Title : 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
Abstract :
- 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 site-directed 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