNAsin (Promega, USA) and 1 µl of Superscript
III enzyme (Invitrogen, USA) was added to the mixture. The mixture was then heated to
42 °C for 30 min and 72 °C for 15 min. The cDNA was stored at -20 °C.

2.3.7
2.3.7.1

Amplification and site-directed mutagenesis of genes
Polymerase Chain Reaction (PCR)
Polymerase chain reaction was used to amplify specific gene sequences using the

appropriate primers. The reaction consisted of a 5 µl of 10 x reaction buffer (Fermentas,
USA), 1 µl of 10 mM dNTP (Promega, USA), 1 µl each of 10 mM of forward and
reverse gene specific primer, 1 µl of Taq polymerase (Fermentas, USA) and an
appropriate amount of template. The reaction was topped up to 50 µl with ultrapure water
(Milipore, USA). The mixture was amplified in a PCR thermocycler machine (Bio-RAD,
USA). The standard protocol for DNA amplification was as follows: 95 °C for 5 min, 30
cycles of 95 °C for 30 sec, 55 °C for 30 sec and 72 °C for 1 min. Finally, 72 °C for 3 min.
The annealing temperature, the number of cycles and the time for each step can be
adjusted for each primer pair to obtain optimal DNA amplification.

2.3.7.2

Site-directed mutagenesis
Site-directed mutagenesis was performed using the QuickChange Lightning kit

(Stratagene, USA). The primers were design according to the recommendation of the

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38


kit’s protocol. Briefly, primers are between 25 to 45 bases in length with a melting
temperature of ≥78 ºC. Primer sequences can be found in Appendix 6. Templates used
for mutagenesis were the pCMVmyc-C and the p1.3-transfer. Each reaction was
assembled by adding 5 µl of 10 x reaction buffer, 5-50 ng of DNA template, 125 ng each
of the forward and reverse primers, 1 µl of dNTP mix and water to a final volume of 50
µl.

Putative

phosphorylation

sites

were

predicted

with

NetPhos

2.0

( Each putative serine phosphorylation site was
mutated to alanine.

2.3.8

DNA purification from PCR reaction and agarose gel
PCR Amplified DNA was purified with the QiaQuick gel extraction kit (Qiagen,

USA) to remove the buffer and enzymes in the solution. Purification of PCR products
were performed in accordance to the QiaQuick gel extraction kit protocol. The DNA
band of interest in an agarose gel was also extracted using the QiaQuick Extraction kit.
The DNA band of interest in the agarose gel was visualized on a UV transilluminator and
was excised with a clean and sharp scalpel. The gel slice was then placed in an
eppenddorf tube with QG buffer and incubated at 50 ºC until melted. The solution was
then placed into a QiaQuick spin column for the extraction and purification of DNA. The
DNA was eluted with 30 to 50 µl of water.

2.3.9

Sequencing
Each sequencing reaction contained 4 µl of BigDye Terminator v3.1 (Applied

Biosystems. USA) and 1 µl of the appropriate primer as listed in Table 2-3.

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Approximately 500 ng of plasmid DNA were added to the mix and deionised water was

added to make up the volume to 10 µl. The reaction tube was placed in a thermocycler
(Bio-RAD, USA) and the reaction protocol was as follows: 95 °C for 5 min, 30 cycles of
95 °C for 30 sec, 55 °C for 30 sec and 72 °C for 1 min. Finally, 72 °C for 5 min. The
product was then purified via ethanol precipitation.
Ethanol precipitation was performed by adding 0.1 x 3 M sodium acetate
(Promega, USA) and 2.5 x 95 % ethanol. The resulting mixture was transferred to a 1.5
ml eppendorf tube and incubated on ice for 20 min. The precipitated DNA was pelleted
down at 16000 x g for 30 min at 4 °C. The supernatant was removed carefully and 500 µl
of 70 % ethanol was added. The precipitate was pelleted down again with similar
conditions and the ethanol was discarded. The DNA pellet in the tube was sent to either
the departmental sequencing laboratory or Sigma-Aldrich for sequencing. Sequences
were analyzed using Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov/blast/).

