Modulation of west nile virus capsid protein and viral RNA interaction through phosphorylation 4

5.0 PHOSPHORYLATION OF WEST NILE VIRUS (WNV) CAPSID (C) PROTEIN AND RNA INTERACTION 5.1 Introduction Having established that phosphorylation on C protein peptides could attenuate its RNA binding ability the questions to address are if WNV C protein is indeed phosphorylated and how does modification of the phosphorylation status affect C protein functions. Known characteristics of C protein like nuclear localisation and oligomerization could be affected by phosphorylation. 5.2 Validation of anti-phosphoserine antibodies Prior to using the anti-phosphoserine antibodies for the detection of phosphorylation, it had to be validated since such reagents are known to be unspecific. Several anti-phosphoserine antibodies were tested but only one (Clone 4A4, Milipore) produced satisfactory results. (The other antibodies either resulted in overexposed blots or no signal was observed.) The antibody was first tested by Western blot on the positive control Calyculin A/Okadaic-treated human A431 carcinoma cell lysate supplied by Milipore. The cell lysate was also treated with λ-phosphatase as a negative control (Fig 51). The antibody was able to differentiate between cell lysate that was either treated or untreated with λ-phosphatase and the results were reproducible. As expected, His-C protein that was expressed in bacteria was unphosphorylated. |Results 104 Figure 5-1. Validation of anti-phosphoserine antibodies. Human A431 carcinoma cell lysate (positive control supplied by Upstate) were either treated (Lanes and 7) or untreated (Lanes and 6) with λ-phosphatase and subjected to both Western blot and SDS-PAGE analysis. At the same time bacteria expressed histidine-tagged West Nile Virus (WNV) capsid (C) protein is also subjected to Western blot and SDS-PAGE analyses (Lanes and 8). Markers are in Lanes and on the Western blot and SDSPAGE, respectively. λ-phosphatase is indicated by a black arrowhead (Lane 7). The antiphosphoserine antibody is able to detect phosphorylated proteins in the A431 carcinoma cell lysate (Lane 1) but not in the λ-phosphatase-treated one (Lane 2). The antibody is not able to detect any serine phosphorylation on the histidine-tagged C (His-C) protein (Lane 3). |Results 105 5.3 Phosphorylation of the West Nile virus (WNV) capsid (C) protein 5.3.1 West Nile virus (WNV) capsid (C) protein is a phosphoprotein In order to determine if WNV C protein is a phospho-protein, C protein was immuno-purified from infected BHK cells using anti-C antibody. The immuno-purified protein was blotted onto a nitrocellulose membrane and probed with anti-phosphoserine and anti-C antibody (Fig. 5-2). Immunoblot (Western blot) analysis showed that WNV C protein is indeed a phospho-protein since anti-phosphorine antibodies detected immunopurified WNV C protein. Bioinformatics analysis revealed putative phosphorylation sites; mutations to myc-C protein were performed to target serine 26, 36, 83, 99 and threoine 100 to abolish phosphorylation. These residues were mutated to alanine. Because serine 26 and 36 were close to each other a myc-C mutant carrying double mutations was constructed (Fig. 53A). Similarly, since serine 83, 99 and theronine 100 were close together a triple mutant was constructed (Fig. 5-3A). In addition a mutant where all putative phosphorylation sites were mutated to alanine was also constructed (Fig 5-3A). In order to resolve whether myc-C protein is phosphorylated, [and hence explain its inability to bind RNA (Fig. 4-6)] plasmids encoding wild type or mutant myc-C proteins were transfected into 293FT cells and BHK cells and myc-C proteins were immuno-purified with anti-myc antibody. The 293FT cell line was used because it is a human cell line, therefore phosphorylation of C protein in this cell line would be more relevant. Nonetheless, myc-C protein should be phosphorylated in both eukaryotic cell lines. The immuno-purified wild type myc-C protein was subjected