CCR5⌬32 is a loss-of-function mutation that abolishes cell surface expression of the human immunodeficiency virus (HIV) coreceptor CCR5 and provides genetic resistance to HIV infection and disease progression. Since CXCR4 and other HIV coreceptors also exist, we hypothesized that CCR5⌬32-mediated resistance may be due not only to the loss of CCR5 function but also to a gain-of-function mechanism, specifically the active inhibition of alternative coreceptors by the mutant CCR5⌬32 protein. Here we demonstrate that efficient expression of the CCR5⌬32 protein in primary CD4؉ cells by use of a recombinant adenovirus (Ad5/⌬32) was able to down-regulate surface expression of both wild-type CCR5 and CXCR4 and to confer broad resistance to R5, R5X4, and X4 HIV type 1 (HIV-1). This may be important clinically, since we found that CD4 ؉ cells purified from peripheral blood mononuclear cells of individuals who were homozygous for CCR5⌬32, which expressed the mutant protein endogenously, consistently expressed lower levels of CXCR4 and showed less susceptibility to X4 HIV-1 isolates than cells from individuals lacking the mutation. Moreover, CD4؉ cells from individuals who were homozygous for CCR5⌬32 expressed the mutant protein in five of five HIV-exposed, uninfected donors tested but not in either of two HIV-infected donors tested. The mechanism of inhibition may involve direct scavenging, since we were able to observe a direct interaction of CCR5 and CXCR4 with CCR5⌬32, both by genetic criteria using the yeast two-hybrid system and by biochemical criteria using the coimmunoprecipitation of heterodimers. Thus, these results suggest that at least two distinct mechanisms may account for genetic resistance to HIV conferred by CCR5⌬32: the loss of wild-type CCR5 surface expression and the generation of CCR5⌬32 protein, which functions as a scavenger of both CCR5 and CXCR4.

We used synthetic peptides to the extracellular loops (ECLs) of CCR5 to examine inhibitory effects on HIV infection/fusion with primary leukocytes and cells expressing recombinant CCR5. We show for the first time that peptides derived from the first, second, or third ECL caused dose-dependent inhibition of fusion and infection, although with varying potencies and specificities for envelope glycoproteins (Envs) from different strains. The first and third ECL peptides inhibited Envs from the R5 Ba-L strain and the R5X4 89.6 strain, whereas the second ECL peptide inhibited Ba-L but not 89.6 Env. None of the peptides affected fusion mediated by Env from the X4 LAV strain. IntroductionInfection by HIV-1 requires surface expression of 2 specific receptors on the target cell: CD4 (the primary receptor) and a specific chemokine receptor (the coreceptor). In the generally accepted model for HIV-1 entry (for reviews, see Wyatt and Sodroski 1 and Eckert and Kim 2 ), the external glycoprotein (gp) 120 subunit of the HIV-1 envelope glycoprotein (Env) initiates infection through specific binding to the first domain of CD4. The CD4 interaction induces a conformational change that exposes a highly conserved coreceptor binding site on gp120, which, along with variable loops on gp120, enables gp120 to bind to the coreceptor. The gp120-coreceptor interaction then triggers conformational changes in the gp41 transmembrane subunit of Env, presumably leading to exposure of the N-terminal fusion peptide and its insertion into the plasma membrane of the target cell to initiate the membrane fusion process.Individual HIV-1 isolates can display markedly distinct phenotypes for infection of different CD4-expressing cell types such as primary macrophages, transformed T-cell lines, or primary T cells. Target cell tropism is determined primarily by the ability of the corresponding Env to interact with one or both of the major coreceptors (CCR5, CXCR4), coupled with the expression patterns of these receptor molecules on the different target cell types (for a review, see Berger et al 3 ). HIV-1 isolates capable of using CCR5 but not CXCR4 (designated R5) 4-8 are typically macrophage tropic and greatly predominate after acute infection and throughout the asymptomatic phase. Isolates able to use CXCR4 9 are first detected during the transition to the symptomatic phase; those that function with CXCR4 but not CCR5 (designated X4) are typically T-cell line tropic, whereas those able to function with both coreceptors (designated R5X4) are dual tropic. All these HIV-1 phenotypes readily infected activated primary CD4 ϩ T cells. The gp120/ corecptor interaction has become a major focus for the development of new anti-HIV agents for treating or preventing HIV-1 infection (for reviews, see Agrawal and Alkhatib 10 and Biscone et al 11 ).The coreceptors are members of the superfamily of G-proteincoupled receptors (GPCRs). These proteins are characterized by 7 transmembrane segments that form a helical bundle, an extracellular region that includes the N-ter...

To analyze HTLV-1 cytotropism, we developed a highly sensitive vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. We used this system in a functional cDNA screening to isolate and confirm that the glucose transporter protein 1 (GLUT-1) is a receptor for HTLV-1. GLUT-1 is a ubiquitously expressed plasma membrane glycoprotein with 12 transmembrane domains and 6 extracellular loops (ECL). We demonstrate for the first time that peptide antibodies (GLUT-IgY) raised in chicken to the large extracellular loop (ECL1) detect GLUT-1 at the cell surface and inhibit envelope (Env)-mediated fusion and infection. Efficient GLUT-IgY staining was detected with peripheral blood CD4(+) lymphocytes purified by positive selection. Further, GLUT-IgY caused efficient inhibition of Env-mediated fusion and infection of CD4(+) T and significantly lower inhibition of CD8(+) T lymphocytes. The specificity of GLUT-IgY antibodies to GLUT-1 was demonstrated by ECL1 peptide competition studies. Grafting ECL1 of GLUT-1 onto the receptor-negative GLUT-3 conferred significant receptor activity. In contrast, grafting ECL1 of GLUT-3 onto GLUT-1 resulted in a significant loss of the receptor activity. The ECL1-mediated receptor activity was efficiently blocked with four different human monoclonal antibody (HMab) to HTLV-1 Env. The ECL1-derived peptide blocked HTLV-1 Env-mediated fusion with several nonhuman mammalian cell lines. The results demonstrate the utilization of cell surface GLUT-1 in HTLV-1 infection of CD4(+) T lymphocytes and implicate a critical role for the ECL1 region in viral tropism.