Polyadenylation of vesicular stomatitis virus mRNA dictates efficient transcription termination at the intercistronic gene junctions.
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'AUACU(7)5' 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 AUACU(7) sequence element is required for polyadenylation of VSV mRNA. Deletion of AUAC or U(7) 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 AUACU(7) 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.
Hwang
L N
LN
Department of Microbiology and Immunology, University of Miami School of Medicine, Florida 33136, USA.
Englund
N
N
Pattnaik
A K
AK
D000241:Adenosine; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D005814:Genes, Viral; D016679:Genome, Viral; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D019251:Nucleocapsid / Q000235:genetics*; D019590:Nucleocapsid Proteins; D010443:Peptide Chain Termination, Translational; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D019590:Nucleocapsid Proteins; C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D015678:Viral Structural Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D000241:Adenosine