Background:  HIV-1 subtype C preferentially utilizes CCR5 co-receptor. A CCR5-to-CXCR4 switch in co-receptor usage occurs frequently among HIV-1 subtype C-infected patients with advanced disease on ART. The molecular changes associated with this switch are not fully understood. The presence of a positively charged amino acid at positions 11 and 25 of the V3 loop of the env gene reliably predicts primary X4- from R5-tropic viruses in subtype B. However, env gene is highly variable, and it is difficult to predict CXCR4 usage based on V3 sequence alone, in non-subtype B viruses. We sought to determine the role of N-linked glycosylation pattern in the V3/V4/V5 regions in co-receptor usage in subtype C viruses.

Methods:  To elucidate the molecular determinants of CCR5-to-CXCR4 switch in co-receptor usage in HIV-1 subtype C, we determined the genotype of the C2-V5 region of the env gene of primary isolates derived from the plasma of 17 HIV-1 subtype C-infected individuals from New Delhi, India. Using web-based protein motif analysis tools, we determined the net charge of the V3 loop and N-linked glycosylation patterns of the V3/V4/V5 regions to predict co-receptor usage. In addition, we analyzed the V3 net charge and N-glycosylation patterns of 20 subtype B and 25 subtype C Env sequences from Zimbabwe available at the Los Alamos HIV database.

Results:  Predicted X4-tropic primary HIV-1 subtype B isolates display a high net positive charge (+5 to +9) in the V3 region and low net N-glycosylation (3) at V3/V4/V5 regions. In contrast, R5-tropic HIV-1 subtype C viruses have a low net positive charge (+2 and +3) and a high net glycosylation (4 to 6). Of 17 subtype C viruses with an intermediate +4 net positive charge, 4 also had a low net N-glycosylation (4) suggesting a switch in co-receptor usage.

Conclusions:  In primary, subtype C isolates from India and Zimbabwe, higher and lower ratio of positive charges in V3 : V3/V4V5 glycosylation sites is associated with CXCR4 and CCR5 tropism, respectively. Loss of N-linked glycosylation sites within the V3/V4/V5 regions in addition to the presence of a positively charged amino acid and an intermediate net positive charge within the V3 loop predicted CXCR4 co-receptor usage in primary HIV-1 subtype C viruses. This may be explained by receptor ligand binding and or immune evasion. N-glycosylation may provide an additional subtype C-specific predictor for characterizing co-receptor usage.