Sequences of vesicular stomatitis virus RNA in the region coding for leader RNA, N protein mRNA, and their junction.

Abstract

The RNAs extracted from purified preparations of the Indiana and New Jersey serotypes of vesicular stomatitis virus were polyadenylylated in vitro by using polynucleotide phosphorylase and sequence determination was carried out by the dideoxynucleotide method using reverse transcriptase and dTSAC primer. On both virus RNAs a short stretch of adenylic acid residues is present between the regions coding for the leader and N protein mRNAs. Other features of the RNA sequences of the two viruses are compared to each other and to published data.Vesicular stomatitis virus (VSV) RNA is a molecule of molecular weight 4 X 106 and has negative polarity. Expression of the genetic information encoded in the virion RNA proceeds via sequential primary transcription from the 3' end of virion RNA of a short nonpolyadenylylated leader sequence followed by polyadenylylated mRNAs corresponding to the five virusspecific proteins (N, NS, M, G, and L) induced in cells during infection. The gene order on the virion is 3'-N-NS-M-G-L-5' (1, 2). The nucleotide sequences of the leader RNAs of both VSV Indiana and New Jersey serotypes have been determined by the analysis of partial ribonuclease digestion products after in vitro 32p labeling of the 5' end with polynucleotide kinase or of the 3' end with RNA ligase (3, 4). Confirmation that the sequence of the VSV Indiana leader RNA is complementary to the 3'-terminal sequence of the virus RNA, at least up to 17 bases from the 3' terminus, has been obtained by sequence determination of virus RNA labeled at the 3' end with RNA ligase (5). The sequence of nucleotides in the ribosome binding sites of three of the VSV Indiana mRNAs, including N mRNA has been determined (6). Because the protected sequences of N and M mRNAs contain the cap, they must include data available spanning the region between the end of the leader RNA and the start of the first mRNA sequence.Recently, application of the dideoxynucleotide method of sequence determination by introducing base-specific stops in cDNA (7) has been successfully employed to obtain extensive sequence data of the regions adjacent to the poly(A) tract in two naturally polyadenylylated virus RNAs, encephalomyocarditis virus RNA (8) and poliovirus RNA (9), and of globin mRNA (10). This paper reports extended sequence information on the 3' end of VSV RNA, including the leader/N mRNA junction, obtained by the dideoxynucleotide method, using as template RNA that had been polyadenylylated in vitro with polynucleotide phosphorylase. MATERIALS AND METHODSViruses and RNAs. VSV of serotypes Indiana (MuddSummers strain) and New Jersey (Ogden) was grown in baby hamster kidney cells (BHK-21) infected with cloned virus stocks and incubated with Eagle's minimal essential medium supplemented with 7% calf serum. The virus was purified as described by Doyle and Holland (11) and the RNA was extracted as described by Perrault (12) and purified by sedimentation through a 5-25% sucrose gradient in 0.2 M NaCl/50 mM Tris-HCI/5 mM EDTA/0.1% Sarkosyl at pH 7.4 (NTE/Sa...

Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN and L proteins

“…Consensus sequences similar to Rl and R2 were also reported for non-segmented negative stranded RNA viruses, i.e. VSV (43,44), respiratory syncytial virus (RSV) (45) and measles virus (46), suggesting their common importance in the transcription and/or replication process of these viruses. It is interesting to note that the sequence, GAA or GGG, which was found between two adjacent genes (or between R2 and RI) and thought to be an intergenic sequence (3,4), was detected after R2 of the L gene.…”

Transcription and replication of rhabdoviruses.

“…2. One striking feature is the junction betwen the leader template and the N gene which consists of only three A residues (117,137,138,188) or four A residues in the New Jersey serotype of VSV (117). Thus, the question of how and why the RNA polymerase precisely stops at the end of the leader template without transcribing the three A residues remains an enigma.…”

“…These observations led to further studies to understand the role of individual proteins in the RNA-synthetic process and the mechanism of RNA transcription in vitro. Recently, using recombinant deoxyribonucleic acid (DNA) technology, each of the mRNAs coding for the viral proteins has been molecularly cloned, and through the work of several laboratories, the nucleotide sequences have been determined (53,80,137,138,184,185,188,194,198). In fact, the complete nucleotide sequences of the genome RNA of the Indiana serotype of VSV and of half of the genome RNA of the New Jersey serotype of VSV are now known (18,55,81,84).…”

Stable hairpin structure within the 5'-terminal 85 nucleotides of poliovirus RNA

“…The 35S peak was again ethanol precipitated, and polyadenylic acid-containing RNA was selected by using oligodeoxythymidylic acid-cellulose (Collaborative Research type III) as previously described (42). Polyadenylic acid-containing RNA was then purified by zonal centrifugation, and 35S RNA was selected and precipitated.…”