Function of N-linked glycans on the human immunodeficiency virus envelope glycoprotein (Immune deficiency).

摘要

Recombinant vesicular stomatitis viruses (rVSVs) expressing human immunodeficiency virus-1 (HIV-1) envelope (Env) glycosylation mutants were generated to determine if removal of carbohydrates in Env affected protein function or the generation of neutralizing antibodies in mice. Mutations that eliminated one to six of the sites for N-linked glycosylation in the V1 and V2 domains were introduced into a gene (89.6G) encoding the primary isolate HIV-1 89.6 Env with its cytoplasmic domain replaced with that of the VSV G glycoprotein.; No single mutation affected membrane fusion activity. However, mutation of multiple sites caused progressive reduction in syncytia formation to the point where a mutant lacking all six glycans was completely defective in membrane fusion, although it retained CD4 binding activity. The mutant proteins were transported to the cell surface and incorporated into VSV virions at similar levels, although there were significant differences in the rate and extent of cleavage for mutants lacking four or six glycans, suggesting altered folding. We found that HIV Env glycosylation mutants in V1 and V2 were no better than wild type Env at inducing antibodies that neutralized wild-type Env. However, the mutant Env proteins did induce slightly higher titers to mutant Env than did wild-type Env.; Finally, we investigated the stability and mechanisms of loss of foreign gene expression in two rVSVs. One recombinant expressing the cellular CD4 protein exhibited remarkable stability of foreign gene expression. Sequencing of the CD4 mRNA in a virus no longer expressing CD4 revealed a single nucleotide deletion causing a frameshift and termination of protein synthesis. A second VSV recombinant expressing the measles virus F protein grew poorly and exhibited extreme instability of expression of the F protein. Expression of F was lost through mutation of the upstream transcription termination site, which fused the F mRNA to the 3′end of the G mRNA. We suggest that the latter mechanism may provide a rapid means for complete elimination of expression of proteins that are toxic to the virus.