Polyadenylation of vesicular stomatitis virus mRNA dictates efficient transcription termination at the intercistronic gene junctions

Leroy N. Hwang, Nathan Englund, Asit K. Pattnaik

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

The intercistronic gene junctions of vesicular stomatitis virus (VSV) contain conserved sequence elements that are important for polyadenylation and transcription termination of upstream transcript as well as reinitiation of transcription of downstream transcript. To examine the role of the putative polyadenylation signal 3'AUACU75' at the gene junctions in polyadenylation and transcription termination, we constructed plasmids encoding antigenomic minireplicons containing one or two transcription units. In plasmid-transfected cells, analyses of the bicistronic minireplicon containing the wild-type or mutant intercistronic gene junctions for the ability to direct synthesis of polyadenylated upstream, downstream, and readthrough mRNAs showed that the AUACU7 sequence element is required for polyadenylation of VSV mRNA. Deletion of AUAC or U7 resulted in templates that did not support polyadenylation of upstream mRNA. Interestingly, we found that the loss of polyadenylation function led to antitermination of the upstream transcript and resulted in a readthrough transcript that contained the upstream and downstream mRNA sequences. Mutations that blocked polyadenylation also blocked transcription termination and generated mostly readthrough transcript. Reverse transcription-PCR of readthrough transcripts and subsequent nucleotide sequencing of the amplified product revealed no extra adenosine residues at the junction of the readthrough transcript. These results indicate that polyadenylation is required for transcription termination of VSV mRNA. The intergenic dinucleotide GA did not appear to be necessary for transcription termination. Furthermore, we found that insertion of the polyadenylation signal sequence AUACU7 alone was sufficient to direct polyadenylation and efficient transcription termination at the inserted site. Taken together, the data presented here support the conclusion that polyadenylation is the major determinant of transcription termination at the intercistronic gene junctions of VSV.

Original languageEnglish (US)
Pages (from-to)1805-1813
Number of pages9
JournalJournal of virology
Volume72
Issue number3
StatePublished - Mar 1 1998

Fingerprint

Vesiculovirus
Vesicular Stomatitis
Polyadenylation
transcription (genetics)
Viruses
Messenger RNA
Genes
genes
plasmids
RNA 3' Polyadenylation Signals
Plasmids
conserved sequences
adenosine
Conserved Sequence
signal peptide
Protein Sorting Signals
Adenosine
Reverse Transcription
nucleotides
reverse transcriptase polymerase chain reaction

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Polyadenylation of vesicular stomatitis virus mRNA dictates efficient transcription termination at the intercistronic gene junctions. / Hwang, Leroy N.; Englund, Nathan; Pattnaik, Asit K.

In: Journal of virology, Vol. 72, No. 3, 01.03.1998, p. 1805-1813.

Research output: Contribution to journalArticle

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