to Western blot analysis and was found to be phosphorylated in both cell lines (Fig. 5-3B). In contrast, |Results 106 the mutant myc-C proteins were found to be hypophosphorylated although phosphorylation was not completely abolished, as was the case when myc-C protein was treated with λ-phosphatase (Fig. 5-3B). Mutagenesis of putative phosphorylation sites could have perturbed the native confirmation of C protein, homology modeling of the S26/36/83/99/T100A mutant was performed to ensure that the mutations did not drastically alter the confirmation of C protein. Using SWISS-MODEL (http://swissmodel.expasy.org/), it was found that the mutations did not make any drastic conformational changes to C protein (Fig. 5-4). Hence the different phenotypes observed most likely were due to hypophosphorylation. |Results 107 IP : Anti-WNVC Ab WB: Anti-WNVC Ab (i) 72 55 26 17 10 (ii) WNV-infection Mock-infection IP : Anti-WNVC Ab WB: Anti-phosphoserine Ab C kD 15 14 kD 72 55 26 17 C kD 15 14 kD 10 IP : Rabbit isotype control WB: Anti-phosphoserine Ab WCL WB: Anti-actin Ab (iii) 72 55 26 17 10 Actin 42 kD (iv) Figure 5-2. West Nile virus C protein is a phosphoprotein. BHK cells were infected with WNV and cell lysates were immuno-precipitated using anti-WNV C protein antibody (Ab) and immunoblotted with anti-phosphoserine Ab (i) or anti-WNV C Ab (ii). The appearance of band corresponding to the size of C protein in WNV-infected samples (i and ii) confirms that WNV C protein is a phospho-protein. (iii and iv) Isotype and actin loading controls are shown. |Results 108 A Mutant Capsid protein constructed Amino acid position 26 36 Amino acid position Amino acid position S26/36A 83 99-100 26 36 83 99-100 S83/99/T100A S26/36/83/99/T100 A B Figure 5-3. Mutagenesis of putative phosphorylation sites. (A) Schematic diagram of constructed mutant myc-C proteins. The putative phosphorylated sites are indicated by a thick black line and the names of each of these mutants are indicated on the right. (B) Phosphorylation of myc-tagged capsid protein in 293FT and BHK cells. Plasmids encoding wild type capsid (Lanes and 8) or mutants (Lanes 4, 5, 6, 10, 11 and 12) were transfected into 293FT or BHK cells. The cell lysates was harvested and immunopurified with anti-myc antibody. Cell lysates from 293FT and BHK cells were either treated (Lanes and 9) or untreated with λ-phosphatase and subsequently subjected to Western blot analysis with anti-c-myc antibody and anti-phosphoserine antibody. Mocktransfected cell lysate are Lanes and 7. This shows that the anti-phosphoserine antibody is specific and the myc-C protein is a phosphoprotein (Lanes and 8). Anti-myc antibodies are used to use to show equal loading of myc-C proteins. |Results 109 Figure 5-4. Homology modeling of mutant C protein. The mutant S26/36/83/99/T100A C protein (Blue) was modeled using SWISS-MODEL (http://swissmodel.expasy.org/) and superimposed on the crystal structure of WNV (Kunjin strain) C protein (Dokland et al., 2004) (Red). The point mutations are indicated in yellow. Root mean square deviation value = 0.1. |Results 110 5.3.2 Protein kinase C phosphorylates myc-capsid (C) protein Bioinformatics analysis revealed that protein kinase C as the most probable kinase that could phosphorylate the C protein at serine 83, 99 and theronine 100. Experiments were performed to confirm if protein kinase C was involved in phosphorylating WNV C protein. To address this, kinase-specific activator or inhibitor was added to BHK cells transfected with wild type myc-C proteins or mutant myc-C proteins carrying mutations S83/99/100A. Protein kinase A activator and inhibitors were used as a negative control. Western blot analysis showed that protein kinase C was involved phosphorylating myc-C protein since protein kinase C inhibitor (BIS) brought the phosphorylation level of the wild type myc-C protein to the same level as the S83/99/100A mutant [Fig. 5-5A (i), Lanes and 9]. In addition, wild type myc-C protein harvested from protein kinase C