Mutational analysis of the N-linked glycosylation sites of the SU envelope protein of Moloney murine leukemia virus.
The role of the N-linked glycosylation sites in the major envelope glycoprotein, SU (gp70), of Moloney murine leukemia virus has been examined. By using site-specific oligonucleotide-directed mutagenesis, each of the seven glycan addition sites has been individually eliminated. Mutations resulting in the loss of a single glycosylation site produced, intracellularly, stable precursor SU-TM proteins which were 4 to 5 kDa smaller than the wild-type virus SU-TM protein. Mutant delta 1,4,7, a trimutant lacking three N-linked glycan addition sites, resulted in a viable, infectious virus with a stable SU-TM protein approximately 12 to 15 kDa smaller than the wild-type SU-TM protein. Five of the seven single-site mutations resulted in viable virus as judged by the release of reverse transcriptase in transient-expression assays and XC syncytium assays. Mutations at two of the sites resulted in a detectable phenotype. Virus mutated at position 2 was temperature sensitive in Rat2 cells; viable virus was produced at 32 degrees C but not at 37 degrees C. Virus mutated at position 3 was noninfectious and yielded virions lacking detectable mature SU protein. The mutation results in the block of transport of the protein to the cell surface and assembly into virion particles.
Felkner
R H
RH
Department of Biochemistry, University of Medicine and Dentistry/Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Roth
M J
MJ
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral; D006031:Glycosylation; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D011233:Precipitin Tests; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics* / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism*
D004279:DNA, Viral; D011134:Polysaccharides; D015682:Retroviridae Proteins, Oncogenic; D014759:Viral Envelope Proteins