A suboptimal src 3' splice site is necessary for efficient replication of Rous sarcoma virus.
Regulation of splicing of Rous sarcoma virus (RSV) RNA primary transcripts is necessary, as with other retroviruses, to allow for the accumulation of unspliced RNA and approximately equivalent amounts of spliced env and src mRNAs. Previous studies have indicated that the env 3' splice site is suboptimal because it has a nonconsensus branchpoint sequence and that this suboptimal splice site is required for virus replication (R. A. Katz and A. M. Skalka, 1990, Mol. Cell Biol. 10:696-704). We show in this report that the RSV src 3' splice site is also suboptimal. Mutagenesis of the src polypyrimidine-rich tract, which is interspersed with purines, to an uninterrupted 14-nt pyrimidine tract resulted in a three- to fourfold increase in the level of src splicing. Concomitant with this increase in src splicing, a cryptic 5' splice site within the env gene was activated. Splicing at this splice site is normally detected in nonpermissive mammalian cells were src splicing is elevated but occurs at low levels in permissive chicken embryo fibroblasts (CEF). In CEF, mutant viruses with the improved src 3' splice site replicated significantly slower than the wild-type virus. Transformation-defective revertants lacking the src 3' splice site were rapidly selected after passage of the chicken cells infected with the mutant virus. Thus, an inefficient src 3' splice site appears to be necessary for the efficient replication of RSV.
Department of Microbiology, University of Iowa, Iowa City 52242.
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D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D017931:DNA Primers; D018076:DNA, Complementary; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral; D016391:Genes, src; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis*; D014162:Transfection; D014779:Virus Replication
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D012367:RNA, Viral