No abstract

HIV-1 entry into CD4 ؉ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Hepatitis C virus (HCV) envelope glycoproteins E1͞E2 can pseudotype retroviral particles and efficiently mediate entry into target cells. Using this experimental system, we determined HCV tropism for different cell types. Only primary hepatocytes and one hepatoma cell line were susceptible to HCV pseudovirus entry, which could be inhibited by sera from HCV-infected individuals. Furthermore, expression of the putative HCV receptor CD81 on nonpermissive human hepatic but not murine cells enabled HCV pseudovirus entry. Importantly, inhibition of viral entry by an anti-CD81 mAb occurred at a step following HCV attachment to target cells. Our results indicate that CD81 functions as a postattachment entry coreceptor and that other cellular factors act in concert with CD81 to mediate HCV binding and entry into hepatocytes.I t is estimated that 170 million people worldwide are infected with the hepatitis C virus (HCV) and are at risk of developing chronic hepatitis or cirrhosis, the latter often leading to hepatocellular carcinoma (1, 2). In the past, difficulties with culturing the virus and expressing fusogenic envelope glycoproteins limited studies of HCV tropism and entry. RT-PCR-and electron microscopy-based approaches were relied on to demonstrate the presence of HCV RNA and proteins in primary hepatocytes and certain hepatoma cell lines (3-8). Furthermore, the existence of extrahepatic HCV reservoirs was suggested by the detection of viral RNA in serum and peripheral blood mononuclear cells (PBMC) (9-11). Recently, a major technical advance in the field has been the discovery that unmodified HCV envelope glycoproteins can pseudotype retroviral particles and mediate entry into target cells (12)(13)(14)(15). This model seems to authentically replicate the early steps of the HCV life cycle, enabling detailed studies of HCV tropism and entry into target cells.The cellular tropism of enveloped viruses is largely determined by selective interactions of viral envelope glycoproteins with specific cell-surface receptors. Entry generally proceeds by a cascade of coordinated events wherein virus binding to a host molecule triggers exposure of cryptic envelope glycoprotein domains that mediate downstream interactions and functions. We and others recently demonstrated that DC-SIGN (dendritic cell-specific ICAM-3 grabbing nonintegrin; CD209) and L-SIGN (DC-SIGNR, liver and lymph node specific; CD209L) function as HCV capture receptors but do not mediate viral entry into target cells (16,17). Candidate HCV entry receptors include CD81, scavenger receptor class B type 1, low density lipoprotein receptor, and glycosaminoglycans (18)(19)(20). CD81 is the most extensively characterized putative HCV receptor. A number of groups have demonstrated that the soluble ectodomain of HCV envelope glycoprotein E2 binds specifically and with relatively high affinity (K d Ϸ10 Ϫ8 M) to human and chimpanzee CD81 (21-24). However, CD81 is widely expressed on human cells and therefore cannot account for the restricted tropism of HCV to hepatocytes...

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.Entry of human immunodeficiency virus type 1 (HIV-1) R5 isolates into target cells is mediated by the successive interaction of the envelope glycoprotein gp120 with CD4 and the CCR5 coreceptor (for a review see reference 1). A cluster of negatively charged and sulfotyrosine residues within the CCR5 amino-terminal domain (Nt) are essential for CCR5-mediated fusion and entry of most HIV-1 R5 isolates (for a review see reference 10). Usage of other residues in the CCR5 Nt occurs in an isolate-dependent manner (10). Recently, it has been shown that HIV-1 JRCSF can adapt to use CCR5 lacking the Nt (22). The mutant HIV-1 JRCSF isolate has an increased affinity for the second extracellular loop (ECL2) of CCR5. Studies with anti-CCR5 monoclonal antibodies (MAbs) and low-molecular-weight inhibitors indicate that ECL2 epitopes are important for gp120 binding to CCR5 and for viral entry (7, 10). We have recently identified a cluster of ECL2 residues that are essential for the entry of non-clade B isolates (28). Residues dispersed throughout ECL1 and ECL3 of CCR5 have also been implicated in HIV-1 entry into target cells (10).The binding of gp120 to the CD4 receptor creates and/or exposes a coreceptor binding site characterized by a hydrophobic core surrounded by a positively charged periphery (for a review see reference 23; 18). The coreceptor binding sites on envelope glycoproteins derived from R5 and X4 isolates are principally composed of conserved residues that are organized into nearly identical tertiary structures (17). Nonetheless, there is exquisite specificity for CCR5 and CXCR4 usage by R5 and X4 isolates, respectively. Soluble gp120-CD4 complexes wherein gp120 is derived from an R5 isolate but not from an X4 isolate bind specifically to CCR5 Nt-based sulfopeptides as well as to cell surface CCR5 (8, 9, 13). The interaction between soluble gp120-CD4 complexes and CCR5 Nt-based sulfopeptides involves residues located primarily in the V3 stem and the C4 region of gp120 (8, 9). Residues in the V3 cr...