ANALYSIS OF MUTATIONS WITHIN THE CYTOPLASMIC DOMAIN OF THE MOLONEY MURINE LEUKEMIA-VIRUS TRANSMEMBRANE PROTEIN

The role of the cytoplasmic tail of the Moloney murine leukemia virus transmembrane protein in the regulation of syncytia was examined. Thre e mutations within the cytoplasmic tail were studied. Linker-insertion in7705-12a is within the viral-associated cytoplasmic tail, linker-in sertion in7748-12a is within the R peptide, and a third mutation expre sses TM lacking the R peptide (Env R(-)). The Env R(-) construct was n onviable in Rat1 cells, however, rapidly reverted to a form containing the R peptide when passaged in NIH/3T3 cells. in7705-12a was temperat ure-sensitive in Rat1 cells, as previously characterized, but was viab le at either temperature in NIH/3T3 cells. in7748-12a was comparable w ith wild-type M-MuLV. The ability of the env constructs to form large multinucleated syncytia with NIH/3T3 and XC cells were examined using transient expression assays, eliminating reversion events due to viral passage and reverse transcription. The Env R(-) constructs formed syn cytia with NIH/3T3 cells. in7705-12a displays enhanced proteolytic cle avage of the R peptide. Neither linker-insertion mutation in7705-12a o r in7748-12a activated fusion with NIH/3T3, despite the abundance of p rocessed TM with in7705-12a. All three mutants were fusion competent w ith Rat XC cells, even in the absence of any cleavage of the R peptide . These results provide insights regarding steric and the temporal aff ects of cleavage of the R peptide and the assembly of a fusion compete nt oligomer. (C) 1997 Academic Press