Molecular basis of attenuation of the live attenuated Japanese encephalitis virus vaccine SA14 14 2

Abstract

Japanese encephalitis virus is a member of Flaviviridae family. Flaviviridae family has four genera which comprises of Flavivirus, Pestivirus, Hepacivirus and Pegivirus. The global burden of disease caused by these viruses is very significant. Japanese encephalitis virus (JEV) is a major cause of encephalitis in children below five years of age in south-east Asian countries. JEV is transmitted to humans primarily by Culex mosquitoes. JEV genome is ~11kb in size. A single polyprotein is encoded from a positive sense RNA which is co- and post-translationally cleaved by viral and cellular proteases to give rise to three structural and seven non-structural proteins. Live attenuated vaccine SA14-14-2 derived from its wild-type parent strain SA14 is a very effective vaccine and extremely safe for use in humans. Earlier reports have attributed attenuation of SA14-14-2 to the absence of NS1 protein, whose synthesis is abrogated in SA14-14-2 by a single silent mutation (G to A) in stem1 of a pseudoknot structure at the 5 end of the NS2a gene. Sequence comparisons of SA14 and SA14-14-2 have revealed that envelope protein has nine and NS1 has four aminoacid changes in the protein sequence with an additional amino acid changes distributed among the remaining proteins. It has been shown that the mutant envelope of SA14-14-2 is sufficient to attenuate the neurovirulence and neuroinvasion displayed by the wild type virus. In this study we therefore investigated the contribution of both the viral glycoproteins, NS1 and envelope to the attenuated phenotype, for which we found ER stress to be a surrogate marker. As glycoproteins need the ER environment for their proper folding and glycosylation, we first compared the levels of ER stress in infected cells. We found elevated levels of ER stress in SA14-14-2-infected cells as compared to P20778 virus. We studied the PERK pathway in greater detail and found that the WT P20778 virus is able to dephosphorylate e- IF2and#945; very efficiently as compared to the vaccine strain SA14-14-2...