Multiple Residues Contribute to the Inability of Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human Immunodeficiency Virus Type 1 Isolates

Infection of CD4-positive cells by human immunodeficiency virus type 1 (HIV-1) requires functional interaction of the viral envelope protein with a coreceptor belonging to the chemokine receptor family of seven-membrane-spanning receptors. For the majority of macrophage-tropic HIV-1 isolates, the physiologically relevant coreceptor is the human CCR-5 (hCCR-5) receptor. Although the murine homolog of CCR-5 (mCCR-5) is unable to mediate HIV-1 infection, chimeric hCCR-5/mCCR-5 molecules containing single extracellular domains derived from hCCR-5 are effective coreceptors for certain macrophage-tropic HIV-1 isolates. Here, we have sought to identify residues in hCCR-5 critical for HIV-1 infection by substitution of mCCR-5-derived residues into the context of functional chimeric hCCR-5/mCCR-5 receptor molecules. Using this strategy, we demonstrate that residues 7, 13, and 15 in the first extracellular domain and residue 180 in the third extracellular domain of CCR-5 are important for HIV-1 envelope-mediated membrane fusion. Of interest, certain substitutions, for example, at residues 184 and 185 in the third extracellular domain, have no phenotype when introduced individually but strongly inhibit hCCR-5 coreceptor function when present together. We hypothesize that these changes, which do not preclude chemokine receptor function, may inhibit a conformational transition in hCCR-5 that contributes to HIV-1 infection. Finally, we report that substitution of glycine for valine at residue 5 in CCR-5 can significantly enhance the level of envelope-dependent cell fusion by expressing cells. The diversity of the mutant phenotypes observed in this mutational analysis, combined with their wide distribution across the extracellular regions of CCR-5, emphasizes the complexity of the interaction between HIV-1 envelope and coreceptor.Infection of cells by human immunodeficiency virus type 1 (HIV-1) requires interaction of the viral envelope protein with not only CD4 but also a second cell surface molecule, termed a coreceptor (reviewed in reference 19). Coreceptor usage varies significantly among different HIV-1 isolates, although all known coreceptors are members of the G-protein-coupled chemokine receptor family of seven-membrane-spanning receptors. The primary coreceptor used by non-syncytium-inducing, macrophage-tropic (M-tropic) HIV-1 isolates, which constitute the majority of primary isolates, is CCR-5 (1, 6, 8, 12, 27). In contrast, syncytium-inducing, T-cell-line-adapted (T-tropic) HIV-1 isolates predominantly use CXCR-4 as a coreceptor (13). Other chemokine receptors utilized by a small percentage of generally dualtropic HIV-1 isolates include CCR-2b and CCR-3 (6, 11). The importance of two orphan chemokine receptors, termed Bonzo/STRL33 and BOB/GPR15, in infection by HIV-1 remains to be established, although these proteins were recently shown to serve as coreceptors for several simian immunodeficiency virus and HIV-2 isolates (2, 9). The critical importance of CCR-5 for infection by primary, M-tropic HIV-1 isolates, however, has been highlighted by the finding that a small percentage of humans lack a functional CCR-5 gene and as a result appear highly, although not completely, resistant to infection by HIV-1 (17, 22). Importantly, primary T cells derived from such individuals are refractory to infection by M-tropic HIV-1 isolates in vitro (17, 22, 27), thus demonstrating that CCR-5 is the physiologically relevant coreceptor for the majority of primary isolates.At present, relatively little is known about how the viral envelope and coreceptor interact, although it appears clear that interaction is dependent upon a prior conformational shift induced by binding of the envelope gp120 subunit to CD4 (24, 26). This in turn is believed to lead to the formation of a ternary complex, consisting of gp120, coreceptor, and CD4, on the surface of the target cell (15, 24, 26). It is unknown how this protein complex then induces the fusion of the viral and host cell membranes, although the envelope gp41 subunit is believed to play a critical role at this stage.An important unresolved question is the identity of the amino acid residues in gp120 and the coreceptor that interact during infection. However, it is well established that HIV-1 tropism, and hence coreceptor usage, is largely controlled by a small number of residues located in the envelope V3 loop (6, 14, 23, 25). Efforts to identify residues in the CCR-5 coreceptor involved in mediating infection have thus far largely focused on the functional analysis of chimeric receptors generated with human CCR-5 (hCCR-5) and a chemokine receptor lacking coreceptor function, such as the murine CCR-5 homolog (mCCR-5) (3, 5, 20, 21). These studies have led to three major conclusions. Firstly, the residues in hCCR-5 involved in mediating HIV-1 infection are diffuse, being located on at least three of the four extracellular domains of CCR-5. Secondly, these residues are functionally redundant, so that several distinct regions of hCCR-5 can suffice independently to confer coreceptor function when substituted into mCCR-5. Lastly, different HIV-1 envelope proteins interact differently with CCR-5, such that CCR-5 residues important for mediating fusion by one envelope protein may be largely irrelevant to the interaction of CCR-5 with a second envelope protein. Overall, these data demonstrate that the envelope–CCR-5 interaction is likely to be highly complex and to involve the interaction of multiple residues in both proteins.As noted above, the mCCR-5 chemokine receptor, despite extensive sequence similarity to hCCR-5, fails to function as an HIV-1 coreceptor (3, 5, 20). Therefore, it is apparent that one or more of the 20 extracellular residues that differ between mCCR-5 and hCCR-5 must contribute to the interaction with the HIV-1 envelope protein. Using mutational analysis in the context of chimeric mCCR-5/hCCR-5 receptors, we have now identified several residues, located in three of the four extracellular domains of hCCR-5, that play roles in mediating infection by HIV-1.