Modulation of west nile virus capsid protein and viral RNA interaction through phosphorylation 5

7.0 DISCUSSION AND CONCLUSION Although the entry and egress of WNV have been studied extensively (Chu et al., 2003; Chu & Ng, 2002a; 2004; Chu et al., 2005; Ng et al., 2001), the processes of assembly are still not well understood. Assembly of the virus can be broken down into two stages – encapsidation of the viral RNA by the C protein to form nucleocapsid and envelopment of nucleocapsid by viral envelope protein (Fig. 7-1). This study is focused on the processes that lead to the formation of the nucleocapsid. The most important process is the interaction between C protein and viral RNA. Oligomerization of C proteins would no doubt also be an important process. Besides these two processes, the cellular localisation of C protein needs to be considered since it has been noted by many investigators (Bulich & Aaskov, 1992; Mori et al., 2005; Oh et al., 2006; Sangiambut et al., 2008) that C protein localises in the nucleus of infected cell but assembly of the virus occurs in the cytoplasm. Hence, it is postulated that the functions of C protein might be modulated to achieve efficient assembly of the nucleocapsid. This study accumulated evidence that phosphorylation of WNV C protein acts as such a modulator of C protein function. | Discussion and conclusion 151 Figure 7-1 General diagram of flavivirus assembly process. This shows the overview of the replication process of a virus life cycle. In the inset, it shows the assembly process of the nucleocapsid, which involves interaction of the capsid protein (hexagon) with RNA (wavy line) and the oligomerization of the capsid protein. Once the nucleocaspid is assembled, it translocates into the lumen of the endoplasmic reticulum (ER), where the envelope protein (circle) would wrap itself around the nucleocapsid. The upper panel was reproduced from a review by Mukhopadhyay et al., 2005 | Discussion and conclusion 152 In order to investigate the role of phosphorylation in the processes of nucleocapsid assembly, a series of experiments were performed. In addition double and triple mutations were introduced at the amino- and carboxyl- terminal of C protein, respectively. This is because it was previously found that flavivirus C protein was still functional when either terminal was deleted suggesting redundancy and functional flexibility (Kofler et al., 2002; Patkar et al., 2007; Schlick et al., 2009). Moreover, analysis of other flavivirus C protein sequences also revealed that putative phosphorylation sites were found on the amino- and/or carboxyl- terminal (Table 4-3). This suggested that even though the exact residues or sites may not be conserved, the regions of phosphorylation on C protein were consistent. In addition, it was also shown that protein kinase C was responsible for phosphorylating C protein (Fig. 5-5). This is the first time C protein of a member of the mosquito-borne flaviviruses has been shown to function as a phospho-protein. Although homology modeling was performed to ascertain that mutations introduced did not perturb the conformation of the C protein it could not be extended to include the mutations at positions 99-100 (Fig. 5-4) since the crystal structure of WNV C protein was solved for amino acids 24 to 96 (Dokland et al., 2004). Therefore only of the mutation sites were shown (Fig. 5-4). As noted earlier, nuclear localisation of flavivirus C protein have been demonstrated by various investigators (Bulich & Aaskov, 1992; Mori et al., 2005; Oh et al., 2006; Sangiambut et al., 2008; Wang et al., 2002) and this seemed to be important for the replication of the virus during the earlmmmmy phase [(Bhuvanakantham et al., 2009), (Fig. 7-2)]. It was recently discovered that importin-α protein facilitated the | Discussion and conclusion 153 nuclear localisation of Dengue and WNV C proteins (Bhuvanakantham et al., 2009). The strength of interaction between importin-α protein and WNV C protein was demonstrated to be enhanced by C protein phosphorylation (Bhuvanakantham et al. 2010). The observation that the cellular distribution of mutant myc-C proteins was more diffused than wild type myc-C protein (Fig. 5-7A) is consistent with previous finding observed with Hepatitis C virus (Lu & Ou, 2002) where phosphorylation enhanced nuclear localisation of C protein. To complement studies using hypophosphorylated mutant C proteins, protein kinase C activators and inhibitors were used to enhance or attenuate wild type C protein phosphorylation (Fig. 5-5). As expected, nuclear localisation of wild type myc-C protein was disrupted in bisindolylmaleimide-treated (PKC inhibitor) cells (Fig. 5-8A). The proportion of cells with wild type myc-C protein localized in the nucleus and cytoplasm in bisindolylmaleimide-treated cells was about the same as cells transfected with mutant S26/36/36/83/99/T100A (Fig. 5-7B). Because the results from both studies (mutagenesis and PKC inhibitor) were consistent, the phenotype observed in the mutants was due to hypophosphorylation of myc-C protein. Nuclear