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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.

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