GLUT-1-independent infection of the glioblastoma/astroglioma U87 cells by the human T cell leukemia virus type 1.

The human glucose transporter protein 1 (GLUT-1) functions as a receptor for human T cell leukemia virus (HTLV). GLUT-1 is a twelve-transmembrane cell surface receptor with six extracellular (ECL) and seven intracellular domains. To analyze HTLV-1 cytotropism, we utilized polyclonal antibodies to a synthetic peptide corresponding to the large extracellular domain of GLUT-1. The antibodies caused significant blocking of envelope (Env)-mediated fusion and pseudotyped virus infection of HeLa cells but had no significant effect on infection of U87 cells. This differential effect correlated with the detection of high-level surface expression of GLUT-1 on HeLa cells and very weak staining of U87 cells. To investigate this in terms of viral cytotropism, we cloned GLUT-1 cDNA from U87 cells and isolated two different versions of cDNA clones: the wild-type sequence (encoding 492 residues) and a mutant cDNA with a 5-base pair deletion (GLUT-1Delta5) between nucleotides 1329 and 1333. The deletion, also detected in genomic DNA, resulted in a frame-shift and premature termination producing a truncated protein of 463 residues. Transfection of the wild-type GLUT-1 but not GLUT-1Delta5 cDNA into CHO cells resulted in efficient surface expression of the human GLUT-1. Co-expression of GLUT-1 with GLUT-1Delta5 produces a trans-inhibition by GLUT-1Delta5 of GLUT-1-mediated HTLV-1 envelope (Env)-mediated fusion. Co-immunoprecipitation experiments demonstrated physical interaction of the wild-type and mutant proteins. Northern blot and RT-PCR analyses demonstrated lower GLUT-1 RNA expression in U87 cells. We propose two mechanisms to account for the impaired cell surface expression of GLUT-1 on U87 cells: low GLUT-1 RNA expression and the formation of GLUT-1/GLUT-1Delta5 heterodimers that are retained intracellularly. Significant RNAi-mediated reduction of endogenous GLUT-1 expression impaired HTLV-1 Env-mediated fusion with HeLa cells but not with U87 cells. We propose a GLUT-1-independent mechanism of HTLV-1 infection of U87 cells. The results may have important implications for HTLV-1 neurotropism and pathogenesis.

Infection of CD4+ T lymphocytes by the human T cell leukemia virus type 1 is mediated by the glucose transporter GLUT-1: evidence using antibodies specific to the receptor's large extracellular domain.

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.

Role for CCR5Delta32 protein in resistance to R5, R5X4, and X4 human immunodeficiency virus type 1 in primary CD4+ cells.

CCR5Delta32 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 CCR5Delta32-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 CCR5Delta32 protein. Here we demonstrate that efficient expression of the CCR5Delta32 protein in primary CD4(+) cells by use of a recombinant adenovirus (Ad5/Delta32) 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 CCR5Delta32, 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 CCR5Delta32 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 CCR5Delta32, 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 CCR5Delta32: the loss of wild-type CCR5 surface expression and the generation of CCR5Delta32 protein, which functions as a scavenger of both CCR5 and CXCR4.

Specific inhibition of HIV-1 coreceptor activity by synthetic peptides corresponding to the predicted extracellular loops of CCR5.

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. Fusion mediated by Envs from several primary HIV-1 isolates was also inhibited by the peptides. These findings suggest that various HIV-1 strains use CCR5 domains in different ways. Experiments involving peptide pretreatment and washing, modulation of the expression levels of Env and CCR5, analysis of CCR5 peptide effects against different coreceptors, and inhibition of radiolabeled glycoprotein (gp) 120 binding to CCR5 suggested that the peptide-blocking activities reflect their interactions with gp120. The CCR5-derived ECL peptides thus provide a useful approach to analyze structure-function relationships involved in HIV-1 Env-coreceptor interactions and may have implications for the design of drugs that inhibit HIV infection.

Multiple determinants are involved in HIV coreceptor use as demonstrated by CCR4/CCL22 interaction in peripheral blood mononuclear cells (PBMCs).

Although a number of chemokine receptors display coreceptor activities in vitro, chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) remain the major coreceptors used by the human immunodeficiency virus type 1 (HIV-1). In this study, we used an envelope-mediated fusion assay to demonstrate low CCR4 coreceptor activity with some primary HIV-1 and simian immunodeficiency virus-1 (mac316) isolates in vitro. The coreceptor activity was sensitive to CCR4-specific antibodies and to the CCR4-specific chemokine ligand macrophage-derived chemokine (MDC)/chemokine ligand 22 (CCL22). Treatment of peripheral blood mononuclear cells (PBMCs; which express high levels of CCR4) with CCL22 caused down-modulation of endogenous CCR4 but had no significant effect on HIV-1 entry, suggesting that CCR4 may not be used as an entry coreceptor. Despite expression of other minor coreceptors on PBMCs, CCR5 and CXCR4 are preferentially used by HIV-1 isolates, as shown by chemokine-inhibition data. To determine the factors involved in this selective use, we analyzed CCR4 coreceptor activity and compared it with CCR5 use in PBMCs. We used a quantitative fluorescence-activated cell-sorting assay to estimate the numbers of CCR4 and CCR5 antibody-binding sites (ABS) on PBMCs. Although CCR4 was found on a higher percentage of CD4(+) cells, CCR5 ABS was twofold greater than CCR4 ABS on CD4(+) cells. Confocal microscopy revealed strong cell-surface CD4/CCR5 but weak CD4/CCR4 colocalization in PBMCs. Binding studies demonstrated that soluble gp120 had greater affinity to CCR5 than CCR4. The results suggested that coreceptor density, colocalization with CD4, and affinity of the viral gp120 to the coreceptor may determine preferential coreceptor use by HIV-1.