localisation of C protein is an integral part of virus replication and the inhibition of nuclear localisation of C protein by deleting or mutating the nuclear localisation signal on the WNV or Dengue virus C protein had deleterious effects on the viruses (Bhuvanakantham et al., 2009). There are suggestions that nuclear localisation of viral C proteins may be a strategy used by viruses to disrupt cytokinesis (Hiscox et al., 2001; Ning & Shih, 2004). In the case of flaviviruses, the C protein was shown to be involved in cell-cycle arrest (Helt & Harris, 2005; Oh et al., 2006) and there was some evidence to suggest correlation between WNV C protein nuclear localisation and cell | Discussion and conclusion 154 cycle arrest (Oh et al., 2006). Nonetheless, whatever the function of flavivirus C protein nuclear localisation may be, disruption or inhibition of this process was detrimental to flavivirus replication (Bhuvanakantham et al., 2009; Mori et al., 2005; Sangiambut et al., 2008). Hence the reduced viral yield of the mutant viruses (Fig. 6-3) could be partially explained by the cellular localisation of C protein during early infection. In addition to the above-suggested functions of C protein in the nucleus, nuclear localisation of C protein during the early phase of infection (Fig. 6-12, v and vi) may also have another function. Translocation of C protein into the nucleus in the first 12 hr of infection may prevent premature C protein and RNA interaction since viral RNA synthesis occurs in the cytoplasm (Fig. 4-8). Investigations into how C protein and viral RNA interacted in a cellular environment showed that there was no co-localisation between myc-C and viral RNA since the RNA remained in the cytoplasm while the mycC protein was translocated in the nucleus (Fig. 4-5). In addition, wild type C protein and viral RNA were shown to localise in the nucleus and cytoplasm of infected cells, respectively (Fig. 4-9). It was only at 18 and 24 hr post-infection that any co-localisation of C protein and viral RNA was observed in the cytoplasm (Fig. 4-9). This showed that C protein did not remain in the nuclei permanently but the location between the nuclei and cytoplasm could be dynamic and regulated. These observations coincided with the gradual dephosphorylation of C protein in infected cells (Fig. 5-10). Having observed the presence of mutant C protein in the cytoplasm of infected cells as early as 12 hr post-infection (Fig. 6-12, i and ii) and that His-C protein could interact with negative strand viral RNA (Fig. 4-2 and Fig. 4-3) there is a real possibility | Discussion and conclusion 155 that mutant C protein could have incorporated negative-sense viral RNA into the virions during early replication phase. To investigate if mutant virus would indeed incorporate relatively more negativesense viral RNA into the virions, viruses were purified for analysis with RT-PCR. The results indicated that on average, there was about 10 times more positive strand RNA than negative strand RNA in a pool of wild type virus (Fig. 6-14 A). In contrast, the ratio between the positive- and negative-strand RNA in the pool of mutant virus is almost 2:1 (Fig 6-14A). This means that almost 1/3 of the viruses in the mutant pool were noninfectious and this could explain the reduced titre seen in the mutant virus infections (Fig. 6-8). The detection of negative strand RNA in a pool of positive strand virus is not all that surprising. On average, for viruses in the Flaviviridae family, the ratio between positive- to negative-strand viral RNA in infected cells is about 10:1 (Quinkert et al., 2005; Richardson et al., 2006) and similar results was also found infected cell lysates from this study (Fig 6-14A), hence it would be logical to assume that the ratio of positive to negative strand viral RNA packaged into wild type virions would also be about 10:1. Similar changes in the ratio of genomic and anti-genomic RNA being incorporated into virions due to mutations or deletions of viral C protein have been reported in the Sendai virus, a single-stranded negative-sense RNA virus (Irie et al., 2008). The change in the ratio between genomic and anti-genomic RNA packaged into the virions was due to aberrant changes in RNA synthesis. However, in the case of WNV, the mutations introduced into the C protein did not cause the ratio of positive- to negative-strand RNA to change in infected cells (Fig. 6-14A). Therefore the changes in | Discussion and conclusion 156 the ratio of positive to negative strand RNA being packaged into the virions is likely due to the relative abundance of the negative-sense RNA during the early phase of infection. Hence the nuclear localisation of C protein could indirectly prevent premature C protein and RNA association, although direct regulation of C protein and RNA interaction was achieved through phosphorylation of C protein. Phosphorylation of C protein has been demonstrated in both RNA and DNA viruses to aid the release of nucleic acid into infected cells and regulate packaging of its genome (Gazina et al., 2000; Ivanov et al., 2001; Law