Assignment of the large oligonucleotides of vesicular stomatitis virus to the N, NS, M, G, and L genes and oligonucleotide gene ordering within the L gene

Abstract

Analyses of prototype vesicular stomatitis (VSV, Indiana serotype) mRNA-32Plabeled viral RNA duplexes have established the assignments of 65 of the 72 large oligonucleotides that are recovered by two-dimensional electrophoresis of RNase T1 digests of the viral RNA. Fifty of the oligonucleotides are recovered in the L RNA duplex, four each in the N, M, and NS duplexes, and three in the G RNA duplex. Studies of three small defective-particle RNA species indicate that they have only L gene oligonucleotides in addition to three of the seven unassigned oligonucleotides. Some L gene ordering of oligonucleotides can be postulated from the defective-particle RNA sequence analyses. Analyses of naturally occurring alternate isolates of VSV Indiana have established that by comparison to the prototype virus strain, the alternate isolates minimally have genome sequence differences in L, G, N, NS and/or unassigned regions of the genome. Changes in the genome have also been induced by in vitro high-level mutagenesis of the prototype virus.

Genome complexities of the three mRNA species of snowshoe hare bunyavirus and in vitro translation of S mRNA to viral N polypeptide

“…686 CASH ET AL. Bishop (5). Twenty large T flasks (150 cm2) of confluent BHK-21 cells, pretreated with 5 ,ug of actinomycin D per ml for 1 h, were infected with wild-type SSH virus (multiplicity of infection, 50).…”

“…After a 30min absorption period at 18°C the monolayers were incubated at 33°C for 7.5 h in Eagle medium containing 20 ,tCi of [I'H]uridine per ml, 5 ,ug of actinomycin D per ml, and 5% newborn calf serum to induce the maximum levels of specifically labeled SSH intracellular cRNA. The infected cells were washed and extracted for nucleic acids by a phenol-m-cresol-chloroform mixture, and the majority of the singlestranded RNA species were separated by 1 M NaCl precipitation (5). The nucleic acids in both the 1 M NaCl pellet (representing 70% of the labeled species) and supernatant fluids (representing 30% of the labeled species) were then recovered.…”

“…Annealing 32P-labeled viral and 3H-labeled infected-cell nucleic acids and recovery of doublestranded RNA duplexes. [ 'H]uridine-labeled infected-cell RNA species and individual 0'P-labeled virion RNA species were annealed together, and the RNase resistance was determined by using procedures described elsewhere (5). After determining the optimal conditions for rendering the ['P]RNA RNase resistant by 0.5 h of incubation at 60°C, a preparative annealing mixture reaction was made and annealed at 60°C for 0.5 h, the RNA was digested with nuclease (RNase T2, RNase T,, or pancreatic RNase; see the text below), and the duplexes were recovered by Sephadex G50 or cellulose column chromatography with or without polyacrylamide slab gel electrophoresis as described previously (5).…”

“…Oligonucleotide fingerprint analyses utilized the method of de Watchter and Fiers as modified by Clewley and associates (6). Duplex RNA preparations were heat denatured before RNase T, digestion (5). Resolution of RNA species by polyacrylamide gel electrophoresis.…”

Comparison of ribonucleotide sequences from the genome of vesicular stomatitis virus and two of its defective interfering particles

“…The solution was incubated or 1 h at 37°C. The RNase T1-resistant oligonucleotides were 5'-end labeled in a 50-pl reaction mixture containing 10 mM potassium phosphate (pH 9.5), 10 mM magnesium acetate, 5 mM dithiothreitol, 10,uM [y-3nP]ATP (1,300 to 1,900 Ci/mmol), and 200 U of T4 polynucleotide kinase per ml. After incubation for 1 h at 37°C, 50 1 of 0.04-mg/ml yeast tRNA-0.4 M sodium acetate was added.…”