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 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 -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 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. | Results 77 Table 4-1 List of RNA fragments synthesized for C protein pull-down assay and Northwestern blot Fragment No. Region of the WNV genome | Results sense 1-1017 sense 960-1974 sense 1920-2934 sense 2880-3894 sense 3830-4839 sense 4790-5804 sense 5758-6772 sense 6728-7742 sense 7698-8702 10 sense 8655-9669 11 sense 9613-10617 12 sense 10053-11057 13 anti-sense 1-1017 14 anti-sense 10053-11057 78 DNA A kb M M 10 11 12 13 14 10 11 12 13 14 RNA B kb 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. | Results 79 Fragment 10 11 12 13 14 RNA His-C 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 antisense 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). | Results 80 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 overrepresented 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 and 12. The difference in the amount of positive- and negative-stranded RNA being pulled down [region 1-1017 (Fragment 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. | Results 81 Amount of RNA pulled down (ng) 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 was pulled down the most often. Fragment 13 and 14 are anti-sense fragments of Fragments and 12. This suggests that C protein binding sites on the WNV RNA are found in the regions of - 2934 and 9613 - 11057 of the WNV RNA genome. | Results 82 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 is detailed in Table 4-2. Table 4-2. List of C protein peptides synthesized and their charge at pH 7.0. Fragment Amino acid Charge at pH 7.0 Amino acid sequence No. position 1-23 6.9 mskkpggpgknravnmlkrgmpr 18-45 5.9 krgmprglsliglkramlslidgkgpir 41-64 3.9 kgpirfvlallaffrftaiaptra 61-84 4.1 aptravldrwrgvnkqtamkhllsf 79-105 8.1 khllsfkkelgtltsainrrstkqkkr 101-123 3.9 kqkkrggtagftillgliacaga In the peptide dot blot assay, each peptide is blotted onto nitrocellulose paper and probed with 3’ UTR of the viral genome since this was used in the previously reported experiment (Khromykh & Westaway, 1996). The peptide dot blot assay showed that peptides that interacted with the biotinylated-WNV RNA strongly were found to be on the amino- and carboxyl- terminal of the C protein (Fig. 4-4). The peptides relative | Results 83 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). His-C 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. | Results 84 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 rhodaminelabelled 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. | Results 85 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. | Results 86 B DAPI Merged i myc-C ii RNA iii iv Volume rendering v | Results 88 BHK cell lysate Transfected cell lysate IP myc-C 72 kDa 55 kDa 3’ UTR RNA probe 26 kDa 17 kDa 10 kDa 72 kDa 55 kDa Anti-myc antibody 26 kDa 17 kDa 10 kDa 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. Immunopurified (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 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. | Results 89 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. | Results 90 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. | Results 91 | Results 0.25 µM actinomycin D 0.5 µM actinomycin D 1.0 µM actinomycin D 1.5 µM actinomycin D 2.0 µM actinomycin D 2.5 µM actinomycin D 92 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 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 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 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 | Results 93 RNA in the cytoplasm [Fig. 4-9B (white arrows)]. Hence, in this assay some colocalisations between the C protein and nascent viral RNA were observed only at the later time points of an infection. | Results 94 Figure 4-8. Labelling of viral RNA in infected cells. Cells were infected with WNV and RNA was labelled with Click-iT RNA labeling kit. The cells were fixed and processed for microscopy at hr (ii), 12 hr (iii), 18 hr (iv) and 24 hr (v) post infection (p.i.). Mockinfected cells were fixed and processed after 24 hr. Distinct punctuate green fluorescent dots (white arrowheads) are seen in the cytoplasm of infected cells (ii-v). In addition diffused cytoplasmic stainings (red arrows) are observed in infected cells at 18 hr (iv) and 24 hr (v) p.i. No distinct green fluorescent dots or diffused cytoplasmic staining is observed with mock-infected cells (i). Scale bar is shown on the bottom right of the images. | Results 95 (i) Mock Mockinfected Infected (iii) 12 hr p.i. (ii) hr p.i. (iv) 18 hr p.i. (v) 24 hr p.i. | Results 96 Figure 4-9. Localisation of C protein and viral RNA in infected cells over 24 hr. (A) BHK cells were either mock-infected (i) or infected with WNV (ii-v) with an M.O.I of and fixed at hr (ii), 12 hr (iii), 18 hr (iv) and 24 hr (v) p.i. Host RNA synthesis were partially inhibited with actinomycin D and viral RNA was labelled with Click-iT RNA labelling kit (green). Viral C protein was probed with anti-C antibody and the anti-C antibody was detected with anti-mouse antibody conjugated to Alexafluor 594. At early timings (6-12 hr p.i.) the C proteins are predominantly found in the nuclei (ii-iii) while at later timings (18-24 hr p.i.) C proteins are found predominantly in the cytoplasm. The arrows indicate co-localisation of viral RNA with C protein. (B) Panels (i) and (iii) show the 3-dimenisonal rendering for panels (iv) and (v) in (A), respectively. Panels (ii) and (iv) are blow ups of an area in (i) and (iii), respectively. The white arrows indicate the colocalisation (yellow) of C protein (red) and and viral RNA (green) in the cytoplasm. | Results 97 A Nucleus RNA Merged Capsid i Mockinfected 10 µm ii hr p.i. iii 12 hr p.i. iv 18 hr p.i. v 24 hr p.i. | Results 98 B i i i ii i i 18 hr p.i. ii iii iii 24 hr p.i. iviv | Results 99 4.5 Phospho-peptides and RNA interaction. Although some co-localisations of C proteins with viral RNAs at 18 hr and 24 hr p.i. were observed (Fig. 4-9), the difference between His-C and myc-C proteins’ ability to interact with viral RNA was still unresolved. Not only were both proteins tagged differently, they were also expressed in different types of cells. The tags on both these recombinant proteins were small and therefore unlikely to affect any significant biochemical alterations to the protein. However, myc-C protein expressed in eukaryotic cells like BHK cells could undergo post-translational modifications. Conversely, His-C protein, expressed in prokaryotic cells would not have undergone post-translational modification. A possible post-translational modification on the myc-C protein that might have affected RNA binding would be phosphorylation. Since phosphate groups are negatively charged they could neutralize or attenuate the intrinsic positive charge of C protein. Hence the difference in RNA binding property between His-C (Fig 4-2) and myc-C (Fig. 4-6) proteins could probably due to phosphorylation. Bioinformatics analysis revealed putative phosphorylation sites on the WNV C protein (Sarafend), of which are serine residues (Table. 4-3). Bioinformatics analysis of the C protein of other flaviviruses including the Wengler and New York strains of WNV also revealed multiple putative phosphorylation sites (Table 4-3). | Results 100 Table 4-3. Putative phosphorylation sites on flavivirus C protein Amino Accession no. No. of Putative Virus acid/Sequence (GenBank) phosphorylation sites position West Nile S26, S36, S83, S99, AY688948 (Sarafend) T100, West Nile D00246/ (Kunjin/New S26, S36, S83, S995 FJ151394 100 York) Japanese AAQ73507 T2, S36, S83 ABB69689 S22, S24, S97, S102 Encephalitis Yellow Fever S24, S34, T58, T71, Dengue AAC40839 S101 Tick-borne AAA86870 S19, S97 Encephalitis | Results 101 Coincidentally, the putative phosphorylation sites reside in regions of the C protein, which exhibited the strongest interaction with viral RNA (Fig. 4-4). Therefore, the question is whether phosphorylation at these sites would attenuate the binding affinity of the C protein to viral RNA. Consequently, peptides representing amino acids 18 to 45 and 79 to 105 of the C protein were synthesized and phosphorylated at the appropriate residues (Table 4-3). For the peptide representing amino acid positions 18-45 of the C protein, serine 26 and 36 were phosphorylated. In addition, for the peptide representing amino acid positions 79-105, serine 83, 99 and threonine 100 were phosphorylated (Table 4-3). These peptides were then used in a dot blot assay to determine if phosphorylation affected RNA binding. Results show that viral RNA did not bind to the phospho-peptides blotted on the membrane but only bound to the non-phosphorylated form of the peptides (Fig. 