N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is extensively glycosylated, containing approximately 23 asparagine (N)-linke d glycosylation sites on its gp120 subunit, In this study, specific glycosy lation sites on gp120 of a dualtropic primary HIV-1 isolate, DH12, were eli minated by site-directed mutagenesis and the properties of the resulting mu tant envelopes were evaluated using a recombinant vaccinia virus-based cell -to-cell fusion assay alone or in the context of viral infections. Of the g lycosylation 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 lo op preferentially inhibited the usage of CCRS; 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 containin g this mutation was severely compromised in peripheral blood mononuclear ce lls, MT-4 cells, and primary monocyte-derived macrophages. A revertant viru s, which acquired second site changes in the V3 loop that resulted in an in crease in net positive charge, was isolated. The revertant virus fully reco vered the usage of CXCR4 but not of CCRS, thereby altering the tropism of t he parental virus from dualtropic to T-tropic, These results suggest that c arbohydrate moieties near the V1/V2 and the V3 loops play critical roles in maintaining proper conformation of the variable loops for optimal interact ion with receptors, Our results, combined with those of previously reported studies, further demonstrate that the function of individual glycans may b e virus isolate dependent.