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. Three mutations within the cytoplasmic tail were studied. Linker-insertion in7705-12a is within the viral-associated cytoplasmic tail, linker-insertion in7748-12a is within the R peptide, and a third mutation expresses TM lacking the R peptide (Env R-). The Env R- construct was nonviable in Rat1 cells, however, rapidly reverted to a form containing the R peptide when passaged in NIH/3T3 cells. in7705-12a was temperature-sensitive in Rat1 cells, as previously characterized, but was viable at either temperature in NIH/3T3 cells. in7748-12a was comparable with 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 syncytia with NIH/3T3 cells. in7705-12a displays enhanced proteolytic cleavage of the R peptide. Neither linker-insertion mutation in7705-12a or in7748-12a activated fusion with NIH/3T3, despite the abundance of processed TM with in7705-12a. All three mutants were fusion competent with Rat XC cells, even in the absence of any cleavage of the R peptide. These results provide insights regarding steric and the temporal affects of cleavage of the R peptide and the assembly of a fusion competent oligomer.
Thomas
A
A
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
Gray
K D
KD
Roth
M J
MJ
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D017931:DNA Primers; D017353:Gene Deletion; D015752:Genes, env; D015726:Giant Cells; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000502:physiology; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D015682:Retroviridae Proteins, Oncogenic / Q000096:biosynthesis / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014779:Virus Replication
D017931:DNA Primers; D011994:Recombinant Proteins; D015682:Retroviridae Proteins, Oncogenic; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C078678:p15E protein, Murine leukemia virus