Modulation of west nile virus capsid protein and viral RNA interaction through phosphorylation 3

4.0 CHARACTERIZATION OF CAPSID C PROTEIN AND VIRAL RNA INTERACTION 4.1 Introduction Due of the positively charged nature, C proteins of the flaviviruses interacts with viral nucleic ac

4.0 CHARACTERIZATION OF CAPSID (C) PROTEIN AND VIRAL RNA

INTERACTION

4.1 Introduction

Due of the positively charged nature, C proteins of the flaviviruses interacts with viral nucleic acids The RNA binding ability of Kunjin virus C protein was confirmed (Khromykh & Westaway, 1996) but the mechanism of C protein and RNA interaction is still poorly defined Hence, this study sought to define C protein binding regions on viral RNA, viral RNA binding regions on C protein and its interaction in cellular environment

4.2 Defining the capsid-binding region on the viral RNA

The capsid-binding region has not been specifically defined and this study sought

to elucidate if such a region exists, if at all Overlapping viral RNA fragments of approximately 1 kb in length, spanning the entire viral genome, was synthesized from

amplified DNA fragments of the WNV infectious clone (Li et al., 2005) The synthesized

RNA was then biotinylated Each of these RNA fragments was used as a probe to detect for capsid-RNA interaction Table 4 -1 shows the list of RNA fragments synthesized The

integrity and size of the amplified DNA (Fig 4-1A) and in vitro synthesized RNA were

checked with gel electrophoresis (Fig 4-1B)

In the Northwestern blot assay, it was found that that all the RNA fragments, including Fragment 13 and 14, which represented the negative strand of Fragment 1 and

12, respectively interacted with the purified His-C protein (Fig 4-2) In addition, there were no apparent differences in the intensity of each band

Table 4-1 List of RNA fragments synthesized for C protein pull-down assay and Northwestern blot

Fragment No Region of the WNV genome

Figure 4-1 Gel electrophoresis of amplified DNA fragments and synthesized viral RNA

(A) Fragments 1-14 (Table 1) were amplified by PCR using West Nile virus infectious

clone as the template Each of these templates is tagged with a T7 promoter for in vitro

RNA synthesis The size and integrity of the PCR product is analysed with DNA gel electrophoresis (B) Purified PCR products from (A) were used as templates to synthesis RNA The integrity and size of the RNA was analysed with denaturing RNA gel electrophoresis The numbers correspond to the fragment number in Table 1 M is the molecular marker for the DNA and RNA gel electrophoresis

Figure 4-2 Northwestern blot with the overlapping WNV RNA fragments Equal

amounts of His-C protein was loaded into each lane and subjected to SDS-PAGE and transferred onto a nitrocellulose membrane The membrane was subsequently probed with each RNA fragment individually (Upper panel) The numbers above corresponded

to the RNA fragment described in Table 4-1 All the RNA fragments including the sense Fragments (13, 14) are shown to interact with His-C protein with no observable differences in band intensities To ensure equal loading, the same volume of His-C protein was loaded into each well and subjecting it to Western blot analysis with anti-His antibody (Lower panel)

anti-RNA

His-C

Fragment 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Since it was not obvious which fragment had a greater affinity for the C protein, each interaction was quantified by allowing the His-C protein and all the RNA fragments

to form complexes in solution and pulling down the RNA with anti-histidine antibody If

C protein had preference for a certain region of the WNV RNA genome, this region would be over-represented when the protein was pulled down The RNA fragments that were pulled down were then quantified with RT-PCR It was found that the over-represented fragments were Fragment 1, 2, 3, 11, 12, 13, 14 (Fig 4-3) although all fragments were detected by RT-PCR It was surprising to find that the C protein had also pulled down a significant amount of the negative-stranded WNV RNA of Fragment 1 and

12 The difference in the amount of positive- and negative-stranded RNA being pulled down [region 1-1017 (Fragment 1 and 13) on the WNV RNA genome] was not significant However it was clear that Fragment 2, representing the region 960-1974 on the WNV RNA genome was over-represented by more than two times relative to other fragments

RNA Fragment Number

Figure 4-3 Capsid protein pull-down of WNV RNA fragments Overlapping RNA

fragments spanning the entire WNV genome including the anti-sense RNA were synthesized (Table 4-1) and mixed with purified His-C protein or histidine-tagged domain III (negative control) of the Dengue E protein (EDIII) Subsequently, anti-His antibodies-conjugated to sepharose beads were used to pull down the proteins As a control, RNA fragments alone were mixed with the antibody-conjugated sepharose beads

to ensure that the RNA was not pulled down unspecifically The beads were then treated with proteinase K to remove the proteins in the solution and the RNA was extracted from the mixture with phenol-chloroform The extracted portion was then processed for detection with RT-PCR for each RNA fragment with specific primers Fragments 1, 2, 3,

