RNA-PROTEIN INTERACTIONS - INVOLVEMENT OF NS3, NS5, AND 3'-NONCODING REGIONS OF JAPANESE ENCEPHALITIS-VIRUS GENOMIC RNA

The mechanism of replication of the flavivirus Japanese encephalitis v irus (JEV) is not well known, The structures at the 3' end of the vira l genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins, UV cr oss-linking experiments were performed to identify the proteins that b ind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR, The quantit ies of these binding proteins increased during the course of JEV infec tion and correlated with the levels of JEV RNA synthesis in cell extra cts, UV cross-linking coupled,vith Western blot and immunoprecipitatio n analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase, The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins, Furthermore, both pro teins could interact with each other and form a protein-protein comple x in vivo, These findings suggest that the 3' NCR of JEV genomic RNA m ay form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.