Early RNA synthesis in Bunyamwera virus-infected cells

Abstract

RNA synthesis in Bunyamwera virus-infected cells was analysed either by sedimentation analysis in SDS-containing sucrose gradients or by hybridization procedures involving annealing with viral genome RNA (vRNA) followed by electrophoretic analysis. Using either procedure, none of the virus-specific RNAs from infected cells was found to be polyadenylated when analysed by oligo(dT)-cellulose chromatography. In addition, viral messenger RNA activity was found to be associated only with non-polyadenylated RNA species when assayed in an in vitro translation system. The infected cell RNAs could be partially resolved by sucrose gradient centrifugation, and virus-specific RNAs of each polarity were present in these preparations which indicated that the characteristic amplification of secondary transcription was occurring. In the presence of cycloheximide or puromycin, no detectable primary RNA transcription occurred. The same inhibitors, when used later in the infection cycle, caused a dramatic and almost complete inhibition of secondary RNA transcription. The inhibition of RNA synthesis caused by these drugs appeared to be fully reversible. Thus, these inhibitors of protein synthesis affect both primary and secondary RNA transcription by Bunyamwera virus indicating that this virus employs transcription mechanisms different from those known for other families of negative-stranded viruses. Hybridization of 32P-labelled vRNA from Bunyamwera virus with RNA extracted from virus-infected cells produced four duplex RNA molecules that were resolved by gel electrophoresis. Analysis by hybridization and oligonucleotide mapping showed that the two larger duplexes contained complementary (c)RNAs that were transcribed from the L and M segments of viral RNA while the cRNAs contained in the two smaller duplexes were both transcribed from the S RNA segment. Based on a comparison of their oligonucleotide fingerprints, the two latter cRNAs showed a considerable sequence overlap.