et al., 2003). To investigate C protein and RNA interaction and how phosphorylation might affect this interaction in vitro, interaction between C protein and viral RNA needs to be characterized. Although the delineation of RNA binding regions on the WNV C protein is not new (Khromykh & Westaway, 1996), the use of overlapping peptides to map out the RNA binding regions is novel since it gave a better definition as to where the RNA binding sites on the C protein are (Fig. 4-4). It was found that the strongest RNA binding regions corresponded with the positive charge of each peptide (Table 4-2). From previous studies (Khromykh & Westaway, 1996; Patkar et al., 2007) it was shown that 3’ and 5’ UTR of the viral RNA were able to bind to WNV C protein but those were not the only regions that could interact with C protein. This study showed that all regions of the viral RNA could interact with His-C protein (Fig. 4-2). In some cases, C protein of other flaviviruses could even interact with singlestranded DNA to form nucleocapsid particles in vitro (Kiermayr et al., 2004). Hence interaction of C protein with nucleic acid may be indiscriminate. Nonetheless, the strongest binding RNA fragment was confirmed when His-C protein pull-down of viral | Discussion and conclusion 157 RNA was performed in conjunction with RT-PCR (Fig. 4-3). From the results in Fig. 4-3, it showed that the encapsidation signal was found in Fragment 2, which is from position 960-1974. What determines C and viral RNA interaction is unclear but it was speculated that secondary RNA structures on the viral could mediate C protein and RNA interaction (Khromykh & Westaway, 1996). The finding that C protein was able to pull down negative-sense viral RNA in vitro (Fig 4-3, Fragment 13 and 14) reinforced the idea that C protein and viral RNA interaction may be indiscriminate. Hence suggesting that a mechanism such as phosphorylation could be employed by the virus as a form of regulation to ensure a more specific interaction between C protein and viral RNA. Indeed, preliminary investigations with phosphorylated peptides of the RNA binding regions of C protein show that phosphorylation attenuated RNA binding (Fig. 410). In light of this, the putative phosphorylation sites located within the RNA binding regions of the C protein (Table 4-3 and Fig. 4-4) did not seem to be coincidental. To study the effect of phosphorylation of C protein on RNA interaction, Northwestern blot was employed. Although this method is typically used to detect protein-RNA interaction, the RNA pull-down assay was also developed in this study to complement the Northwestern blot. In the Northwestern blot assay, myc-C protein did not show any interaction with viral RNA (Fig. 5-6A). In contrast, using the RNA pull-down assay, the wild type myc-C showed evidence of interacting with RNA since it was pulled down (Fig. 5-6B, Lane 1). The advantage of using the RNA pull-down assay over Northwestern blot is that both C protein and RNA are free to associate or dissociate in solution thus mimicking interaction as in a cellular environment. However the lack of | Discussion and conclusion 158 phosphorylation seen on wild type myc-C protein that was pulled down could be due to two reasons (Fig. 5-6B, Lane 1, bottom panel). It could be because there exists a population of unphosphorylayted myc-C protein in the cell lysate that was pulled down. Or it could be that there was not enough myc-C protein being pulled down to give a phosphorylation signal on the Western blot (Fig. 5-6B, Lane 1, bottom panel). Nonetheless these assays demonstrate that phosphorylation attenuates RNA binding. In addition, they also confirm the functional redundancy found in WNV C protein (Kofler et al., 2002; Patkar et al., 2007; Schlick et al., 2009) with regards to the attenuation of RNA binding (Fig. 5-6A, Lane 2). Phosphorylation at either terminal of the C protein only slightly attenuated the RNA binding ability of the C protein and hence this is consistent with findings that assembly of infectious virions could still occur even when large sections of the either the amino- or carboxyl- terminal were deleted (Patkar et al., 2007; Schlick et al., 2009). A straightforward explanation as to why phosphorylation of C protein would attenuate its affinity for viral RNA in WNV and also other viruses (Gazina et al., 2000; Ivanov et al., 2001; Law et al., 2003) is that phosphates are negatively-charged. Phosphorylation would thus reduce the positive charge on C protein and reduce its affinity for negatively-charged nucleic acids. However, phosphorylation does not always lead to attenuation of nucleic acid binding. In some cases there were no effect (Maroto et al., 2000) and in other cases phosphorylation enhanced protein binding to nucleic acid (Green et al., 1992). In these instances, phosphorylation of protein caused a conformational change which exposed the nucleic acid binding sites on the protein for interaction with RNA/DNA (Green et al., 1992). Although physical steric changes could | Discussion and conclusion 159 arise from phosphorylation