activator-treated (PMA) cells had a higher level of phosphorylation than myc-C protein harvested from DMSO-treated cells [Fig5-5B (i) Lanes, and 8]. In contrast, myc-C protein from protein kinase A inhibitor- and activator-treated cells (H89 and Forskolin) did not exhibit a reduced nor an increased level of phosphorylation [Fig. 5-5A (i) Lane 5, Fig. 5-5B (i) Lane 5] when compared to myc-C protein harvested from DMSO-treated cells [Fig 5-5A (i) Lane 2, Fig 5-5B (i) Lane ]. In order to confirm that protein kinase C can indeed phosphorylate C protein, in vitro phosphorylation of C protein was performed using His-C protein since His-C protein was shown to be unphosphorylated (Fig. 5-1). When protein kinase C was added to and incubated with His-C protein in vitro, His-C protein was found to be phosphorylated (Fig. 5-5C, Lane 1) but hypophosphorylated in the presence of protein kinase inhibitor (BIS) (Fig 5-5C, Lane 2). |Results 111 Figure 5-5. Phosphorylation of C protein by protein kinase C. (A and B) BHK cells treated with PKA/PKC inhibitors (A) or activators (B) were transfected with plasmid encoding wild type myc-C or mutant myc-C S83/99/T100A protein. Both wild type and mutant proteins were subjected to co-immunoprecipitation using anti-myc Ab followed by immunoblotting with anti-phosphoserine Ab. The phosphorylation signal of C protein is reduced [Lane 8, A(i)]/increased [Lane 2, B(i)] following treatment with PKC inhibitor/activator, respectively. The numbers at the left of the immunoblot represent the molecular weight of the protein ladder. (ii) Immunoprecipitation control. (iii) Actin loading control. (C) (i) In vitro phosphorylation of His-tagged C protein is performed with PKC in the absence (Lane 1) or presence (Lane 2) of kinase inhibitor. The presence of band in Lane indicates that PKC phosphorylates C protein. Phosphorylation of Histagged C protein by PKC is reduced by PKC inhibitor (Lane 2). (ii) Input controls showing equal loading of His-tagged C protein. |Results 112 IP: Anti-Myc Ab WB: Anti-Myc Ab myc-C S83/99/T100A myc-C WT protein ladder Mock transfected myc-C WT S83/99/T100A myc-C S83/99/T100A Mock transfected IP: Anti-Myc Ab WB: Anti-phosphoserine Ab myc-C S83/99/T100A S83/99/T100A Mock Transfected myc-C WT PKA(-)H89 PKC(-)BIS DMSO A 72 55 (i) (ii) 26 17 10 72 55 26 17 10 C 15 kD 72 WCL WB: Anti-actin Ab (iii) C 15 kD Actin 42 kD 43 26 10 |Results 113 Figure 6-5. Cytopathic effect of wild type (WT) or mutant viruses on BHK cells. BHK cells were grown in T75 cm2 and infected with either WT or mutant WNV. The cells were stained with crystal violet after 28 hr. Cells exhibiting cytopathic effect were lifted off from the monolayer hence leaving empty spaces between cells. |Results 135 6.3.3 Ultrastructural studies To further characterise the mutant viruses, electron microscopy was performed to study the effect of the viruses on the intracellular morphology of BHK cells. It was observed that wild type viruses caused more vacuolation (contributing to the obvious cytopathic effect observed in Fig. 6-5) in infected cells than mutant viruses (Fig. 6-6A). To quantify the average number of vacuoles in infected cells, ten cells infected with each of the different wild type and mutant viruses were selected at random and the number of vacuoles in each cell was counted (Fig. 6-6B). Only cells infected with mutant viruses S83/99/T100A and S26/36/83/99/T100A showed significant lesser vacoulation compared to cells infected with wild type virus (Fig 6-6B). In addition the vacuoles in wild type infected cells were observed to be slightly larger than those in mutant infected cells (Fig. 6-6A) |Results 136 Figure 6-6. Ultrastructural studies on the effect of wild type and mutant viruses infection on cells. (A) BHK cells infected with either wild type (WT) or mutant viruses and they were processed for electron microscopy at 12 hr post-infection (Section 2.9.2). Formations of vacuoles (arrows) were observed. N is the nucleus of the cell. (B) The number vacuoles from each infected cell by the different viruses were counted. A total of ten cells were selected and the average number of vacuoles was calculated. Cells infected with mutant viruses S83/99/T100A and S26/36/83/99/T100A showed significantly less vacoulation than cells infected with wild type virus. *, ** represents p < 0.05. |Results 137 WT A S26/36/A N S83/99/T100A S26/36/83/99/T100A N N Average number of vacuoles B |Results ** * 138 6.4 Complementation with myc-capsid (C) protein In order to ensure that the phenotypes observed with the mutant viruses were not due to extraneous mutations on other sites other than the ones introduced on the capsid protein, a trans-complementation study was performed with myc-C protein. Prior to infection with viruses, cells were transfected with plasmid encoding myc-C protein and the growth kinetics of the viruses were assayed. It was observed that although the virus yield was almost restored (from 20 hr to 28 hr p.i.), the lag in virus production was still apparent (Fig, 6-7). While this could still mean that other mutations, other than the ones introduced on the C protein, could have contributed to the observed phenotype. This seemed unlikely since virus titres were restored close to 70% of the WT virus titre 20 hr post-infection. A likely explanation for the lag in virus replication could be the inability of C protein to phosphorylate during virus entry early replication events. The ability to phosphorylate C protein at early infection phase may have a crucial role since nuclear localisation, which is enhanced by phosphorylation is critical for virus production (Bhuvanakantham et al., 2009). 6.5 Growth kinetics of virus produced by transfection of infectious RNA In order to by-pass the early events of infection, wild type and mutant viruses were produced through the transfection of infectious RNA into BHK cells and virus yield assayed at regular intervals. Since the S26/36/83/99/T100A mutant virus exhibited the greatest phenotypic change, it was chosen for subsequent experiments to study the effects of C protein hypophosphorylation. The growth kinetics of viruses produced through the transfection of infectious RNA showed that the lag in virus replication observed earlier |Results 139 (Fig. 6-3 and 6-7) was abrogated (Fig. 6-8) but virus titre was still not restored completely (Fig. 6-8). It was observed that the restoration of mutant virus titre occurred only when plasmid encoding wild type myc-C protein was co-transfected with infectious RNA (Fig 6-9). The lag in virus replication also diminished. Figure 6-7. Growth kinetics and virus titre in BHK cells complemented with myc-C protein. Growth kinetics of wild type (WT) and mutant viruses in BHK cells transfected with plasmid encoding wild type myc-C protein. The transfected BHK cells were subsequently infected with WT or mutant viruses at an M.O.I of for 24 hr. Virus titres were assayed at regular intervals. Mutant viruses replication is slower than WT but virus titre of mutant viruses is almost restored. |Results 140 Figure 6-8. Growth kinetics and virus titre of wild type (WT) and mutant viruses produced from the transfection of infectious viral RNA. BHK cells were transfected with µg of infectious wild type (WT) or mutant infectious viral RNA. Virus titre was assayed at regular intervals. The lag in mutant virus replication has mostly been abolished through transfection of infectious RNA. |Results 141 Figure 6-9. Growth kinetics and virus titre of viruses produced by co-transfection of infectious wild type (WT) or mutant viral RNA with complementation of WT myc-C protein in BHK cells. BHK cells were co-transfected with either µg of WT or mutant infectious viral RNA and plasmid encoding WT myc-C protein. Virus titre was assayed at regular intervals. The lag in mutant virus has been abolished through the transfection of infectious RNA. In addition, mutant virus titre has also been restored. |Results 142 6.6 Viral RNA and protein synthesis Since there was an observed difference in the rate of replication of mutant viruses produced through infection and transfection of infectious RNA, the rate of RNA synthesis and protein synthesis could likewise also be affected. Total RNA synthesis from whole cell lysate was measured by RT-PCR with primers 17 and 44 (Table 2-3, partially encodes for the E protein) and the E protein is used as a proxy for viral protein translation. 