11, 12, 13 and 14 are overrepresented in the pull down although RT-PCR was able to detect trace amounts of other RNA fragments Amongst all the fragments, Fragment 2 was pulled down the most often Fragment 13 and 14 are anti-sense fragments of Fragments 1 and 12 This suggests that C protein binding sites on the WNV RNA are found in the regions of 1 - 2934 and 9613 - 11057 of the WNV RNA genome

4.3 Defining the West Nile virus (WNV) RNA-binding region on the capsid (C)

protein

Although the Kunjin virus RNA-binding region on the C protein had previously been identified to be at the amino- and carboxyl-terminal (Khromykh & Westaway, 1996) greater definition of this region can be achieved by using overlapping peptides of approximately 23-26 amino acids in length The list of each peptide and charge at pH 7.0

Charge at pH 7.0 Amino acid sequence

intensity of each blot corresponded to the charge of each peptide As expected the stronger the positive charge, the greater the intensity of the blot As a control the His-C protein was also blotted on to the membrane and it was functionally capable of binding to viral RNA (Fig 4-4) This assay showed that the strongest RNA binding regions of the C protein were between amino acids 1-23 and 79-105 Thus this gives a better definition of where the RNA binding sites are located (Khromykh & Westaway, 1996)

Figure 4-4 Dot blotting peptides of the C protein to detect interaction with viral RNA

The peptides listed 1-6 in Table 4-2 as well as purified His-C protein were dot blotted onto a nitrocellulose membrane The membrane was dried and blocked with yeast tRNA Subsequently, the membrane was incubated with 3’ UTR of the WNV RNA labelled with biotin conjugated to alkaline phosphatase The blot was washed and developed with an alkaline phosphatase substrate The amount of RNA bound to each peptide corresponds

to the charge of each peptide at pH 7.0 (Table 4-2) and it shows that RNA binding regions are found on the amino- and carboxyl-terminal

1 2 3 4 5 6 His-C

4.4 Capsid (C)-RNA interaction in vivo

It has been reported that WNV C protein is imported into the nuclei when a cell is

infected (Bhuvanakantham et al., 2009; Wang et al., 2002) Given that C protein is highly positively charged and that C protein-RNA interaction has been shown to occur in vitro

(Fig 4-2), investigation was initiated on how such an interaction would occur in a cellular environment and if the C protein could translocate viral RNA into the nucleus Plasmid encoding myc-tagged C (myc-C) protein was co-transfected with rhodamine-labelled viral RNA and visualized with either fluorescence or confocal microscopy It was shown that, though myc-C protein was localized in the nuclei, the RNA remained in the cytoplasm (Fig 4-5A) Confocal microscopy further showed no evidence of viral RNA being translocated into nucleus by myc-C protein (Fig 4-5B) Co-localisation of myc-C protein and synthesized WNV RNA in the nucleus or cytoplasm was not detected (Fig 4-5A) This was unanticipated since the highly positively charged nature of the C protein was expected to interact with the negatively charged RNA

To eliminate the possibility that the absence of interaction observed in the cells was not due some inhibitory factors in the cellular environment, it was decided that a Northwestern blot assay with myc-C protein be performed The RNA was not able to bind to myc-C protein immobilized on the nitrocellulose membrane (Fig 4-6) This demonstrated that the absence of interaction seen in the cellular environment was not due

to due to inhibitory factors in the cells, rather the nature of the myc-C protein It is possible that myc-C protein, being expressed in BHK cells, could be phosphorylated In contrast, His-C protein was translated in bacteria cells and proteins expressed by prokaryotic cells are not known to be phosphorylated

Figure 4-5 Cellular localisation of myc-C protein and synthesized viral RNA in BHK

cells (A) Visualization of myc-C protein and transfected viral RNA in BHK cells with fluorescence microscopy Plasmid encoding myc-C protein and synthesized full length viral RNA were either transfected individually (i-iv and v-viii) or co-transfected (ix-xii) into BHK cells and processed for immunofluorescence microscopy 24 hr post transfection No co-localisation of myc-C protein (green) and RNA (red) are observed Most of myc-C protein is localised in the nucleus (blue) while the RNA is observed in the cytoplasm (B) Visualization of myc-C protein and transfected viral RNA in BHK cells with confocal microscopy In order to be certain that co-localisation of myc-C protein and RNA did not occur and that the RNA was not translocated into the nucleus, BHK cells co-transfected with plasmid encoding myc-C protein and full length viral RNA were viewed using confocal microscopy Individual channels showing the nucleus (i), myc-C protein (ii), RNA (iii) as well as the combined image (iv) are shown In addition, volume rendering of the combined image is shown (v) Confocal microscopy shows that the RNA

is not translocated into the nucleus nor is there any observable co-localisation of myc-C protein and viral RNA Scale bars are shown on the bottom right of the images