2.3.10 Cloning, propagation and purification of plasmids
2.3.10.1

Cloning of capsid protein gene

The viral capsid gene was amplified from the WNV (Sarafend) infectious clone
using primers 1 and 2 as listed in Table 2-3. The amplified DNA fragment was loaded
into an agarose gel for electrophoresis. The DNA fragment of interest was excised from
the gel and purified as described in Section 2.3.8. The DNA fragment was eluted in 43 µl
of deionised water and digested with 1 µl of restriction enzymes Nde1 and Xho1
(Promega. USA) supplemented with 5 µl of 10 x reaction buffer (Promega, USA). The
mixture was incubated at 37 °C for 4 hr and purified with QiaQuick Column described in

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Section 2.3.8. Similarly, 1 µg of the pET28A vector was also digested with Nde1 and
Xho1 (Promega, USA) and purified. Approximately 7.5 µl of the digested PCR product
and 0.5 µl of the linerized pET28 vector were mixed together in the presence of 1 µl of
T4 DNA ligase (Promega, USA) and 1 µl of Ligase buffer (Promega, USA) and
incubated overnight at 4 °C. After incubation, 5 µl of the mixture was added into an
eppendorf tube containing 100 µl of competent cells (Invitrogen, USA) that was thawed
on ice. The resulting mixture of cells and DNA was incubated on ice for 20 min and heatshocked at 42 °C for 45 sec. The cells were pelleted at 4000 x g for 1 min and the
supernatant was discarded. The cells were resuspended in 100 µl of LB broth and plated
onto agar plate containing 50 µg/ml of kanamycin. The plate was incubated at 37 °C
overnight and several colonies were picked for PCR colony screening (Section 2.3.10.2).

2.3.10.2

Colony PCR

Transformed bacteria colonies were picked up with a 10 µl pipette tip and
transferred to a new agar plate containing either ampicilin or kanamycin while part of the
colony was added into a tube containing reagents for a PCR reaction as described in
Section 2.3.7.1. The colony was subjected to PCR amplification as described in Section
2.3.7.1 and the amplified DNA fragment was analyzed with gel electrophoresis (Section
2.3.4.2). Positive colonies were picked from the new agar plates and sequenced (Section
2.3.9).

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2.3.10.3 Propagation of plasmids

Bacteria colonies transformed with the plasmid of interests were incubated in 2 ml
LB broth for 24 hr at 36 °C in an orbital incubator (Sartorius, Germany). Subsequently
1.9 ml of the culture was transferred into a 500 ml conical flask containing 100 ml of LB
broth and incubated at the same conditions. The remaining 0.1 ml of culture was kept at 4
°C as a seed stock.

2.3.10.4

Isolation and purification of plasmid

Plasmid from the bacteria culture of less than 5 ml or 100 ml was isolated and
purified with a QIAprep Miniprep kit (Qiagen, USA) or a PureLink HiPure Midiprep kit
(Invitorgen, USA), respectively. The bacteria cells were sedimented at 4000 x g for 15
min and the plasmid was extracted from the cells by lysing the cells in lysis buffer
supplied by the abovementioned kits. The plasmid was then isolated and purified from
the lysate by passing the lysate through a purification column supplied by the kits. The
identity of the plasmid was confirmed through sequencing as described in Section 2.3.9.

2.3.11

Real-time PCR
Real-time PCR (RT-PCR) is performed using the ABI prism 7000 (Applied

biosystems, USA) machine. Total volume of an RT-PCR reaction is 20 µl, which
comprise of 10 µl SYBR® II green dye (Invitrogen, USA), 0.4 µl of 10 uM appropriate
primer pairs, 1 µl of cDNA (Section 2.3.6) and 8.2 µl of ultrapure water (filtered by MiliQ, USA) all of which were loaded into a well on a 96-well RT-PCR plate (Thermo

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42

Scientific, USA). The reaction protocol was as follows: 40 cycles of 95 °C for 30 sec, 55
°C for 30 sec and 72 °C for 1 min.

