Moloney murine sarcoma virus MuSVts110 DNA: cloning, nucleotide sequence, and gene expression

We have cloned Moloney murine sarcoma virus (MuSV) MuSVts110 DNA by assembly of polymerase chain reaction (PCR)-amplified segments of integrated viral DNA from infected NRK cells (6m2 cells) and determined its complete sequence. Previously, by direct sequencing of MuSVts110 RNA transcribed in 6m2 cells, we established that the thermosensitive RNA splicing phenotype uniquely characteristic of MuSVts110 results from a deletion of 1,487 nucleotides of progenitor MuSV-124 sequences. As anticipated, the sequence obtained in this study contained precisely this same deletion. In addition, several other unexpected sequence differences were found between MuSVts110 and MuSV-124. For example, in the noncoding region upstream of the gag gene, MuSVts110 DNA contained a 52-nucleotide tract typical of murine leukemia virus rather than MuSV-124, suggesting that MuSVts110 originated as a MuSV-helper murine leukemia virus recombinant during reverse transcription rather than from a straightforward deletion within MuSV-124. In addition, both MuSVts110 long terminal repeats contained head-to-tail duplications of eight nucleotides in the U3 region. Finally, seven single-nucleotide substitutions were found scattered throughout MuSVts110 DNA. Three of the nucleotide substitutions were in the gag gene, resulting in one coding change in p15 and one in p30. All of the remaining nucleotide changes were found in the noncoding region between the 5' long terminal repeat and the gag gene. In NIH 3T3 cells transfected with the cloned MuSVts110 DNA, the pattern of viral RNA expression conformed with that observed in cells infected with authentic MuSVts110 virus in that viral RNA splicing was 30 to 40% efficient at growth temperatures between 28 and 33 degrees C but reduced to trace levels above 37 degrees C.