6.6.1 Viral RNA synthesis in infected or transfected cells Cell lysates from infected or transfected cells were harvested at regular time intervals and the RNA quantified by RT-PCR. It was observed that RNA synthesis in cells infected with wild type virus was higher than cells infected with mutant virus (Fig 6-10A). In contrast, RNA synthesis of cells transfected with wild type or mutant viruses showed only a slight little difference (Fig. 6-10B). 6.6.2 Production of viral envelope (E) protein This difference between infected cells and transfected cells was also reflected in the production of viral E protein. The differential rate of protein synthesis between wild type and mutant viruses were only apparent in infected cells and not transfected cells (Fig. 6-11). |Results 143 A B Figure 6-10. Viral RNA synthesis in cells (A) infected with virus or (B) transfected with viral RNA. (A) BHK cell lysate infected with either wild type (WT) or mutant virus were harvested at 6, 12, 18 and 24 hr post-infection. RT-PCR was performed on the cell lysates with primers specific for part of the viral E protein to quantitate the amount of viral RNA at each time point. (B) BHK cell lysate transfected with wild type (WT) or mutant infectious viral RNA were harvested at 24, 36, 42 and 48 hr post-transfection. As in (A) RT-PCR was performed to quantitate the amount of viral RNA. The lag in viral RNA synthesis observed in (A) has been abolished when infectious RNA were transfected into the cells instead. |Results 144 A Hr postinfection 12 WT M WT 18 M WT 24 M WT M Mock infected Anti-E Ab Anti-actin Ab B Hr posttransfection 24 WT 30 M WT 36 M 42 WT M WT M Mock infected Anti-E Ab Anti-actin Ab Figure 6-11. Production of E protein in (A) infected or (B) transfected cells. (A) BHK cells were infected with either wild type (WT) or mutant S26/36/83/99/T100A (M) viruses and the cell lysate was harvested at regular time intervals and subjected to Western blot analysis with anti-E protein and anti-actin antibodies. (B) BHK cells were transfected with either wild type (WT) or mutant S26/36/83/99/T100A (M) infectious RNA and the cell lysate was harvested and analyse as in (A). The difference in E production at each time point is only apparent in infected and not transfected cells. |Results 145 6.7 Localisation of mutant capsid (C) protein in infected cells. The inefficient nuclear localisation of C protein could explain the reduced viral titre hence cellular localisation relative to viral RNA was investigated using mutant S26/36/83/99/T100A virus. As in Fig. 4-9, BHK cells were infected with the mutant virus, and viral RNA and C protein were probed at regular time intervals to visualize their location after infection. It was observed that at hr post-infection [Fig. 6-12A (i)], C protein was seen in both the cytoplasm and nuclei in infected cells, whereas in the wild type virus infection, the C protein was found in the nuclei [Fig. 6-12A (v)]. By 12 hr post-infection, mutant C protein was no longer seen in the nuclei. In contrast, the wild type C protein was still found predominantly in the nucleus. By 18 hr post-infection onwards, C protein is found predominantly in the cytoplasm for both wild type and mutant virus infections. The number of cells with C protein (wild type or mutant) found exclusively in the nucleus or in both nucleus and cytoplasm was quantitated by randomly selecting 50 cells. The cells were then assigned into the above-mentioned categories – in nucleus only or in both nucleus and cytoplasm. The results obtained at 24 hr post-infection (Fig. 6-12B) were similar to data obtained with