2.4

Analysis of protein samples

2.4.1

Sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Electrophoresis and separation of protein samples were done via the Laemmli

discontinuous gel system (Laemmli, 1970). The electrophoresis and gel casting apparatus
was from Bio-Rad, USA. To cast the 12 % separation gel (Appendix 7A), 4.5 ml of the
gel mixture was pipetted into the gel casting apparatus and isopropanol was layered on
top of the mixture. The gel was allowed to polymerize before the propanol was decanted.
A layer of 5 % stacking gel (Appendix 7B) was layered on top of the polymerized
separation gel. Finally combs were dropped into the stacking gel and the gel was allowed
to polymerize. The comb was removed and the wells flushed with upper-tank running
buffer (Appendix 7C) before loading the protein samples.
Protein samples were prepared by adding 6 x loading buffer (Bio-rad, USA) and
boiled at 100 °C for 1 min. Each well is loaded with protein samples of up to 30 µl. Prestained or unstained molecular weight markers (Promega, USA) were added to one well
in each gel. The upper-tank of the gel electrophoresis was filled to the brim with uppertank running buffer and the lower tank was filled with lower-tank running buffer
(Appendix 7D). Electrophoresis was performed using a constant voltage of 100 V for
approximately 1.5 hr. The electrophoresis was performed in a cold room. After
electrophoresis, the proteins were either stained or transferred to a nitrocellulose
membrane.

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2.4.2

Staining of SDS-PAGE gels
Gels were stained in coomassie blue solution (Appendix 7E) over-night and de-

stained sequentially with de-stain buffer I and de-stain buffer II (Appendix 7F and 7G).

2.4.3

Immnoblot (Western blot)
Transfer of proteins onto a membrane was accomplished through a dry or wet

transfer method. The dry method involved the use of the iBlot transfer apparatus
(Invitrogen, USA). The gel was placed onto the BOTTOM blotting stack and then
overlayed with the TOP blotting stack (Invitrogen, USA). The assembly was then placed
in the iBlot machine for 7 min. The membrane, which the proteins were transferred to,
was removed from the assembly after 7 min. The wet transfer involved the use of 0.45
µm nitrocellulose membrane (Bio-rad, USA). The membrane was cut into an appropriate
size and briefly dipped in methanol for 15 sec and then pre-soaked in transfer buffer
(Appendix 8A) together with filter paper and sponges. The gel was placed between the
wet nitrocellulose membrane and filter paper. This was then sandwiched between 2
pieces of sponge. The assembly was then put into a transblot cell and lowered into a
transfer chamber (Bio-RAD, USA) containing pre-chilled transfer buffer in such a way
that the nitrocellulose membrane was adjacent to the positive electrode. The transfer was
carried out at 300 mA for 2 hr in a cold room.

After the transfer of the proteins, the membrane was soaked in Tris-buffered
Saline Tween-20 [TBST (Appendix 8B)] with 5 % skimmed milk or 3 % bovine serum
albumin [BSA (Gibco, USA)] for 1 hr. The blot was then washed 3 times with TBST for
15 min each. Primary antibodies were prepared in TBST with 5 % skimmed milk

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(Anlene, Australia) or 3 % BSA and added to the membrane. The membrane was
incubated overnight with the antibody in a cold room on an orbital shaker (Bellco, USA).
The next day the antibody was removed and washed 3 times with TBST for 15 min each.
The membrane was then incubated for 1 hr at room temperature with the appropriate
secondary antibody (Thermo Scientific, USA) conjugated to either alkaline phosphatase
(AP) or horseradish peroxidase (HRP). In most cases, antibodies conjugated to AP is
diluted 1:1000 while antibodies conjugated to HRP is diluted 1: 10000. After 1 hr, the
membrane was washed 4 times with TBST for 15 min each. Membranes incubated with
AP-conjugated antibodies were developed with the AP substrate (Chemicon, USA) while
membranes probed with HRP-conjugated antibodies were developed with enhanced
chemiluminescence Western blotting substrate (Thermo Scientific, USA). Luminescence
was detected with a CL-XPosure film (Thermo Scientific, USA) and the film was into a
X-ray film developer (SRX-101A, Konica Minolta, Japan) after exposure.

