Modulation of West Nile Virus Capsid Protein and Viral RNA Interaction through Phosphorylation
West Nile virus (WNV) is a mosquito-borne flavivirus, which can cause fatal meningoencephalitis. Although studies have been done to examine virus life cycle, the mechanisms that regulate its assembly are unknown. This study aims to characterize the interaction between WNV viral RNA and the capsid (C) protein and also elucidate how the processes of nucleocapsid assembly are regulated. Initial in vitro C protein and viral RNA interaction studies showed that this interaction was not specific. This suggested the presence of a regulatory mechanism to regulate C protein and RNA interaction. There are 5 putative phosphorylation sites on C protein hence; functions of C protein could be regulated by phosphorylation. Western blot analysis with anti-phosphoserine antibodies confirmed that C protein was phosphorylated. Experiments using kinase inhibitors and activators identified protein kinase C to be one of the kinases responsible for the phosphorylation of C protein. Mutations were introduced into C protein to abolish phosphorylation. The effects of hypophosphorylation with regards to the nucleocapsid assembly processes like RNA binding; nuclear localisation and oligomerization were investigated. Phosphorylation of C protein attenuated RNA binding and this study showed that C protein was dephosphorylated later during infection to facilitate C protein and viral RNA interaction. It is also known that WNV C protein localises predominantly in nuclei of infected cells. Hypophosphorylation of C protein was shown to disrupt this process. Tracking of the cellular localisation of C protein during an infection showed that C protein localised in the nucleus during the early phase of infection but was found in the cytoplasm during late phase of infection. This correlated with the gradual dephosphorylation of C protein in infected cells. Capsid protein also oligomerized to form the nucleocapsid but hypophosphorylation did not affect the formation of oligomers. However, the rate of oligomerization was retarded by phosphorylation. These data showed that hypophosphorylation favoured the processes of nucleocapsid assembly. The biological significance of these hypophosphorylated mutants was further investigated by introducing the same mutations into a WNV infectious clone. Characterization of the mutant viruses showed that mutant viruses produced a lower viral yield and also experience a lag in viral production compared to wild type virus. Complementing mutant virus infected cells with wild type C protein partially restored viral yield, however, the lag in virus production was still apparent. The lag in mutant virus replication was only abolished through the transfection of infectious viral RNA into cells. This suggested that phosphorylation was critical for the early events of viral replication linked to nuclear localisation. In addition, it was found that, while wild type virus packaged 10 times more positive-stranded viral RNA than negative-stranded viral RNA, mutant virus packaged only twice as much positive-stranded viral RNA to negative-stranded viral RNA. This study shows that WNV C functions as a phospho-protein and proposed the dynamics of phosphorylation and dephosphorylation of C protein prevents the premature assembly of the nucleocapsid. This allows assembly to occur during the late phase of an infection where large pools of positive-stranded virus RNA are present.
flavivirus, capsid, phosphorylation, West Nile virus, regulation
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