Transcript Initiation and 5′-End Modifications Are Separable Events during Vesicular Stomatitis Virus Transcription

Abstract

In this report we describe a novel, bipartite vesicular stomatitis virus (VSV) replication system which was used to study the effect of mutations in the transcription start sequence on transcript initiation and 5-mRNA modifications. The bipartite replication system consisted of two genomic RNAs, one of which (VSV⌬G) was a recombinant VSV genome with the G gene deleted and the other (GFC) contained the G gene and two non-VSV reporter genes (green fluorescent protein [GFP] and chloramphenicol acetyltransferase [CAT]). Coinfection of cells with these two components resulted in high-level virus production and gave titers similar to that from wild-type-VSV-infected cells. Mutations were introduced within the first 3 nucleotides of the transcription start sequence of the third gene (CAT) of GFC. The effects of these changes on the synthesis and accumulation of CAT transcripts during in vivo transcription (e.g., in infected cells), and during in vitro transcription were determined. As we had reported previously (E. A. Stillman and M. A. Whitt, J. Virol. 71:2127-2137, 1997), changing the first and third nucleotides (NT-1 and NT-3) reduced CAT transcript levels in vivo to near undetectable levels. Similarly, changing NT-2 to a purine also resulted in the detection of very small amounts of CAT mRNA from infected cells. In contrast to the results in vivo, the NT-1C mutant and all of the second-position mutants produced near-wild-type amounts of CAT mRNA in the in vitro system, indicating that the mutations did not prevent transcript initiation per se but, rather, generated transcripts that were unstable in vivo. Oligo (dT) selection and Northern blot analysis revealed that the transcripts produced from these mutants did not contain a poly(A) ؉ tail and were truncated, ranging in size from 40 to 200 nucleotides. Immunoprecipitation analysis of cDNA-RNA hybrids with an antibody that recognizes trimethylguanosine revealed that the truncated mutant transcripts were not properly modified at the 5 end, indicating the transcripts either were not capped or were not methylated. This is the first demonstration that transcript initiation and capping/methylation are separable events during VSV transcription. A model is proposed in which polymerase processivity is linked to proper 5-end modification. The model suggests that a proofreading mechanism exists for VSV and possibly other nonsegmented minus-strand RNA viruses, whereby if some transcripts do not become capped during transcription in a normal infection, a signal is transduced such that the polymerase undergoes abortive elongation and the defective transcript is terminated prematurely and subsequently degraded.

Reviewing Chandipura: A Vesiculovirus in Human Epidemics

“…Presence of core promoter element associated with each transcription unit was further investigated by mutagenic analysis of bipartite minigenome replicon to identify 3¢-UYG-5¢ tri-nucleotide at the beginning of each five protein coding genes as necessary element for transcription initiation and capping of the mRNA (Stillman and Whitt 1999). In this regard, vesiculovirus uses promoter elements that are downstream of initiation site and resembles with downstream promoter elements found within mammalian transcriptional repertoire (Kadonaga 2002).…”

“…However, unlike cellular guanylyltransferase, L protein incorporates GDP rather than GMP in the capped structure as Gp(alpha)p(beta)-p(alpha)A. The 5¢end modification events were proposed to be successive to transcription initiation, whereby, nascent mRNA termini maintains contact with transcribing polymerase until modified (Stillman and Whitt 1999). Notably, leader RNA lacks 5¢ cap structure.…”

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

“…SeV and VSV RdRp become processive independent of nascent chain assembly upon initiation (and capping) of the N mRNA, and they do so while retaining their ability to respond to stutter signals. How this processivity is acquired is unknown, but work with VSV has indicated that processivity is acquired only after the modification of the nascent mRNA 5Vend when it is approximately 50 nt long (Stillman and Whitt, 1999). Similarly, VSV RdRp does not respond to a polyA/stop site within the first 50 nucleotides of the gene-start site (Whelan et al, 2000).…”

mRNA Cap Methylation Influences Pathogenesis of Vesicular Stomatitis VirusIn Vivo

“…Following cap formation, VSV mRNAs are sequentially methylated at the ribose 2=-O position and G-N-7 position (8,21), an order which is opposite that of other known methylation reactions. Unlike traditional cap-forming enzymes (22), the VSV capping and methylation machinery require cis-acting signals in the RNA (8,(23)(24)(25) and a minimal nascent RNA chain length of 31 nucleotides (nt) (26). In response to a gene end sequence, L polyadenylates and terminates mRNA synthesis by the programmed stuttering of polymerase on a U7 tract (27).…”