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

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

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 Cell Passage level Origin

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 75cm2flask 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

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 %

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

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 Shield, 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 %)

(Axis-of 1 ml each (Axis-of the OptiPrep medium on top (Axis-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

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)

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 Gene or regions cloned Vector backbone Purpose P28-His-C

Amino-terminal Histidine-tagged mature capsid protein

Pet28 A (Novagen)

Expression of C protein in bacteria cells pCMVmyc-C

Amino-terminal tagged mature capsid

cMyc-protein

pCMV-myc (Clontech)

Expression of C protein in bacteria cells p1.3-transfer The first 1.3kb of the WNV genome PBR322 (Promega)

A carrier vector for the mutagenesis of the C protein pWTIC WNV infectious clone PBR322 (Promega) 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

Table 2-3 List of the names of primers used and its purpose

1 His-C F

3 Sense RNA 3 UTR F

4 Sense RNA 3 UTR R Synthesize sense 3’ UTR viral RNA

5 Sense RNA 5 UTR F

6 Sense RNA 5 UTR R Synthesize sense 5’ UTR viral RNA

7 Sense RNA 5 UTR+C F

8 Sense RNA 5 UTR+C R Synthesize sense 5’ UTR+C viral RNA

9 A-sense RNA 3 UTR F

10 A-sense RNA 3 UTR R Synthesize anti-sense 3’ UTR viral RNA

11 A-sense RNA 5 UTR F

12 A-sense RNA 5 UTR R Synthesize anti-sense 5’ UTR viral RNA

13 A-sense RNA 5 UTR+C F

14 A-sense RNA 5 UTR+C R Synthesize anti-sense 5’ UTR+C viral RNA

42 A-sense RNA 12R viral RNA

54 A-sense RNA RT 2 Rev

55 A-sense RNA RT 12 Rev

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

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)

2.3.4 Agarose electrophoresis

2.3.4.1 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

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 withQiaPrep 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 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

(DEPC)-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

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 Amplification and site-directed mutagenesis of genes

2.3.7.1 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

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 (http://www.cbs.dtu.dk/services/NetPhos/) 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

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

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 shocked 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)

heat-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)

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 Mili-

Q, USA) all of which were loaded into a well on a 96-well RT-PCR plate (Thermo

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 Pre-stained or unstained molecular weight markers (Promega, USA) were added to one well

in each gel The tank of the gel electrophoresis was filled to the brim with tank 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

upper-2.4.2 Staining of SDS-PAGE gels

Gels were stained in coomassie blue solution (Appendix 7E) over-night and stained sequentially with de-stain buffer I and de-stain buffer II (Appendix 7F and 7G)

de-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

(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

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 BL2-

CodonPlus 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

Table 2-4 The amount of DNA and Lipofectamine 2000 required to transfect different amount of cells

Culture Vessel (cm2) DNA (µg) Lipofectamine 2000 (µl) Opti-Mem (µl)

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)

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 immuno-purified 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

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

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 µl of LabelIT® 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