Transcript initiation and 5 '-end modifications are separable events during vesicular stomatitis virus transcription

In this report we describe a novel, bipartite vesicular stomatitis virus (V SV) replication system which was used to study the effect of mutations in t he transcription start sequence on transcript initiation and 5'-mRNA modifi cations. The bipartite replication system consisted of two genomic RNAs, on e of which (VSV Delta G) was a recombinant VSV genome with the G gene delet ed 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 v irus production and gave titers similar to that from wild-type-VSV-infected cells. Mutations were introduced within the first 3 nucleotides of the tra nscription start sequence of the third gene (CAT) of GFC. The effects of th ese changes on the synthesis and accumulation of CAT transcripts during in vivo transcription (e.g., in infected cells), and during in vitro transcrip tion 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 nucle otides (NT-1 and NT-3) reduced CAT transcript levels in vivo to near undete ctable levels. Similarly, changing NT-2 to a purine also resulted in the de tection 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 mut ants produced near-wild-type amounts of CAT mRNA in the in vitro system, in dicating that the mutations did not prevent transcript initiation per se bu t, rather, generated transcripts that were unstable in vivo. Oligo (dT) sel ection and Northern blot analysis revealed that the transcripts produced fr om these mutants did not contain a poly(A)(+) tail and were truncated, rang ing in size from 40 to 200 nucleotides. Immunoprecipitation analysis of cDN A-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 methylat ed. This is the first demonstration that transcript initiation and capping/ methylation are separable events during VSV transcription. A model is propo sed in which polymerase processivity is linked to proper 5'-end modificatio n, The model suggests that a proofreading mechanism exists for VSV and poss ibly dither nonsegmented minus-strand RNA viruses, whereby if some transcri pts do not become capped during transcription in a normal infection, a sign al is transduced such that the polymerase undergoes abortive elongation and the defective transcript is terminated prematurely and subsequently degrad ed.