Biomedical subjects

E A Stillman

Publications and source records attributed to E A Stillman.

5 recordsLinked to original sources

PubMed1999

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 (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 (VSVDeltaG) 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.

Animals↗

PubMed1998

The length and sequence composition of vesicular stomatitis virus intergenic regions affect mRNA levels and the site of transcript initiation.

In this study, we used a dicistronic vesicular stomatitis virus (VSV) minigenome to investigate the effects of either single or multiple nucleotide insertions placed immediately after the nontranscribed intergenic dinucleotide of the M gene on VSV transcription. Both Northern blot and primer extension analysis showed that the polymerase responded to the inserted nucleotides in a sequence-specific manner such that some insertions had no effect on mRNA synthesis from the downstream G gene, nor on the site of transcript initiation, whereas other insertions resulted in dramatic reductions in transcript accumulation. Some of these transcripts were initiated at the wild-type site, while others initiated within the inserted sequence. We also examined the transcriptional events that occurred when a natural, 21-nucleotide intergenic region located between the G and L genes from the New Jersey (NJ) serotype of VSV was inserted into the minigenome gene junction. In contrast to the normal 25 to 30% attenuation observed for downstream transcription at gene junctions containing the typical dinucleotide (3'-GA-5') intergenic region, the NJ variant showed greater than 75% attenuation at the gene junction. In addition, the polymerase initiated transcription at two major start sites, one of which was located within the intergenic sequence. Collectively, these data suggest that the polymerase "samples" the intergenic sequences following polyadenylation and termination of the upstream transcript by scanning until an appropriate start site is found. One implication of a scanning polymerase is that the polymerase presumably switches states from a processive elongation mode to a stuttering mode for polyadenylation to one in which no transcription occurs, before it reinitiates at the downstream gene. Our data support the hypothesis that sequences surrounding the intergenic region modulate these events such that appropriate amounts of each mRNA are synthesized.

Base Composition↗

PubMed1997

Mutational analyses of the intergenic dinucleotide and the transcriptional start sequence of vesicular stomatitis virus (VSV) define sequences required for efficient termination and initiation of VSV transcripts.

We have used dicistronic vesicular stomatitis virus (VSV) minigenomes to dissect the functional importance of the nontranscribed intergenic dinucleotide and the conserved transcription start sequence found at the beginning of all VSV genes. The minigenomes were generated entirely from cDNA and contained the G and M protein genes, flanked by the leader and trailer regions from the Indiana serotype of VSV. All mutations were made either within the nontranscribed M-G intergenic dinucleotide or within the transcription start sequence of the downstream G gene. Immunofluorescence microscopy and immunoprecipitation analysis of the mutated minigenomes indicated that the first three nucleotides of the transcriptional start sequence are the most critical for efficient VSV gene expression, whereas the nontranscribed, intergenic dinucleotide and the other conserved nucleotides found at the 5' mRNA start sequence can tolerate significant sequence variability without affecting G protein production. RNA analysis indicated that nucleotide changes in the transcriptional start sequence which resulted in reduced G protein expression correlated with the amount of transcript present. Therefore, this conserved sequence appears to be required for efficient transcript initiation following polyadenylation of the upstream mRNA. While the minimum sequence for efficient transcription (3'-UYGnn-5') is similar to that of other rhabdoviruses, it is not homologous to the start sites for viruses from the Paramyxoviridae or Filoviridae families. Using Northern blot analysis, we also found that some nucleotide changes in the nontranscribed intergenic region resulted in higher levels of read-through transcription. Therefore, the nontranscribed intergenic dinucleotide plays a role in transcript termination.

Animals↗

PubMed1995

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Animals↗

PubMed1995

Replication and amplification of novel vesicular stomatitis virus minigenomes encoding viral structural proteins.

We have developed a system in which vesicular stomatitis virus (VSV) minigenomes encoding viral structural proteins can be expressed from plasmids. These RNAs can be replicated, transcribed, and packaged into infectious particles when coexpressed with the other VSV proteins. The minigenomes contain either the glycoprotein (G protein) gene (GMG [stands for G minigenome]) or both the G and matrix (M) protein genes (GMMG [stands for G/M minigenome]) from the Indiana serotype of VSV flanked by the trailer and leader regions from the wild-type VSV genome. Northern (RNA) blot analysis showed that the minigenome RNAs were replicated and that a positive-sense replicative intermediate was synthesized when coexpressed with the nucleocapsid (N) protein and the two VSV polymerase proteins (phosphoprotein [P] and the large catalytic subunit [L]) in vivo. In addition, functional mRNAs were transcribed from the minigenome templates, and the appropriate encoded proteins were expressed. Expression of the G and M proteins from GMMG resulted in the assembly and release of infectious particles that could be passaged on cells expressing the N, P, and L proteins only. Amplification occurred during successive passages, and after four passages approximately 30% of the cells expressed both the G and M proteins. Analysis of the RNAs produced in the GMMG-infected cells also showed that the minigenomes accurately reproduced all of the replicative and transcriptional events that normally occur in a VSV-infected cell. GMMG is therefore a novel type of defective particle which encodes functional viral proteins critical to its own propagation.

Animals↗