Functional studies of the role of nonstructural proteins in Alphavirus and Flavivirus replication

Alpha- and Flaviviruses are enveloped, plus-strand RNA viruses that are transmitted mainly by mosquitoes and pose serious health concern globally. These viruses cause severe illnesses like encephalitis and hemorrhagic fever. The viral genome encodes nonstructural or replication proteins that, replicate the viral RNA in the cell cytoplasm, and mediate most of the host-virus interactions crucial for RNA replication. Alphaviruses encode 4 nonstructural proteins (nsP1-4) and flaviviruses encode 7 nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). The alphavirus nonstructural protein nsP2 from the prototype Sindbis virus (SINV), and the flavivirus nonstructural protein, NS5 from dengue virus serotype 2 (DENV-2) were studied for roles in the virus life cycle. SINV nsP2 is a multifunctional protein with an N-terminal helicase domain and a C-terminal protease domain. Structurally the nsP2 protease domain contains an independently folded domain at the C-terminus with structural similarities with 2\u27-O-methyltransferases (MTase), but this domain is not a functional enzyme. To investigate the functional significance of the MTase-like domain, conserved residues were mutagenized and characterized for effects on protease activity, minus- and plus-strand RNA synthesis. The mutations of surface exposed residues led to protease independent reduction in minus-strand RNA synthesis and therefore replication. The results suggested a role for the MTase-like domain as a scaffold in the establishment of a functional minus-strand replication complex. SINV nsP2 is also involved in virus-mediated shutdown of host cellular machinery to counter the host antiviral response. Residues in the MTase-like domain, and the inter-domain loop between the protease and the MTase-like domain were involved in differential regulation of nsP2-mediated inhibition of cellular transcription and translation. These results displayed the functional versatility of the nsP2 MTase-like domain during virus infection. Flavivirus NS5 is the viral methyltransferase and RNA-dependent RNA polymerase. A system was established for, large scale purification of flavivirus NS5 from a bacterial expression system, and biochemical analysis of the protein by means of an in vitro polymerase assay. In infected cells, DENV-2 NS5 is differentially phosphorylated and phosphorylation is thought to trigger critical changes in NS5 functions during infection. Phosphorylation of NS5 in mammalian cells was investigated and a cGMP-dependent protein kinase G was identified as a cellular kinase that phosphorylates NS5 and is critical for virus replication and growth