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- IF2α very efficiently as compared to the vaccine strain SA14-14-2. We also observed a significant increase in the chaperone levels in cells infected with the vaccine strain SA14-14-2 as compared to the WT virus. As expected, the persistent phosphorylation of eIF2α led to increased autophagic flux in SA14-14-2-infected cells. To study the contribution of NS1’ protein to the above ER stress, lentivirus expressing NS1’ was generated and used to transduce porcine kidney cell line. While we did not observe significant difference in the PERK pathway or autophagy, we did observe the constitutive expression of CHOP in NS1’ lentivirus transduced cells. CHOP mediates the dephosphorylation of p-eIF2α by GADD34 directed p110 phosphatase in a negative feedback loop. We also observed that dephosphorylation of e-IF2α in WT JEV-infected cells expressing NS1’ is independent of PERK and this dephosphorylation may be achieved by the efficient recruitment of CHOP. Hence, the role of NS1’ in WT JEV-infected cells is to relieve the cell from ER stress mediated by the phosphorylation of eIF2α as an antiviral response. Our results from the studies of envelope protein (E) of the WT and the vaccine strain using a panel of monoclonal antibodies (MAbs) suggested that it is assembled very differently on the vaccine virus particle relative to WT virus. Comparison of the half-lives of the metabolically labelled E protein from the infected cell lysates revealed that the SA14-14-2 E is degraded much rapidly compared to the WT with half-lives of 5.9h and 1.2h, respectively. Extensive investigations using DTT to reduce the disulphide groups and alkylation of –SH groups using N-ethyl maleimide (NEM) revealed that the intracellular SA14-14-2 E protein folded extremely slowly. These findings led us to conclude that the folding kinetics and assembly of the E protein from the two viruses are very different. We concluded that this non-native folded state of SA14-14-2 E is the prime cause of increased ER stress in infected cells, leading to autophagy and thereby viral attenuation.