Abstract
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.