Experiments are proposed to study the composition and activity of flavivirus replication complexes using West Nile virus (WNV), strain E101. Flaviviruses are small, plus-strand RNA viruses, that account for a significant amount of human disease worldwide. After uncoating, the genome RNA, which is the only flavivirus mRNA, must first be translated to produce the viral replicase protein(s) needed for viral RNA synthesis. Currently nothing is known about the flavivirus replication complex proteins. Since two large, viral non-structural proteins, ns5 (96K) and ns3 (67K), are encoded in the viral genome, it is possible that the plus and minus strand replication complexes differ. We propose to identify the WNV replication complex proteins after extensive purification of the complexes by gradient centrifugation, column chromatography and immunoprecipitation. Reconstitution experiments will attempt to demonstrate protein function. Host proteins associated with these complexes will also be studied using cross-linking and RNA binding techniques. The involvement of at least one cellular protein in flavivirus RNA replication is indicated by the existence of a murine flavivirus- resistance gene. This resistance is inherited as an autosomal dominant trait. Cells from resistant animals synthesize WNV genomic RNA inefficiently and preferentially amplify viral DI RNAs. Comparison of cellular protein components of the WNV replication complexes obtained from resistant and congenic susceptible mouse cells may lead to identification of the products of the alleles of the flavivirus resistance gene. We also propose to identify possible transcription control signals on the WNV RNA by investigation of 3' and 5' terminal sequence conservation among the genomic RNAs of different flaviviruses and WNV DI RNAs. The function of these signals will be tested using a template-dependent in vitro polymerase assay and genetically engineered RNA transcripts produced from full-length clones of WNV DI RNA. Sequence and functional analysis of a WNV replication-efficient mutant will provide additional information on the regulatory elements involved in WNV transcription.
