N-linked glycosylation in the V3 region of HIV type 1 surface antigen modulates coreceptor usage in viral infection

The V3 hypervariable region of HIV-1 surface protein has been identified as a major determinant for viral tropism and coreceptor usage. However, the r ole of the highly conserved N-linked glycan at the V3 loop remains controve rsial. To further examine its role in viral infection, we introduced a cons ervative amino acid substitution (asparagine to glutamine) in the V3-proxim al glycosylation motif (Asn-X-Ser/Thr) in the surface glycoprotein of a CXC R4-using virus (BRU), a CCR5-using virus (SF162), and a dual-tropic virus ( 89.6). The effect of the mutation was determined by complementation assays, and by infectivity on CEMx174 and U373-MAGI cells expressing either CXCR4 or CCR5. The mutation resulted in decreased CXCR4 usage by SHIV89.6, but in creased usage by BRU. Similarly, it abrogated CCR5 usage by SHIV89.6, but h ad no effect on SF162. This effect was not dependent on the specific amino acid substitution used, because a threonine-to-alanine mutation in the same motif in 89.6 Env yielded identical results as the asparagine-to-glutamine mutation. These findings support the notion that multiple factors, includi ng glycosylation at V3, contribute to coreceptor usage and that the particu lar effects exerted by the N-linked glycan itself appear to be isolate depe ndent.