Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins

Abstract

The genome RNA of the flavivirus West Nile (WN) virus has been transcribed into cDNA, the cDNA has been cloned, and the nucleotide sequences coding for the structural proteins have been determined (Castle et al., 1985; Wengler et al., 1985). We have now determined the nucleotide sequence coding for all viral nonstructural proteins which comprises 7929 nucleotides. Together with our earlier sequence analyses these data show that a long open reading frame (ORF) containing 10,290 nucleotides is present on the genome of WN virus. The two largest nonstructural proteins which can be detected in flavivirus-infected cells are the proteins NV5 and NV4 which have an apparent molecular mass of 97,000 and 74,000 Da, respectively. Both proteins were isolated by preparative polyacrylamide gel electrophoresis, and partial amino acid sequences of peptides derived from these proteins were determined. These analyses allow us to localize the nucleotide regions which code for these proteins and show that the region coding for the NV5 protein is located at the 3'-terminus of the long ORF. Together with our earlier analyses these data show that the protein sequences of virus-specific proteins are present on the viral polyprotein translated from the long ORF in the order V2-NV2-V3-(nonstructural proteins of up to 75,000 Da)-NV4-(nonstructural proteins of up to 45,000 Da)-NV5. Our data indicate that virus-specific structural and nonstructural proteins which are synthesized from a single long ORF accumulate in large amounts in infected cells. A possible role of the presence of these molecules, which are associated to cellular membranes, in the accumulation of membrane vesicles which characteristically occurs in flavivirus-infected cells is discussed.