in WNV C protein, it is unlikely that the attenuation of RNA binding observed with WNV C protein was due to conformational changes. This is because phosphorylation of WNV C peptides, which are not known to have secondary structures, also attenuated its RNA binding capacity (Fig. 4-10). Hence this data supports the idea that WNV C protein and RNA interaction is due to electrostatic forces rather than physical steric forces. However, the enhanced affinity for viral RNA and inefficiency of nuclear localisation of hypophosphorylated C protein was insufficient to explain the lag in virus replication observed in mutant viruses (Fig. 6-3). Although complementation with wild type myc-C protein partially restored the virus yield it did not abolish the lag in virus replication (Fig. 6-7). Since in vitro results suggested that dephosphorylation favours the formation of nucleocapsid (enhanced RNA binding and cytoplasmic localisation of C protein), the ability to phosphorylate C protein must be crucial for the early events of viral replication. In order to circumvent the early events of virus replication, infectious RNA was transfected into the cells and asked if the lag in virus replication could be abolish. Indeed, when infectious viral RNA was used to produce mutant virus, the lag observed earlier was abolished (Fig. 6-8). 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Leidner, U & Wengler, G (19 85) Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins Virology 1 45, 227-236 CDC: West Nile Virus - Surveillance and control case count of West Nile Virus disease 2004, accessed on 04 January 2010, < http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount04_detailed.ht... 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References 166 glycoprotein gene of Euro-African West Nile viruses J Gen Virol 78 ( Pt 9), 2293-2297 Bhuvanakantham, R., Cheong, Y K & Ng, M L (2010) West Nile virus capsid protein interaction with importin and HDM2 protein is regulated by protein kinase C-mediated phosphorylation Microbes Infect 12, 6 15- 6 25 Bhuvanakantham, R., Chong, M K & Ng, M L (2009) Specific interaction of capsid protein and importin-alpha/beta... influences West Nile virus production Biochem Biophys Res Commun 389, 63-69 Bhuvanakantham, R & Ng, M L (20 05) Analysis of self-association of West Nile virus capsid protein and the crucial role played by Trp 69 in homodimerization Biochem Biophys Res Commun 329, 246- 255 Bradford, M M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye... hypothesis that C protein functions as a phosphoprotein and that the dynamic process of phosphorylation and deposphorylation of C protein serves to modulate the processes of nucleocapsid assembly | Discussion and conclusion 164 Figure 7-2 Model of how phosphorylation modulates the functions of C protein to bring about the assembly of the virus The diagram is divided into two general phases of the virus replication... to West Nile virus Indian J Med Res 106, 2 25- 228 Irie, T., Nagata, N., Yoshida, T & Sakaguchi, T (2008) Paramyxovirus Sendai virus C proteins are essential for maintenance of negative-sense RNA genome in virus particles Virology 374, 4 95- 5 05 Ivanov, K I., Puustinen, P., Merits, A., Saarma, M & Makinen, K (2001) Phosphorylation down-regulates the RNA binding function of the coat protein of potato virus. .. M., Mandl, C W., Messner, P & Heinz, F X (2004) Isolation of capsid protein dimers from the tick-borne encephalitis flavivirus and in vitro assembly of capsid- like particles J Virol 78, 8078-8084 Kofler, R M., Heinz, F X & Mandl, C W (2002) Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence J Virol 76, 353 4- 354 3... (2009) In vitro assembly of nucleocapsid-like particles from purified recombinant capsid protein of dengue-2 virus Arch Virol 154 , 6 95- 698 Lu, W & Ou, J H (2002) Phosphorylation of hepatitis C virus core protein by protein kinase A and protein kinase C Virology 300, 20-30 Ma, L., Jones, C T., Groesch, T D., Kuhn, R J & Post, C B (2004) Solution structure of dengue virus capsid protein reveals another... inhibition of viral RNA and protein synthesis in mutant WNV infection (Fig 6-10A and 6-11A) The high affinity of hypophosphorylated C protein to viral RNA may be inefficient in dissociating encapsidated RNA from the C protein hence causing the viral RNA less available for translation and replication Whether phosphorylation destabilizes oligomers of WNV C is unknown but observation with hypophosphorylated and . Wengler, G. (19 85) . Sequence analysis of the viral core protein and the membrane-associated proteins V1 and NV2 of the flavivirus West Nile virus and of the genome sequence for these proteins. Virology. localisation of C protein could indirectly prevent premature C protein and RNA association, although direct regulation of C protein and RNA interaction was achieved through phosphorylation of C protein. . protein and RNA interaction and how phosphorylation might affect this interaction in vitro, interaction between C protein and viral RNA needs to be characterized. Although the delineation of