4-10). Hence, phosphorylation could be a factor in attenuating the interaction between C protein and RNA by mitigating the positive charge on the C protein. Therefore it was likely that the difference between His-C protein and myc-C protein with regards to viral RNA binding was due to phosphorylation. | Results 102 Amino acid 18-45 of C protein Amino acid 79-105 of C protein Unphosphorylated Phosphorylated Unphosphorylated Phosphorylated ng of peptide ng of peptide Figure 4-10. Phospho-peptide dot blot with RNA. Peptides corresponding to amino acid positions 18-45 (Lanes and 2) and 79-105 (Lanes and 4) of the C protein were synthesized either without (Lanes and 3) or with (Lanes and 4) phosphorylation at serine 26, 36, 83, 99 and threonine 100. Approximately ng or ng of these peptides were dot-blotted onto a nitrocellulose membrane and dried. The membrane was then blocked with yeast tRNA and probed with 3’UTR of the viral RNA. The unphosphorylated peptides were able to bind more viral RNA than the phosphorylated form of the same peptide in a dose-dependent manner. | Results 103 [...]... Results 100 Table 4 -3 Putative phosphorylation sites on flavivirus C protein Amino Accession no No of Putative Virus acid/Sequence (GenBank) phosphorylation sites position West Nile S26, S36, S 83, S99, AY688948 5 (Sarafend) T100, West Nile D00246/ (Kunjin/New S26, S36, S 83, S995 FJ15 139 4 100 York) Japanese AAQ 735 07 3 T2, S36, S 83 ABB69689 4 S22, S24, S97, S102 Encephalitis Yellow Fever S24, S34, T58, T71,... 3 and 4) of the C protein were synthesized either without (Lanes 1 and 3) or with (Lanes 2 and 4) phosphorylation at serine 26, 36 , 83, 99 and threonine 100 Approximately 5 ng or 2 ng of these peptides were dot-blotted onto a nitrocellulose membrane and dried The membrane was then blocked with yeast tRNA and probed with 3 UTR of the viral RNA The unphosphorylated peptides were able to bind more viral. .. 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... His-C protein and myc-C protein with regards to viral RNA binding was due to phosphorylation | Results 102 Amino acid 18-45 of C protein Amino acid 79-105 of C protein Unphosphorylated Phosphorylated Unphosphorylated Phosphorylated 5 ng of peptide 2 ng of peptide 1 2 3 4 Figure 4-10 Phospho-peptide dot blot with RNA Peptides corresponding to amino acid positions 18-45 (Lanes 1 and 2) and 79-105 (Lanes 3. .. 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 | Results 93 RNA in the cytoplasm [Fig 4-9B (white arrows)]... Localisation of C protein and viral RNA in infected cells over 24 hr (A) BHK cells were either mock-infected (i) or infected with WNV (ii-v) with an M.O.I of 1 and fixed at 6 hr (ii), 12 hr (iii), 18 hr (iv) and 24 hr (v) p.i Host RNA synthesis were partially inhibited with actinomycin D and viral RNA was labelled with Click-iT RNA labelling kit (green) Viral C protein was probed with anti-C antibody and the... colocalisation (yellow) of C protein (red) and and viral RNA (green) in the cytoplasm | Results 97 A Nucleus RNA Merged Capsid i Mockinfected 10 µm ii 6 hr p.i iii 12 hr p.i iv 18 hr p.i v 24 hr p.i | Results 98 B i i i ii i i 18 hr p.i ii iii iii 24 hr p.i iv iv | Results 99 4.5 Phospho-peptides and RNA interaction Although some co-localisations of C proteins with viral RNAs at 18 hr and 24 hr p.i were... (Fig 4-2) and myc-C (Fig 4-6) proteins could probably due to phosphorylation Bioinformatics analysis revealed 5 putative phosphorylation sites on the WNV C protein (Sarafend), 4 of which are serine residues (Table 4 -3) Bioinformatics analysis of the C protein of other flaviviruses including the Wengler and New York strains of WNV also revealed multiple putative phosphorylation sites (Table 4 -3) | Results... the C protein and nascent viral RNA were observed only at the later time points of an infection | Results 94 Figure 4-8 Labelling of viral RNA in infected cells Cells were infected with WNV and RNA was labelled with Click-iT RNA labeling kit The cells were fixed and processed for microscopy at 6 hr (ii), 12 hr (iii), 18 hr (iv) and 24 hr (v) post infection (p.i.) Mockinfected cells were fixed and processed... 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 . 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. 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 - 2 934 and 96 13 - 11057 of. the WNV RNA genome. | Results 83 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