Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

HIV and chemokines: ligands sharing cell-surface receptors.

At the cell surface, chemokine receptors and CD4 act in concert to bind to human immunodeficiency virus (HIV) and trigger its entry into and infection of cells. Several different chemokine receptors can act as co-receptors for HIV entry, although either CCR5 or CXCR4 is used by all HIV-1 strains studied so far. The capacity of different HIV strains to exploit different chemokine receptors influences their cell tropism, cytopathicity and pathogenicity. Chemokines, the natural ligands for these receptors, can block HIV entry and are thus potential starting points for the design of novel therapeutic agents against HIV infection.

Human T cell leukemia viruses use a receptor determined by human chromosome 17.

Human T cell leukemia viruses (HTLV-I and HTLV-II) can infect many cell types in vitro. HTLV-I and HTLV-II use the same cell surface receptor, as shown by interference with syncytium formation and with infection by vesicular stomatitis virus (VSV) pseudotypes bearing the HTLV envelope glycoproteins. Human-mouse somatic cell hybrids were used to determine which human chromosome was required to confer susceptibility to VSV(HTLV) infection. The only human chromosome common to all susceptible cell hybrids was chromosome 17, and the receptor gene was localized to 17cen-qter. Antibodies to surface antigens known to be determined by genes on 17q did not block the HTLV receptor.

Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist.

The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency virus-type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1-infected individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane (AOP)-RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1 strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication. Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong candidates for the therapy of HIV-1-infected individuals.

A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.

Immune escape and tropism of HIV.

HIV-1 cell tropisms are partly determined by the hypervariable loops V1/V2 and V3 in gp120, which also contain epitopes for neutralizing antibodies. Mutations conferring tropism changes can result in escape from neutralization and vice versa. We examine whether variant viruses that can colonize new cell types and simultaneously escape neutralizing antibodies have an enhanced advantage in vivo.

A chimeric MIP-1alpha/RANTES protein demonstrates the use of different regions of the RANTES protein to bind and activate its receptors.

Human RANTES (CCL5) and MIP-1alpha (CCL3) bind and activate several CC chemokine receptors. RANTES is a high-affinity ligand for CCR1 and CCR5, and it binds CCR3 with moderate affinity and CCR4 with low affinity. MIP-1alpha has similar binding characteristics to RANTES except that it does not bind to CCR3. Here we have generated a chimera of human MIP-1alpha and RANTES, called MIP/RANTES, consisting of the eight amino terminal residues of MIP-1alpha preceding the CC motif, and the remainder of the sequence is RANTES. The chimera is able to induce chemotaxis of human monocytes. MIP/RANTES has >100-fold reduction in binding to CCR1 and does not bind to CCR3 but retains full, functional binding to CCR5. It has equivalent affinity for CCR5 to MIP-1alpha and RANTES, binding with an IC(50) of 1.12 nM, and is able to mobilize calcium and induce endocytosis of CCR5 in PBMC in a manner equi-potent to RANTES. It also retains the ability to inhibit R5 using HIV-1 strains. Therefore, we conclude that the amino terminus of RANTES is not involved in CCR5 binding, but it is essential for CCR1 and CCR3.

Development of HIV-1 group-specific neutralizing antibodies after seroconversion.

OBJECTIVE: To study the induction of group-specific (gs) neutralizing antibodies to HIV-1 after seroconversion. DESIGN AND METHODS: Serum samples taken sequentially from seven Dutch homosexual men and four British haemophiliacs (anonymous sample, therefore sex not known) before and after seroconversion were tested for neutralizing antibodies effective against five diverse HIV-1 strains. Strains of HIV-1 tested included isolates from the United States, Europe and Africa. RESULTS: The gs neutralizing antibody response varied between individuals. Only five of the 11 individuals studied produced detectable neutralizing antibodies to laboratory-adapted HIV-1 strains (for example, IIIB) within 32 weeks of seroconversion. Most individuals initially produced antibodies effective against US/European isolates; the response then generally broadened to include the more diverse strains, i.e., African. CONCLUSIONS: These results suggest that the gs neutralizing target for HIV-1 is poorly immunogenic in vivo and is probably not highly conserved among diverse HIV-1 strains.

Neutralizing monoclonal antibodies to the AIDS virus.

The production of neutralizing monoclonal antibodies (MAbs) will permit the exact localization of neutralizing epitopes on the AIDS virus, HIV-1. We describe the properties of seven MAbs to the envelope of the LAV-1 isolate. Five MAbs recognise the central portion of gp110, amino acids 279-472, and four of these are capable of high-titre neutralization of HIV-1, by infection inhibition, syncytial inhibition and vesicular stomatitis virus (VSV) pseudotype neutralization. One of the two MAbs to gp41 inhibits syncytium formation. Neutralization, live cell immunofluorescence and immunoprecipitation of gp110 are type-specific and restricted to HIV-1 isolates closely related to LAV-1.

