Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
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We examined the mos-specific intracellular RNA species in 6m2 cells, an NRK cell line nonproductively infected with the ts110 mutant of Moloney murine sarcoma virus. These cells present a normal phenotype at 39 degrees C and a transformed phenotype at 28 or 33 degrees C, expressing two viral proteins, termed P85gag-mos and P58gag, at 28 to 33 degrees C, whereas only P58gag is expressed at 39 degrees C. It has been previously shown that 6m2 cells contain two virus-specific RNA species, a 4.0-kilobase (kb) RNA coding for P58gag and a 3.5-kb RNA coding for P85gag-mos. Using both Northern blot and S1 nuclease analyses, we show here that the 3.5-kb RNA is the predominant viral RNA species in 6m2 cells grown at 28 degrees C, whereas only the 4.0-kb RNA is detected at 39 degrees C. During temperature shift experiments, the 3.5-kb RNA species disappears after a shift from 28 to 39 degrees C and is detected again after a shift back from 39 to 28 degrees C. By Southern blot analysis, we have detected only one ts110 proviral DNA in the 6m2 genome. This observation, as well as previously published heteroduplex and S1 nuclease analyses which showed that the 3.5-kb RNA species lacks about 430 bases found at the gag gene-mos gene junction in the 4.0-kb RNA, suggests that the 3.5-kb RNA is a splicing product of the 4.0-kb RNA. The absence of the 3.5-kb RNA when 6m2 cells are grown at 39 degrees C indicates that the splicing reaction is thermosensitive. The splicing defect of the ts110 Moloney murine sarcoma virus viral RNA in 6m2 cells cannot be complemented by acute Moloney murine leukemia virus superinfection, since no 3.5-kb ts110 RNA was detected in acutely superinfected 6m2 cells maintained at 39 degrees C. The spliced Moloney murine leukemia virus env mRNA, however, is found in acutely infected cells maintained at 39 degrees C, suggesting that the lack of ts110 viral RNA splicing at 39 degrees C is not due to an obvious host defect. In sharp contrast, however, 6m2 cells chronically superinfected with Moloney murine leukemia virus produce a 3.5-kb RNA species at 39 degrees C as well as at 28 degrees C and contain proviral DNAs corresponding to the two viral RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Temperature-sensitive viral RNA expression in Moloney murine sarcoma virus ts110-infected cells. 298 39

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.
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PMID:Molecular basis underlying phenotypic revertants of Moloney murine sarcoma virus MuSVts110. 300 54