2.4.4

Quantitation of proteins
Protein was quantitated with the Bradford assay (Bradford, 1976). A BSA

standard ranging from 10 µg/ml to 1000 µg/ml was prepared and added to the assay

reagent (Bio-rad, USA). At the same time, the protein sample to be measured was also
added to the assay reagent and incubated for 1 min. Each concentration of the BSA
protein standards and sample were then placed in spectrometer to measure absorbance at
595 nm.

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2.5

Expression and purification of proteins

2.5.1

Expression of histidine-tagged capsid (C) protein in bacteria cells
The WNV C protein was cloned downstream of a histidine tag. The plasmid

encoding the histidine-tagged C (His-C) protein was transformed into the BL2CodonPlus competent E.coli bacteria cells. The cells were grown in LB broth at 37 °C
until the optical density of the culture reached 0.6. Expression of His-C was then induced
with a range of isopropyl β-D-1-thiogalactopyranoside [IPTG (Sigma-Aldrich)]
concentrations and the culture was harvested at an optimal timing. The cells were pelleted
at 4000 x g for 15 min and resuspended in lysis buffer (Appendix 9A). The cells were
lysed on ice by sonication. After sonication, the insoluble inclusion body and the
supernatant were separated by centrifugation at 16000 x g for 15 min. Both the inclusion
body and supernatant were analyzed by SDS-PAGE.

2.5.2

Expression of myc-tagged capsid (C) protein in mammalian cells
Expression of myc-tagged C (myc-C) protein is achieved by transfecting

pCMVmyc-C (Table 2-2) into BHK or 293FT cells cultured in 24-well plates, 6-well
plates or T75 flasks. Transfection reagent used is Lipofectamine 2000 (Invitrogen). The
amount of plasmid DNA, Lipofectamine 2000 and Opti-Mem media (Invitrogen, USA)
for the 24-well plate, 6-well plate and T75 flask is listed in Table 2-4

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Table 2-4 The amount of DNA and Lipofectamine 2000 required to transfect different
amount of cells.
DNA (µg) Lipofectamine 2000 (µl)
Opti-Mem (µl)
Culture Vessel (cm2)
24 well

0.8

2

50

6 well

4

10

250

T75 flask

25

60

1500

The appropriate DNA and Lipofectamine 2000 were diluted separately with an
appropriate amount of Opti-Mem media and incubated at room temperature (25 °C) for 5
min. Both the diluted DNA and Lipofectamine 2000 solutions were mixed and incubated
at room temperature for 30 min. After incubation, the DNA-liposome complexes were
added to the cell monolayers. The cells were then transferred into a 37 °C incubator for 4
hr. The supernatant was removed after 4 hr and fresh 2 % FCS culture medium was
added to the cells.

2.5.3

Purification of histidine-tagged capsid (C) protein
Purification of the histidine-tagged C (His-C) protein was performed by metal ion

affinity membrane chromatography (MIAMC). The bacteria cell lysate from Section
2.5.1 was pre-filtered through 0.45 µm and 0.22 µm filters (Sartorius, Germany)
consecutively. Two Sartobind IDA-75 Metal Chelate Adsorber membrane units
(Sartorius, Germany) were connected in series with a 0.22 µm pre-filter and coupled with
a positive displacement peristaltic pump (Cole-Parmer, USA). The flow rate of the pump

was set at 5 ml/min. The membranes were prepared by pumping 5 ml of equilibrium
buffer (Appendix 9B) through it followed by 5 ml of 0.2 M Nickel [Sulphate (NiSO4)

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Appendix 9C] or [Copper Sulphate (CuSO4) Appendix 9C] solution in equilibrium
buffer. The membranes were washed with 5 ml of loading buffer (Appendix 9D) before
the pre-filtered cell lysate were passed through the membranes. The flow through of the
cell lysate was collected for analysis. Subsequently, the membranes were washed with
wash buffer (Appendix 9E) comprising an appropriate concentration of imadzole. Each of
the washes was collected for analysis. The captured His-C protein was eluted with elution
buffer (Appendix 11E) containing an appropriate concentration of imidazole. To
regenerate the membranes, they were washed with 10 ml of 1 M Sulphuric acid [(H2SO4)
Merck, Germany] followed immediately with 10 ml of equilibrium buffer containing 0.02
% w/v Sodium nitrite [(NaN2) Merck, Germany]. This purification procedure was
repeated for subsequent batches of bacteria cell lysates. The purified protein was
concentrated using Vivaspin 5000 MW centricon (Sartorius, Germany) and quantitated
with Bradford assay (Section 2.4.4).

2.5.4

Purification of myc-tagged capsid (C) protein
Transfected BHK or 293FT cells were harvested and lysed with M-Per

mammalian lysis buffer (Thermo Scientific) supplemented with protease inhibitor
(Roche, Germany) and phosphatase inhibitor (Roche, Germany). The cells were lysed at
room temperature for 30 min. Cell debris were removed from the supernatant by spinning

the cell lysate at 16000 x g for 10 min. Myc-tagged C (myc-C) protein is immunopurified with 20 µl of sepharose beads conjugated to anti-myc antibodies (Sigma-Aldrich,
USA). The beads were washed in 3 x in PBS and incubated with the cell lysate overnight
at 4 °C. The beads were sedimented at 800 x g for 1 min and washed thrice with PBS.

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After washing, PBS was removed and the beads were resuspended in Tris-HCl buffer, pH
7.4 supplemented with protease and phosphatase inhibitor. The immuno-purified protein
was stored at -20 °C

2.6

Protein-RNA interaction assays

2.6.1

Preparation of RNA

2.6.1.1

RNA synthesis
RNA was synthesised with T7 RibomaxTM kit (Promega, USA). The WNV

infectious clone was used as template for the synthesis of full length viral RNA. To
synthesize specific regions of the viral RNA, DNA sequences encoding those regions
were amplified by PCR (Section 2.3.7.1) from the WNV infectious clone with the
appropriate primers as listed in Table 2-3. These DNA sequences were used at templates

for RNA synthesis. RNA synthesis reaction was assembled and purified according to the
manufacturer’s protocol. Briefly, the synthesis reaction consisted of 20 µl of 5 x T7
transcription buffer, 30 µl of 25 mM rNTPs, 5 - 10 µg of linearized DNA template in 40
µl of water and 10 µl of T7 enzyme mix.

Before the RNA was extracted with

phenol:chloroform:isoamyl alcohol [(25:24:1) Invitrogen, USA], the DNA template with
the RQ1 DNase. RNA was quantitated with NanoDrop (Thermo Scientific, USA).
Integrity and size of synthesized RNA is visualized by RNA gel electrophoresis.

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2.6.1.2

Biotinylation of RNA
An appropriate amount of RNA was labelled with LabelIT® Biotin labelling kit

(Mirus). Reaction assembly and purification of labelled RNA was performed according to
manufacturer’s protocol. Briefly, the labeling reaction consisted of 5 µl of 10 x labeling
buffer, 5 µl of 1 mg/ml of RNA, 5 àl of LabelITđ reagent in 35 µl of water. The labelled
RNA was purified by ethanol precipitation as described in the second paragraph of
Section 2.3.9. Labelling of the RNA was confirmed by dot blotting the RNA on to a
polyvinylidene fluoride (PVDF) membrane and detected with streptavidin conjugated to
alkaline phosphatase to ensure that the RNA was labelled.

2.6.2

Protein-RNA blot (Northwestern blot)
In a protein-RNA blot, proteins were transferred onto a nitrocellulose membrane

(Section 2.4.3) and probed with biotinylated-RNA (Section 2.6.1.2). The membrane was
blocked with probe buffer (Appendix 10A) with 250 µg/ml of tRNA (Invitrogen) for 1 hr
at room temperature. The probe buffer was removed and incubated overnight with an
appropriate amount (See Chapter 3) of RNA in probe buffer at 4 °C. After incubation, the
buffer was removed and washed thrice with probe buffer. Bound RNA on the membrane
was detected with the Chemiluminescent Nucleic Acid Detection Module (Thermo
Scientific, USA) or luminescence was detected and visualized as described in Section
2.4.3.
.

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