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J Virol, March 1998, p. 1805-1813, Vol. 72, No. 3
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Polyadenylation of Vesicular Stomatitis Virus mRNA
Dictates Efficient Transcription Termination at the Intercistronic
Gene Junctions
Leroy N.
Hwang,
Nathan
Englund, and
Asit K.
Pattnaik*
Department of Microbiology and Immunology,
University of Miami School of Medicine, Miami, Florida 33136
Received 23 September 1997/Accepted 12 November 1997
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.
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, University of Miami School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33136. Phone: (305) 243-6711. Fax:
(305) 243-4623. E-mail:
apattnaik{at}mednet.med.miami.edu.
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