Variants of human immunodeficiency virus type 1 that efficiently use CCR5 lacking the tyrosine-sulfated amino terminus have adaptive mutations in gp120, including loss of a functional N-glycan.

The human immunodeficiency virus type 1 (HIV-1) infection pathway involves a sequential process whereby reversible binding to the CD4 receptor induces a conformational change in the viral gp120-gp41 trimeric complexes that exposes or induces formation of previously inaccessible epitopes in both gp120 and gp41 and dramatically enhances gp120 affinity for a coreceptor (46, 57, 65, 69, 71, 73, 75). Energetic studies have suggested that the gp120 subunit has a high entropy or conformational flexibility that inhibits binding of antibodies or other ligands and that binding to CD4 substantially reduces this flexibility, thus enhancing the subsequent binding of gp120 to additional ligands by an energetic (entropic) mechanism (36, 47). The sequential reversible binding of gp120 to CD4 and to a coreceptor is believed to lower the activation energy for irreversible conformational changes in the metastable gp41 subunits. These conformational changes pull the virus tightly onto the cell surface and induce fusion of the viral and cellular membranes (9, 24). The gp120 glycoprotein contains an inner core of conserved sequences and several variable loops that are heavily N-glycosylated and that form a protective surface shell (8, 32, 41, 55, 60, 74, 77, 78). X-ray crystallographic studies have revealed the structural organization of the gp120 core complexed with a CD4 amino-terminal domain and the monoclonal antibody (MAb) 17b, which occludes a portion of the coreceptor binding region (37, 38). This gp120 core is folded into inner and outer domains connected by a bridging sheet, with discontinuous residues from these domains forming the CD4 binding site (36-38, 61, 62). Residues in the bridging sheet consisting of conserved region 4 (C4), the V1/V2 base, and the V3 loop comprise a coreceptor binding site (36-38, 45, 61, 62, 70, 77, 78), with the highly variable V3 loop controlling the coreceptor specificity (13, 14, 68, 75). Coreceptors are members of a large family of G-protein-coupled chemokine receptors, with CCR5 and CXCR4 being the principal coreceptors used by HIV-1 in vivo (2, 13, 18, 19, 21, 29). Typically, HIV-1 transmission involves an R5 virus that uses CCR5 (40, 58, 64), whereas X4 variants that can use CXCR4 often form during disease progression (15, 20, 30, 44). Mutagenesis and biochemical studies have demonstrated that the tyrosine-rich and highly acidic amino terminus of CCR5 is essential for binding R5 gp120s and for R5 HIV-1 infections (22, 25, 35, 56, 63). The tyrosines at positions 10, 14, and 15, which are modified posttranslationally by sulfation, appear to be most critical (22, 25, 27, 34, 35). Indeed, sulfated peptides corresponding to the CCR5 amino terminus competitively block R5 HIV-1 infections of cells that contain CCR5 (16, 28) and restore HIV-1 infectivity for cells that express CCR5(Δ18), an amino-terminal deletion mutant of CCR5 lacking amino acids 2 to 19 (26). Additional studies have implicated other regions of CCR5 in coreceptor function, with extracellular loop 2 (ECL2) being most clearly shown (3, 5, 34, 35, 49, 63, 67). MAbs specific for the CCR5 amino terminus block gp120 binding but do not prevent HIV-1 infections, whereas MAbs such as 2D7 that are specific for ECL2 have smaller effects on gp120 binding yet are potent inhibitors of infection (39, 48). In addition, we recently analyzed a G163 residue near the transmembrane 4/ECL2 junction that is critical for HIV-1 infections (34, 67). A G163R substitution, which occurs naturally in African green monkey CCR5, has no effect on simian immunodeficiency virus infections or on CCR5 signaling but potently inhibits binding of R5 HIV-1 gp120 molecules and HIV-1 infections. In agreement with these conclusions, we found that variants of the R5 HIV-1 strain JR-CSF that were selected for replication in HeLa-CD4/CCR5(Y14N) cells were weakly able to use CCR5(Δ18) [or CCR5(Δ16), which lacks amino acids 2 to 17] and were more sensitive than the wild-type virus to inhibition by the G163R mutation in ECL2 (51). These results suggested that the adapted viruses, which contained mutations situated exclusively in the gp120 V3 loop, had a reduced dependency on the CCR5 amino terminus and a correspondingly increased reliance on ECL2 (51). A further implication is that these sites in CCR5 have additive or collaborative roles in infection, so that an absence or defect at one site can be compensated for by increased dependency on the other. We have extended our previous studies by selecting HIV-1JR-CSF variants that efficiently replicate in HeLa-CD4/CCR5(Δ18) or HeLa-CD4/CCR5(Δ16) cell clones that express relatively low concentrations of these mutant coreceptors. The adapted viruses obtained from these stringent selections remained dependent on CCR5 and contained functionally adaptive mutations not only in the V3 loop but also in the V2 base and in a site for N-linked glycosylation in the V4 region of gp120. Surprisingly, removal of this same V4 N-glycan caused HIV-1JR-CSF to become less sensitive rather than more sensitive to the CCR5 ECL2 mutation G163R. Our results suggest that loss of this V4 N-glycan converts HIV-1JR-CSF into a hair-trigger form that is no longer reliant on both the CCR5 amino terminus and ECL2. Rather, the adapted viruses can use the CCR5 amino terminus very efficiently although they are no longer dependent on it.