Initiation of vesicular stomatitis virus mutant polR1 transcription internally at the N gene in vitro

Abstract

The vesicular stomatitis virus (VSV) polymerase is thought to initiate transcription of its genome by first copying a small leader RNA complementary to the 3 end of the template. The polR VSV mutants, in contrast to wild-type virus, frequently read through the leader termination site during transcription in vitro. To shed light on polymerase termination and reinitiation events at the crucial leader-N gene junction, we employed RNase protection assays to precisely measure molar ratios of leader, N, and readthrough transcript accumulation in vitro. Wild-type virus synthesized essentially equimolar amounts of leader and N transcripts, but, unexpectedly, the polR1 mutant yielded about twice as much N mRNA as leader (ratio of 1.9 ؎ 0.1). Primer extension assays ruled out an increase in abortive N transcript synthesis for polR1. Transcription entailed multiple rounds of synthesis, with transcript ratios remaining the same after 0.5 or 2 h of synthesis, ruling out a significant contribution from polymerases "pre-positioned" at the N gene. No significant degradation of either leader or N transcripts was observed after incubating purified products with virions. Our data lead us to conclude that transcription can initiate internally at the N gene, at least in the case of polR1 VSV. We propose, however, that productive internal initiation of transcription is a fundamental property of the VSV polymerase and that of related viruses. A model postulating two distinct polymerase complexes, one for leader synthesis and one for internal initiation, is presented.

Reviewing Chandipura: A Vesiculovirus in Human Epidemics

“…Experimental support for this model was gathered when an internal polymerase entry site was described to explain VSV-DI particle synthesis (Schubert et al 1979). Furthermore, a mutant version of polymerase was shown to be able to mediate internal entry at the beginning of N gene in vitro (Chuang and Perrault 1997). While, this model lacks supports of experiments done on wild type virus, it was also unable to explain experimentally observed polar transcription in vesiculoviruses.…”

Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

“…(4) A spontaneous mutation in the VSV N protein (polR) was found that (i) strongly increases vRdRp read-through of the le/N junction, producing incomplete le/N transcripts of various lengths, and (ii) these mutants synthesizes more N mRNA than le RNA in vitro, in contrast to the wild-type reaction (Chuang and Perrault, 1997). This is the best evidence so far that vRdRp can directly initiate the N mRNA in vitro, at least when the N ass of the template is mutated.…”

The Length and Sequence Composition of Vesicular Stomatitis Virus Intergenic Regions Affect mRNA Levels and the Site of Transcript Initiation

“…Although the precise mechanisms responsible for transcript initiation and termination have not been defined, several different models have been proposed over the last two decades to explain certain features of VSV transcription (2). There is some evidence that the viral polymerase may initiate at internal sites on the genome (7,37), and it has been suggested that the polarity observed during VSV transcription is the result of elongation of internally initiated polymerase complexes being dependent on transcription of the upstream gene (37). However, the most widely accepted model is that the viral polymerase initiates transcription de novo from the extreme 3Ј terminus of the genomic RNA (12) and then genes are sequentially transcribed by a start-stop mechanism (12,20).…”