The human and simian immunodeficiency viruses HIV-1, HIV-2 and SIV interact with similar epitopes on their cellular receptor, the CD4 molecule.

The cellular receptor for HIV-1 is the leucocyte differentiation antigen, CD4. Blocking of HIV-1 infectivity can be achieved with monoclonal antibodies (MAbs) to some, but not all epitopes of this antigen. We demonstrate here, by inhibition of virus infection, blocking of syncytium formation and inhibition of pseudotype infection with a panel of CD4 MAbs, that HIV-1, HIV-2 and simian immunodeficiency virus (SIV) isolates share the same cellular receptor, the CD4 glycoprotein. It is also shown that very similar epitopes of this molecule are involved in virus binding. We infer from these data that the binding sites on these viruses are highly conserved regions, and may therefore make good targets for potential vaccines. In addition, we show that cell surface expression of CD4 is similarly modulated after infection of cell lines by all the viruses.

HIV-2 antisera cross-neutralize HIV-1.

The neutralization properties of three independent HIV-2 isolates were examined in comparison with four diverse HIV-1 strains. Human sera containing antibodies specific to HIV-2 can cross-neutralize HIV-1. By contrast, HIV-1 sera are group-specific and have no neutralizing effect on HIV-2. Therefore, HIV-2 antigens may be important components for the development of broadly cross-protective AIDS vaccines.

Human immunodeficiency viruses: neutralization and receptors.

The envelope glycoproteins of HIV, gp120 and gp41, contain epitopes recognized by neutralizing antibodies. Studies of human sera from infected individuals indicate that group-specific neutralization antigens common to most isolates of HIV-1 exist, and that some HIV-2 antisera cross-neutralize HIV-1. Neutralization epitopes for HIV-1 have been identified and mapped, including a group-specific antigen on gp41, and a type-specific antigen on gp120. Neutralization "escape" mutants have been selected in vitro with a neutralizing mab to the type-specific antigenic loop. The CD4 antigen binds HIV-1 gp120 with high affinity and acts as the receptor on human and simian T-lymphocytes and monocytes for all strains of HIV-1, HIV-2, and SIV tested. Following binding to the CD4 receptor, HIV becomes internalized by a pH-independent process. The principle binding domain for gp120 is located in the N-terminal V domain of CD4. Anti-idiotypic sera to CD4 mabs recognizing the same site weakly neutralize HIVs of many strains, and soluble, recombinant forms of CD4 strongly neutralize HIV. Neither anti-CD4 mabs nor sCD4 inhibit the low level of plating of HIV observed on tumour cells in culture of glial (brain) and muscle origin, indicating that CD4 is not essential for infection of these cell types.

Enhancement of soluble CD4-mediated HIV neutralization and gp 120 binding by CD4 autoantibodies and monoclonal antibodies.

We have identified 6 sera containing autoantibodies to CD4 in 174 human immunodeficiency virus-type (HIV-1) positive sera tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) using sCD4, and none in 34 HIV type 2 sera. These autoantibodies do not bind to cellular CD4, but react with sCD4 to increase its binding in ELISA to monoclonal antibodies and the HIV surface glycoprotein gp120. The effect of CD4 autoantibodies is mimicked by monoclonal antibodies to the third and fourth domains of CD4. The enhanced sCD4 binding to gp120 in ELISA is reflected by a reduction in the concentration of sCD4 required to neutralize HIV-1 and HIV-2 infection in tissue culture when CD4 autoantibodies or the relevant monoclonal antibodies were present.

The V3 loops of the HIV-1 and HIV-2 surface glycoproteins contain proteolytic cleavage sites: a possible function in viral fusion?

Located close to the crown of the V3 type-specific neutralization loop of the human immunodeficiency virus type 1 (HIV-1) (IIIB) SU glycoprotein gp120, are several potential sites that should be susceptible to proteolytic cleavage by enzymes of trypsinlike or chymotrypsinlike specificity, or by aspartic proteinases. The linkages potentially sensitive to chymotryptic/aspartic proteinase cleavage are retained also within the equivalent domain of HIV-2 (ROD) gp105. We show that thrombin and tryptase cleave HIV-1 gp120 specifically at the tryptic site (GPGR decreases AFVT), and that cathepsin E, an endosomal aspartic proteinase, cleaves at the chymotrypsinlike site (GPGRAF decreases VT). HIV-2 gp105 is also cut by cathepsin E at a site (QIML decreases MSGH) in its V3 loop. Cleavage of HIV-1 gp120 by thrombin is enhanced by sCD4 binding, but is prevented by transient exposure of gp120 to nonionic detergent. Thrombin treatment of HIV-1 gp120 destroys the binding sites for some neutralizing monoclonal antibodies (MAbs) on the V3 loop, but does not affect the affinity of gp120 for sCD4. Conversely, binding of neutralizing MAbs to the HIV-1 V3 loop prior to addition of thrombin or cathepsin E blocks the cleavage reactions, and the binding of some HIV-positive sera to gp120 blocks thrombin cleavage. Analysis of published sequences suggests that all HIV-1, HIV-2, and simian immunovirus (SIV) isolates contain potential proteolytic cleavage sites at similar positions in their V3 loops or equivalent domains. We suggest that cleavage of the V3 loop by a cell surface or endosomal proteinase occurs during the HIV-cell fusion reaction, and that neutralizing antibodies directed against the V3 loop might act by inhibition of this reaction.

Coreceptor ligand inhibition of fetal brain cell infection by HIV type 1.

The capacity of a panel of HIV-1 isolates to infect primary mixed fetal brain cell cultures was estimated and their sensitivity to inhibition by a range of coreceptor ligands assessed. Our results show that (1) HIV-1 strains that predominantly use CCR5 or only CXCR4 are able to infect microglia in primary brain cell cultures, and (2) ligands to these two coreceptors can inhibit brain cell infection. CCR5 ligands (including AOP-RANTES, a potent inhibitor of CCR5-dependent infection), however, blocked infection only weakly, raising the possibility that alternative unidentified coreceptors are also used. Interestingly, vMIP-II, a chemokine encoded by the Kaposi sarcoma-associated herpes virus (KSHV), reduced brain cell infection by all HIV-1 strains tested, including both R5 and X4 viruses. Our results therefore indicate that novel drugs targeted to the major HIV-1 coreceptors will influence HIV replication in the brain, if they cross the blood-brain barrier.

Chemokine receptors--future therapeutic targets for HIV?

To date, triple drug therapies for HIV have resulted in spectacular reductions in the number of virus particles and often remarkable recovery from disease in infected people. There is still, however, a great need for improved therapies. A battery of drugs aimed at different stages in the life cycle of HIV will enable switching of treatments if resistant viruses emerge or if patients are unable to tolerate particular therapies. Intense efforts are now underway to produce drugs that target chemokine receptors used by HIV to gain entry into cells. HIV needs two receptors on the host cell surface for efficient attachment and infection. HIV first interacts with CD4 but requires a coreceptor to penetrate the cell membrane. The first coreceptor, identified in 1996, is a member of the family of chemokine receptors, members of the G-protein coupled 7TM superfamily, which are involved in the trafficking of leukocytes in immune surveillance and inflammation. Such a therapeutic approach would differ from those used successfully to date, which focus largely on proteins coded by the HIV virus itself, and which are required for the replicative cycle of the virus. Many small, orally bioavailable molecules that block various 7TM receptors are used to treat a panoply of diseases including ulcers, allergies, migraines, and schizophrenia. These molecules are the cornerstone of the pharmaceutical industry's contribution to the fight against so many diseases, and it is hoped that a small molecule inhibitor of coreceptors can be developed that will become an invaluable drug in the fight against AIDS.

Detection of HIV envelope specific antibodies by immunoelectron microscopy and correlation with antibody titer and virus neutralizing activity.

Human antisera positive for HIV were evaluated on HTLV-IIIB producing cells by two different immunoelectron microscopic (IEM) techniques. In preembedding immunoferritin IEM a heavy label was observed with early budding HIV. Under the same conditions cell released 'mature' particles were almost negative, which could be explained by the direct observation that most of the surface glycoprotein knobs are lost spontaneously during virus maturation. Using freshly infected cultures after agarose embedding, immunogold labelling of ultrathin cryosections allowed us to detect and differentiate internal core as well as virus envelope antigens. A good qualitative correlation between neutralization titers and IEM labelling intensity was observed. This type of immunocryoultramicrotomy appears to be useful for the detection of antigens in and on the virion. It might turn out valuable for the characterization of the env gp120 epitopes of HIV.

Vesicular stomatitis virus pseudotypes of retroviruses.

Pseudotype viruses are phenotypically mixed virions containing the genome or nucleocapsid of one enveloped virus and the surface or envelope (env) glycoproteins of another. This chapter will concentrate on vesicular stomatitis virus (VSV) pseudotypes retaining the VSV nucleocapsid but with alternative env glycoproteins derived from retroviruses.