Chemokine receptors and related seven-transmembrane-segment (7TMS) receptors serve as coreceptors for entry of human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) into target cells. Each of these otherwise diverse coreceptors contains an N-terminal region that is acidic and tyrosine rich. Here, we show that the chemokine receptor CCR5, a principal HIV-1 coreceptor, is posttranslationally modified by O-linked glycosylation and by sulfation of its N-terminal tyrosines. Sulfated tyrosines contribute to the binding of CCR5 to MIP-1 alpha, MIP-1 beta, and HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, is also sulfated. Tyrosine sulfation may contribute to the natural function of many 7TMS receptors and may be a modification common to primate immunodeficiency virus coreceptors.

Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.

The chemokine receptor CXCR4 is critical for many biological functions, such as B-cell lymphopoiesis, regulation of neuronal cell migration, and vascular development (1-3). In addition, CXCR4 together with another chemokine receptor CCR5 are two principal co-receptors for the cellular entry of the human immunodeficiency virus type 1 (HIV-1) 1 (4 -7). The stromal cell-derived factor-1 (SDF-1␣) is the only known natural ligand of CXCR4 and plays important roles in migration, proliferation, and differentiation of leukocytes (8, 9). The viral macrophage inflammatory protein II (vMIP-II) encoded by human herpesvirus 8 (10) is an antagonistic chemokine ligand of CXCR4 (11, 12). vMIP-II also interacts with other chemokine receptors such as CCR5 and CCR3 and inhibits HIV-1 entry mediated by these co-receptors.CXCR4 and other chemokine receptors belong to the superfamily of seven transmembrane G-protein-coupled receptors (GPCRs) (13). These membrane proteins transmit signals from extracellular ligands to intracellular biological pathways via heterotrimeric G-proteins and have been a major class of therapeutic targets for a wide variety of human diseases (14). As such, characterizing the mechanism of biological recognition between these receptors and their ligands is essential for understanding the physiological or pathological processes that they mediate and devising novel strategies for clinical intervention. For CXCR4, studies have been carried out by a number of laboratories using chimeric chemokine receptors and site-specific mutants to study multiple domains of CXCR4 that are important for interacting with chemokine ligands and HIV-1 (15-23). However, because there is no high resolution crystal structure available for CXCR4 (or any other chemokine receptor) alone or complexed with ligands, the structural and biochemical basis of ligand binding and signaling through these important membrane receptors remains poorly understood.To further define the structure-function relationship of the chemokine receptor-ligand interaction, theoretical computer modeling and site-directed mutagenesis were combined to predict plausible structural models for chemokine receptors and their complexes with ligands, such as interleukin-8 receptor ␤ (24) and CCR5 (25,26). Structural models of CXCR4 and its complex with ligands were also proposed (27, 28). Complementary to modeling and mutational analyses of the receptors,

SurnrrlliryEngagement of the CD3/T cell antigen receptor complex on small, resting T cells is insufficient to trigger cell-mediated cytotoxicity or to induce a proliferative response. In the present study, we have used genetic transfection to demonstrate that interaction of the B7-BB1 B cell activation antigen with the CD28 T cell differentiation antigen costimulates cell-mediated cytotoxicity and proliferation initiated by either anti-CD2 or anti-CD3 monoclonal antibody (mAb). Moreover, a B7-negative Burkitt's lymphoma cell line that fails to stimulate an allogeneic mixed lymphocyte response is rendered a potent stimulator after transfection with B7. The mixed leukocyte reaction proliferative response against the B7 transfectant is inhibited by either anti-CD28 or B7 mAb. We also demonstrate that freshly isolated small, resting human T cells can mediate anti-CD3 or anti-CD2 mAb-redirected cytotoxicity against a murine Fc receptor-bearing mastocytoma transfected with human B7. These preexisting cytotoxic T lymphocytes in peripheral blood are present in both the CD4 and CD8 subsets, but are preferentially within the CD45RO + "memory" population. While small, resting T cells apparently require costimulation by CD28/B7 interactions, this requirement is lost after T cell activation. Anti-CD3 initiates a cytotoxic response mediated by in vitro cultured T cell clones in the absence of B7 ligand. The existence of functional cytolytic T cells in the small, resting T cell population may be advantageous in facilitating rapid responses to immune challenge. CD28 is a disulfide-linked homodimer that is expressed on the majority of human peripheral blood T cells (6, 7). mAbs against CD28 in conjunction with phorbol ester induce T cell proliferation (7) and augment proliferation induced by anti-CD3 or anti-CD2 mAb (8-12). Anti-CD28 mAb-induced proliferation is IL-2 dependent (7, 8) and possibly results from stabilization of messenger RNA for IL-2 and other cytokines (13).Recently, it has been demonstrated that a B cell activation antigen, B7 (14) or BB1 (15), is a natural ligand for CD28 and that this receptor/ligand interaction mediates heterotypic adhesion (16). Interaction of CD28 and B7 results in augmentation of T cell proliferation and cytokine production, and antibodies against CD28 or B7 inhibit alloantigen and mitogen-induced proliferative responses (16,17). B7 is weakly expressed on resting B cells and monocytes, and is elevated after stimulation with pokeweed mitogen, anti-Ig, anti-HLA class II, and EBV (14,15,17). Therefore, it is likely that T cell activation via CD28 will depend on the capacity of the APC to upregulate expression of B7. The importance of the CD28/B7 interaction in polyclonal mitogenic responses prompted us to investigate whether the binding orB7 to CD28