RNA synthesis of vesicular stomatitis virus. X. Transcription and replication by defective interfering particles

Abstract

In cells coinfected by standard vesicular stomatitis virus (VSV) and defective interfering (DI) T particles, small RNA consisting of 46 nucleotides was synthesized in molar excess over other VSV-specific RNAs. Although its rate of synthesis increased over time, small RNA accumulated linearly, suggesting that the molecule is unstable. In contrast, replication of the genome RNA of DI T particles was relatively constant after 3 h of infection, resulting in the intracellular accumulation of stable genomic and antigenomic RNA of DI T particles. Coinfection of cells with DI T particles and selected temperature-sensitive mutants from all five complementation groups of VSV indicated that the replication of DI genomes was controlled separately from the synthesis of small RNA. Also, when viral RNA replication was inhibited by cycloheximide, small RNA continued to be synthesized as long as there were enough templates present. These results indicate that small RNA is synthesized by the enzyme(s) involved in VSV transcription and that its dependence on RNA replication is due to the requirement for template amplification.

Interference among defective interfering particles of vesicular stomatitis virus

“…some trapped RNA. Agarose gel patterns of ted from the RNA obtained from cells infected with only d in various standard VSV have been published (19,34,35). ird VSV to When cells were triply infected with any two r any one of of the DI particles and standard VSV, 40S RNA re effective synthesis was still inhibited, but there were e. This was varying degrees of replication of the genomes of t of intracel-DI particles.…”

“…RNA was harvested at 6 h after infection and separated on a 1.5% agarose gel. (a) DI 0.52 particles at 0 h, 32P at 1 to 6 h; (b) DI 0.52 particles at 0.5 h, 32P at 1.5 to 6 h; (c) cycloheximide at 0 to 1 h, DI 0.52 particles at 0.5 h, 32p at 1.5 to 6 h; (d) DI 0.52 particles at 1 h, 32P at 2 to 6 h; (e) cycloheximide at 0 to 1.5 h, DI 0.52 particles at 1 h, 32p at 2 to 6 h. (34,35). Sma hybridized against unlabeled virio pared from standard VSV or DI well as against total unlabeled intra< from singly or coinfected cells.…”

“…Infection of ceUs and extraction of cytoplasmic RNA. Infection of 4 x 106 cells at 34°C with the indicated multiplicities for each experiment was done essentially as described (9,35). When RNA was examined, the cells were treated with actinomycin D at 5 p.g/ml at the beginning of infection and 0.1 mCi of 32p per ml at the indicated times.…”

“…Cytoplasmic extracts were made with the nonionic detergent Nonidet P-40 (17). Phenolchloroform extractions of the RNA from the cytoplasm were done as described previously (9,35).…”

“…Separation of RNA. For the separation of RNA species ranging from 30 to 12,000 nucleotides, two different gel systems (10%o polyacrylamide and 1.5% agarose) were used as previously described (35). To provide quantitative comparisons of the amounts of RNA, either the autoradiogram was scanned or the bands were excised from the gels and the radioactivity was counted.…”