Many flaviviruses regularly cause significant human morbidity and mortality. Flavivirus-induced disease ranges from a mild febrile illness to hepatitis, hemorrhagic syndromes, and encephalitis. Vaccines are not available for most flaviviruses. Dengue virus, which can cause severe hemorrhagic disease is spreading northward from the Caribbean basin as the range of the mosquito vectors is extended by global warming. We propose a research project aimed at defining the cis-acting sequences and secondary structure elements of the flavivirus genome. Such signals are essential for regulating the replication, transcription and packaging steps of the virus life cycle. Through comparison of the 3 and 5' sequences of a number of flaviviruses, nine short conserved sequences in the 3' noncoding region and three short conserved sequences in the 5' noncoding region have been identified. Further, terminal secondary structures have been found at both the 3' and 5' ends of the genome, but the two structures differ markedly. The sizes and shapes of these terminal structures are conserved in the absence of sequence conservation in their stem regions. The conserved sequence elements present within the secondary structures are located in loop regions. To date none of these possible cis-acting elements have been functionally tested. Host factors have been identified as essential components of several RNA virus replication complexes. The involvement of at least one cellular protein in flavivirus RNA replication is strongly suggested by the existence of a flavivirus-specific resistance gene in mice. Cells from resistant animals are fully infectible, but synthesize flavivirus genomic RNA inefficiently and preferentially amplify viral DI RNAs. Experiments are proposed to utilize small synthetic viral RNAs to search for high affinity binding cell proteins by gel-shift and UV-induced cross-linking assays. Probes representing the 3' and 5' termini of WNV plus-and-minus-strand RNAs will be tested. Comparison of host factors obtained from congenic resistant and susceptible mouse cells should allow identification of the resistance gene product. Host factors of interest will be sequenced directly or cloned into lambda-gt11. Clones will be screened with host factor-specific antibody and selected clones sequenced. Host factor binding sites on the viral RNA will be mapped.