hypophosphorylated mutant myc-C protein [S26/36/83/99/T100A (Fig. 5-7B)] and bisindolylmaleimide-treated myc-C protein (Fig 5-8B), signifying that localisation of C protein is dynamically modulated by phosphorylation. In the mutant virus-infected sample, there were also less cells with C protein localised exclusively in the nucleus from the onset of infection up to 12 hr postinfection as compared to wild type infected sample (Fig. 6-12B). This difference between mutant infected cells and wild type infected cells was no longer apparent. |Results 146 A Hr Post-infection 12 hr (i) 18 hr 24 hr (ii) (iii) (iv) (vi) (vii) (viii) WT S26/36/83/99/T100A hr (v) Percentage of cells B * * Figure 6-12. Localisation of WNV RNA and C protein during an infection. BHK cells were infected with mutant and wild type (WT) viruses and stained for viral RNA (green) and C (red) protein at 6, 12, 18, 24 hr post-infection. The lower panel is reproduced from Fig. 4-9. In contrast to WT C protein, localisation of mutant C proteins are seen in the cytoplasm as early as 12 hr post-infection. (B) The cellular distribution of C protein is quantified by randomly choosing 50 cells and assigning them into either category – C protein found only in the nucleus or C protein found in the nucleus and cytoplasm. The proportion of cells with mutant C protein localised in both the nucleus and cytoplasm at hr post-infection (6 hr) and 12 hr post-infection (12 hr) were significantly greater than the proportion of cells with wild type C protein localised in both the nucleus and cytoplasm at the same timing post-infection. * represents p < 0.05 |Results 147 6.8 Packaging of genomic RNA The above results (Fig. 6-8 and 6-9) suggested that defects in early events of virus replication are responsible for the lag in virus titre and that a C protein amenable to phosphorylation was important for virus replication (Fig. 6-7 and 6-9). Since it was observed that hypophosphorylated C proteins have a strong affinity for viral RNA and lacks the efficiency, compared to phosphorylated C proteins, to localise in the nucleus, it is tempting to suggest that the hypophosphorylated C protein being in the cytoplasm as early as 12 hr post-infection, could have packaged non-infectious or non-genomic RNA into the nucleocapsid. To investigate this hypothesis, the ratio of positive-strand and negative-strand RNA from purified virus was measured using RT-PCR. Before strand-specific RNA could be measured, the virus particles harvested from cell culture need to be purified away from extraneous nucleic acids and proteins in the supernatant. Hence a density gradient using OptiPrep medium was performed and the fraction in which the virus aggregated after centrifugation was determined (Figure 6-13). In addition to purification, the purified virus was also treated with microccal nuclease (MNase) to remove any nucleic acid associated on the exterior of the virus before being processed for RT-PCR. To control for unexpected changes in replication of positivesense and negative -sense viral RNA in the cells, the cell lysate from infected cells were also used for the relative quantification of positive to negative sense RNA. RT-PCR of the wild type virus showed that the positive-strand RNA was about 10 times in excess of the negative-strand RNA (Fig 6-14A). However, the relative amount of negative- to positive-strand RNA from the mutant S26/36/83/99/T100A virus is about 2:1 (Fig 6-14A). These results were further confirmed with DNA gel electrophoresis of the |Results 148 RT-PCR products (6-14B) showing that there was a higher proportion of negativestranded viral RNA being packaged into the mutant virus. Densitometry analysis of the band intensities also corroborate with the RT-PCR data (Fig. 6-14A). In the densitometry analysis, the intensity of the (+) strand for the WT was 6232.447 arbiturary units while the (-) strand was 698.70 arbiturary units. For the mutant virus, the intensity of the (+) strand was 2317.134 while the (-) strand was 