Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSVts110.
We examined the molecular basis for phenotypic reversion in cells infected with a transformation mutant of murine sarcoma virus, MuSVts110. In MuSVts110-infected NRK cells (6m2 cells), the manifestation of the transformed phenotype at 33 degrees C and the normal phenotype at 39 degrees C is governed by thermosensitive splicing of the MuSVts110 primary transcript, a 4.0-kilobase (kb) RNA which contains the gag and mos genes joined out of frame. At 33 degrees C, selectively, the 4.0-kb RNA is processed to a spliced 3.5-kb RNA in which the gag and mos genes are rejoined in a continuous open reading frame, thus allowing synthesis of the P85gag-mos-transforming protein. In contrast, the MuSVts110 revertant cell lines (designated 54-5A4 and 204-3) appear transformed at all growth temperatures from 33 to 39 degrees C and express a P100gag-mos-transforming protein from an apparently unprocessed 4.0-kb viral RNA. In the current study we established both by S1 nuclease analysis and primer extension sequencing that the revertant 54-5A4 and 204-3 4.0-kb viral RNAs suffered a 5-base deletion at the intron-exon border of the 3' splice site. The effect of this deletion is twofold. First, because of the damage to the 3' splice site, the revertant viral 4.0-kb RNAs cannot be processed to the spliced 3.5-kb RNA and, consequently, cannot be translated to P85gag-mos. Second, the 5-base deletion excises an in-frame stop codon positioned at the intron-exon border in the parental RNA and restores the original mos gene reading frame. The net effect is to produce a continuous open reading frame from the gag, alternate mos, and authentic mos gene reading frames which are fused together in the revertant 4.0-kb RNA. This continuous open reading frame can be translated into the P100gag-mos-transforming protein at any growth temperature.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral; D003062:Codon; D004279:DNA, Viral / Q000235:genetics; D015683:Gene Products, gag; D005814:Genes, Viral; D007668:Kidney; D008980:Moloney murine sarcoma virus / Q000235:genetics*; D009857:Oncogenes; D010641:Phenotype; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D051381:Rats; D012191:Retroviridae Proteins / Q000235:genetics*; D009053:Sarcoma Viruses, Murine / Q000235:genetics*; D013489:Suppression, Genetic; D013696:Temperature
D003062:Codon; D004279:DNA, Viral; D015683:Gene Products, gag; D012333:RNA, Messenger; D012367:RNA, Viral; D012191:Retroviridae Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases