In vitro translation of 42 S virus-specific RNA from cells infected with the flavivirus west nile virus

Abstract

Virus-specific proteins synthesized in BHK cells infected with the flavivirus West Nile (WN) virus and in vitro using the virus-specific infectious 42 S plus-strand RNA as messenger RNA, have been studied. Mapping of the tryptic peptides indicates that the viral core protein V2 and the viral glycoprotein V3 do not share common sequences. No tryptic peptides identifiable by mapping were obtained from the viral membrane-associated protein V1. Seven apparently virus-specific intracellular proteins were detected by comparative SDS-PAGE of mock-infected and virus-infected [ 35S]methionine-labeled cell lysates: p i 15, p i 20, p i 27, p i 37, p i 49, p i 71, and p i 100 (the index i indicates the intracellular origin of the proteins, the number gives the apparent molecular weight in 10 3 daltons). The proteins p i 15 and p i 49 represent the intracellular forms of the viral structural proteins V2 and V3, respectively. p i 15 and V2 are not identical but differ slightly from each other. V1 has not been detected in infected cells. Mapping has shown that the other intracellular proteins (with the possible exception of p i 37, which has not been analyzed) are unrelated to either V2 or V3 and do not share common sequences. They represent nonstructural proteins. The total molecular weight of the apparently unrelated nonstructural and structural proteins is about 290,000 daltons. Data obtained in a number of laboratories have shown that a virus-specific 42 S RNA molecule, which is structurally indistinguishable from the viral genome, probably functions as mRNA for all virus-specific proteins in vivo. This RNA has been isolated from WN virus-infected cells and translated in vitro in the wheat germ and the rabbit reticulocyte lysate system. Digestion of the total [ 35S]methionine-labeled proteins synthesized in vitro in either of both systems with trypsin followed by peptide mapping has shown that the great majority of the resulting peptides are present in the structural proteins V2 and V3 and vice versa. No evidence was obtained for the in vitro synthesis of nonstructural proteins. Proteins synthesized in the reticulocyte lysate were fractionated by SDS-PAGE and isolated polypeptides studied by peptide mapping. Polypeptides of molecular weights between 11,500 and 90,000 daltons were obtained. Their peptide maps indicate that all polypeptides are translated from a single initiation sequence. The map of the smallest in vitro synthesized polypeptide P retic 11.5, having a molecular weight of 11,500 daltons, was almost identical to that of V2. The map of the largest protein synthesized in significant amounts in vitro, the protein P retic 90, was very similar to the map of a mixture of the viral proteins V2 and V3. The analyses suggest the following gene order on the 42 S RNA: 5′-terminus-V2-V3-(V1, p i 20, p i 27, p i 37, p i 71, p i 100)-3′-terminus. The order of genes indicated in brackets remains to be determined. Some implications of these results concerning the possible mode of translation of flavivirus-specific 42 S RNA are discussed.