901.234. Figure 6-13. Optimisation of density gradient purification of WNV. WNV virus was purified using OptiPrep density gradient. The gradient was prepared using 20 %-50 % Optiprep medium and allowed to set overnight. Virus supernatant was layered on top of 20 % Optiprep. A representative plaque assay of the amount of virus in each section of the medium is shown on the right. Viruses were typically found between 30%-40% of the gradient. |Results 149 Relative amount of anti-sense/sense viral RNA A B Mock Infected WT + - + S26/36/83/99/T100A - + DNA Marker - Figure 6-14. Relative quantitation of viral sense and anti-sense RNA in wild type (WT) or mutant virus. (A) Wild type (WT) or the S26/36/83/99/T100A mutant (Mutant) viruses were used to infect BHK cells. RT-PCR was performed on infected cell lysate (C) at 24 hr post-infection, purified and microccal nuclease-treated virions (V) and the infectious clone DNA plasmid (IC Plasmid) to detect for sense and anti-sense viral RNA using primers 17 and 44 (Table 2-3). The amount of sense RNA is set to an arbitrary value of and the amount of anti-sense RNA is expressed as a fraction of the sense RNA. The ratio of positive- to negative-stand RNA in WT virus-infected cell lysate and WT virions are 10:1. In contrast the ratio of positive- to negative-strand RNA in mutant virus-infected cell lysate and mutant virions are 10:1 and 2:1, respectively. (B) The RT-PCR products from WT, mutant were amplified with the same primers and the amplified products were visualized on DNA gel electrophoresis. The band intensities were analysed by densitometry using ImageJ. The (+) and (-) represent the sense and anti-sense products, respectively. The band intensities corresponded with the results obtained by RT-PCR in (A). |Results 150 [...]... |Results 2 1 14 5 .4 Effects of phosphorylation on myc -capsid (C) protein 5 .4. 1 RNA binding of myc -capsid (C) protein is attenuated by phosphorylation Since it was shown that myc-C protein as well as the West Nile virus C protein from infected cells are phosphorylated the next question to ask is if phosphorylation can attenuate myc-C protein and RNA interaction as demonstrated with C peptides (Fig 41 0) Hence... BHK 48 hr 8 9 10 11 Mock transfected 19 GFP 24 hr 20 GFP 48 hr 21 GFP 48 hr GFP 24 hr myc-C S26/36/83/99/T100A myc-C S83/99/T100A 24 hr myc-C S26/36/83/99/T100A myc-C S26/36A myc-C WT myc-C S26/36/83/99/T100A myc-C S83/99/T100A myc-C S26/36A myc-C WT Mock transfected 293FT 48 hr 55 kDa 43 kDa 34 kDa 26 kDa 17 kDa 55 kDa 43 kDa 34 kDa 26 kDa 17 kDa 22 125 5.5 Phosphorylation of West Nile virus (WNV) capsid. .. West Nile virus (WNV) capsid (C) protein diminishes over time If phosphorylation plays a regulatory role in modulating the functions of C protein then the phosphorylation status of C protein should be dynamic If the role of phosphorylation is to prevent premature association of viral RNA or even cellular RNA with C protein during the early stages of infection then C protein can be expected to be phosphorylated... Conversely, at later stages of infection when most of the C protein would interact with viral RNA to form the nucleocapsid, phosphorylation is expected to dimes Hence, BHK cells were infected with WNV and C protein was harvested from the infected cell lysate at 6, 12, 18 and 24 hr post infection The relative level of phosphorylation of C protein at these time points was analysed by Western blot There was... P . 1 04 5.0 PHOSPHORYLATION OF WEST NILE VIRUS (WNV) CAPSID (C) PROTEIN AND RNA INTERACTION 5.1 Introduction Having established that phosphorylation on C protein peptides could attenuate its RNA. (His-C) protein (Lane 3). |Results 106 5.3 Phosphorylation of the West Nile virus (WNV) capsid (C) protein 5.3.1 West Nile virus (WNV) capsid (C) protein is a phosphoprotein. phosphorylation on myc -capsid (C) protein 5 .4. 1 RNA binding of myc -capsid (C) protein is attenuated by phosphorylation Since it was shown that myc-C protein as well as the West Nile virus C protein from