The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linked glycosylation sites on its gp120 subunit. In this study, specific glycosylation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eliminated by sitedirected mutagenesis and the properties of the resulting mutant envelopes were evaluated using a recombinant vaccinia virus-based cell-to-cell fusion assay alone or in the context of viral infections. Of the glycosylation sites that were evaluated, those proximal to the V1/V2 loops (N135, N141, N156, N160) and the V3 loops (N301) of gp120 were functionally critical. The glycosylation site mutations near the V1/V2 loop compromised the use of CCR5 and CXCR4 equally. In contrast, a mutation within the V3 loop preferentially inhibited the usage of CCR5; although this mutant protein completely lost its CCR5-dependent fusion activity, it retained 50% of the wild-type fusion activity with CXCR4. The replication of a virus containing this mutation was severely compromised in peripheral blood mononuclear cells, MT-4 cells, and primary monocyte-derived macrophages. A revertant virus, which acquired second site changes in the V3 loop that resulted in an increase in net positive charge, was isolated. The revertant virus fully recovered the usage of CXCR4 but not of CCR5, thereby altering the tropism of the parental virus from dualtropic to T-tropic. These results suggest that carbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interaction with receptors. Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may be virus isolate dependent.
Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4ϩ T lymphocytes and cells of monocyte-macrophage lineage. The cellular tropism of HIV-1 is determined largely at the level of virus entry, which depends on a series of interactions between viral envelope glycoprotein and cellular receptors. The gp120 surface glycoprotein subunit is thought to interact first with CD4 (the primary receptor) and then with one (or more) of the recently identified coreceptors, which include chemokine receptors CXCR4 and CCR5 (for reviews, see references 15, 16, 26, and 32). These interactions are thought to trigger conformational changes in the complex multimeric viral envelope glycoprotein structure, allowing the hydrophobic domain of transmembrane glycoprotein gp41 subunit to interact with the cellular membrane and induce viruscell fusion.While the exact mechanism of membrane fusion is still unclear, the interactions between gp120 and cellular receptors are slowly beginning to be understood. The detailed molecular nature of the interactions between gp120 and CD4 has been elucidated from analysis of a crystal structure of gp120 core complexed with CD4 (two N-terminal domains) and the 17b neutralizing monoclonal antibody (Fab fra...