Figure 4-6 Northwestern blot analysis of myc-C protein Plasmid encoding myc-C

protein was transfected into BHK cells and harvested 24 hour post-transfection purified (IP) myc-C protein (Lane 1), transfected BHK cell lysate (Lane 2) and untransfected BHK cell lysate (Lane 3) were subjected to SDS-PAGE and the proteins were transferred onto a nitrocellulose membrane The membrane was probed with 3’UTR RNA of the WNV genome and developed The membrane was then stripped and probed with anti-myc antibody Molecular weight of myc-C protein is slightly less than 17 kDa and the molecular weight markers are shown on the right Although the RNA probe is able to interact with other cellular proteins in the cell lysate (Upper panel, Lanes 2 and 3), none of these proteins are less than 17 kDa in size, hence the RNA is unable to interact with myc-C proteins on the membrane

1 2 3

Anti-myc antibody

3’ UTR RNA probe

Recognising that the conditions of the previous study were artificially created since RNA was transfected together with a myc-C protein expressing plasmid, another

assay was developed to study C protein and WNV RNA interaction in vivo In this assay,

nascent viral RNA was fluorescently labelled in infected cells instead and at the same viral C protein was probed with anti-C antibodies during the course of an infection In order to label nascent viral RNA, Click-IT RNA labelling kit was used The kit contained

an RNA analog, which was added to the growth media in the cell culture Hence, the RNA analog will be incorporated into newly synthesized RNA However, this labeling method also labels cellular mRNAs that are transcribed in the nucleus In order to reduce the labelling of cellular mRNA, actinomycin D was added to the cells to arrest cellular transcription in the nucleus However, high concentrations of actinomycin D is cytotoxic

to the cells, hence an optimal concentration was needed to reduce host mRNA synthesis without affecting cellular viability This was achieved by adding a range of actinomycin

D concentrations to BHK cells while observing cellular morphology and RNA labelling The optimal concentration of actinomycin D, which reduced cellular RNA labelling without visible effects of cytotoxicity is 1.0 µM (Fig 4-7) It can be seen that fluorescence was detected only in the nucleus and this was consistent with cellular transcription in the nucleus and that the addition of actinomycin D reduced the number of cells exhibiting fluorescence

Figure 4-7 Optimal concentration of actinomycin D needed to disrupt cellular mRNA

synthesis without affecting BHK cell morphology In order to find the optimal concentration of actinomycin D needed to disrupt cellular mRNA synthesis in uninfected cells, a range of actinomycin D concentrations (0.5 - 2.5 µM) were added into the growth media and nascent RNA was labelled and stained (green) with Click-iT RNA labeling kit (green) The nucleus of the cell was stained with DAPI The slides were viewed with fluorescence microscopy and the fluorescent image was overlaid onto the DIC image of the slide RNA labelling of nascent mRNA decreases with increasing concentration of actinomycin D At 1.0 µM of actinomycin D BHK cells begin to appear elongated

0.25 µM actinomycin D 0.5 µM actinomycin D

1.5 µM actinomycin D 1.0 µM actinomycin D

2.5 µM actinomycin D 2.0 µM actinomycin D

With the optimal actinomycin D concentration determined, the efficiency of labeling viral RNA in infected cells was tested Cells were infected with WNV, fixed and processed for fluorescent microscopy at 6 hr, 12 hr, 18 hr and 24 hr post-infection (p.i.)

As a control, mock-infected cells were also labelled for nascent RNA and fixed at 24 hr p.i Distinct and punctate green fluorescent dots in the cytoplasm were observed in all infected cells but the number and intensity of the fluorescent dots increased over time [Fig 4-8 (white arrows)] At 18 hr and 24 hr post infection, diffused cytoplasmic staining was observed [Fig 4-8 (red arrows)], whereas no distinct punctate dots in cytoplasm were observed in the mock-infected cells Hence, it was concluded that the distinct punctuate green fluorescent dots found in cytoplasm of the cells were sites of viral RNA synthesis and at later time points (18 hr and 24 hr) viral RNA had begun to diffuse into the cytoplasm Because the RNA labelling kit also labels host RNA, fluorescently labelled RNA in the nuclei were excluded as theses were sites of host mRNA synthesis

To observe C protein and viral RNA localisation in infected cells, the cells were infected with WNV and fixed at 6 hr, 12 hr, 18 hr and 24 hr p.i Capsid protein in infected cells was detected with anti-C protein antibodies Anti-C antibodies were subsequently stained with anti-mouse antibody conjugated to Alexafluor 594 It was observed that while the C protein was stained mostly in the nuclei at 6 hr p.i [Fig 4-9A (ii)], it was found in the cytoplasm and nucleus at later time points [Fig 4-9A (iii-v)] Although in some cells at 12 hr post-transfection, the RNA and C protein were localized

in the cytoplasm, co-localisation of cytoplasmic RNA and C protein was only apparent

in cells at 18 hr and 24 hr post-infection [Fig 4-9A (iv-v)] A 3-dimensional rendering of Fig 4-9A (iv-v) showed that there were distinct co-localisation of the C protein and viral