Unique mode of transcription in vitro by vesicular stomatitis virus

Abstract

In addition to the five mRNA species and 47 nucleotide long leader RNA synthesized by purified virions of vesicular stomatitis virus, at least three discrete low molecular weight RNA species having approximate chain lengths of 28, 42 and 70 nucleotides can be detected in vitro. Each of these RNA species displays a unique and characteristic T1 fingerprint profile and contains (p)ppAA as its 5' terminus. By partial sequence analyses, two of the small RNA products, 42 and 28 bases long, were found to contain 5' terminal sequences identical to those in the N and NS mRNAs, respectively. Ultraviolet inactivation studies demonstrate that each of these RNA species has a target size in agreement with its molecular weight indicating independent initiation. Kinetic studies show that the small RNA species are synthesized within 1 min, while mRNA chain completion occurs later in the sequential order N-NS-M-G. These results indicate that viral mRNA synthesis occurs in vitro by multiple initiations at different promoter sites on the genome RNA, and that the elongation and completion of the individual mRNAs depend on prior transcription of 3' proximal genes. We present a model for viral mRNA synthesis in vitro.

Interaction of L and NS Proteins of Vesicular Stomatitis Virus with its Template Ribonucleoprotein during RNA Synthesis in vitro

“…Several models were proposed for this sequential mode of VSV mRNA transcription in vitro (Banerjee et al, 1977). Some recent observations have suggested a model (Testa et al, 1980) in which the transcriptase is located at putative multiple promoter sites on the template RNP and initiates RNA chains. RNA chain elongation, however, occurs only after its T-proximal gene is transcribed.…”

“…Each RNA species was eluted from the gel, quantified, and identified by fingerprint analyses. The bands designated leader RNA, RNA I and RNA III were confirmed to be leader RNA, small initiated N mRNA and NS mRNA, respectively, as described previously (Testa et al, 1980). RNA II was confirmed to be the G-start RNA species of 28 bases, synthesized within the N gene as observed by Schubert et al (1982) and RNA IV, which is a mixture of three distinct bands, was confirmed to be the initiated N mRNA sequence of chain lengths 11 to 14 as previously observed by Pinney & Emerson (1982a) (data not shown).…”

“…We next studied the effect of arabinosyl ATP (ara-ATP) on the synthesis of RNA and the small initiated mRNA sequences during reconstitution (Chanda & Banerjee, 1980). The RNA synthesized at two different concentrations of ara-ATP (0.25 and 0.5 raM) was analysed by polyacrylamide gel electrophoresis.…”

“…Reconstitution reactions were carried out as described in Fig. 1 except that ara-ATP was included in the reaction mixture: (a) no ara-ATP, (b) 0-25 mM-ara-ATP, (c) 0.5 mM-ara-ATP (Chanda & Banerjee, 1980). Similar transcription reactions were also carried out using RNP purified from Triton-disrupted virus in the absence (d), or in the presence of 0.25 mM-ara-ATP (e), or 0-5 mM-ara-ATP (f).…”

Structural Analysis of Animal Virus Genomes: The Fifth Fleming Lecture

“…The details of how NS mRNA transcription starts are not known, that is whether the new RNA is generated by a nucleolytic processing event, a stop/start reinitiation, or an initiation by a separate polymerase molecule. No one current model satisfactorily encompasses all observations (Ball & White, 1976;Herman et al, 1978;Testa et al, 1980). We do know the start position of the new mRNA, and that it is capped and methylated by some part of the virus's transcription apparatus.…”