Functional analysis of the non-enzymatic properties of the dengue virus non-structural protein 5

The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. The DENV RNA genome is translated into a polyprotein that is cleaved into three structural and seven nonstructural proteins. Nonstructural protein 5 (NSS) has enzymatic activities required for capping and replication of the viral genome. NSS is phosphorylated and with some serotypes can accumulate in the nucleus of infected cells. However the role of NSS phosphorylation or nuclear trafficking in the virus lifecycle, is not well understood. Using a combination of proteomic and bioinformatic approaches, potential NSS phosphorylation sites were identified and analysed for their role in the virus lifecycle using a DENV reverse genetic system. Mutation of the NSS amino acid Thr-39S was found to decrease NSS nuclear localisation and viral replication in a cell specific manner. Mutations preventing and mimicking phosphorylation at Thr-39S had similar effects on NSS localisation and viral growth, showing that the effects were not caused by phosphorylation. Mutation of Thr-39S did not influence NSS mediated suppression of interferon signalling. For the first time this thesis revealed that there are serotype specific differences in NSS localisation. DENV-1 and DENV-4 NSS are predominantly localised to the cytoplasm, whereas DENV-2 and DENV-3 NSS accumulate in the nucleus. The role of the NSS nuclear localisation signal (NLS) in mediating these differences was investigated. NSS of all DENV serotypes bound to importin-a, the protein responsible for active nuclear import, but exchanging the NLSs of DENV-2 and 4 NSS didn't fully confer the properties of the NLS to the background protein, suggesting that protein context was also important for NSS nuclear localisation. Chimeric DENV-2 containing sequences encoding the DENV-3 NSS or polymerase subdomain were produced, whilst DENV-2 containing the DENV-4 NSS sequence failed to replicate. The two DENV-2/3 chimeric viruses showed decreased replication especially in the insect cell line C6/36; continued passaging resulted in the accumulation of mutations in NS1 and NS2B that increased viral replication.EThOS - Electronic Theses Online ServiceGBUnited Kingdo