Chemokine Receptors and HIV-1: An Attractive Pair? (1996)

Binding of virus particles to the host cell surface initiates a cascade of events that culminates with the introduction of the viral genetic material into the host cell. For enveloped viruses, this cascade begins with the envelope proteins on the virus binding to specific cellular receptors and is completed following viral glycoprotein–mediated fusion of the viral and target membranes. Presently, the best model for viral envelope–mediated membrane fusion is that of influenza hemaglutinnin (HA). HA mediates membrane fusion by first binding to its cognate receptor on the host membrane. After endocytosis, pH-induced conformational changes lead to insertion of a hydrophobic stretch of residues (the so-called fusion peptide) of HA into the host membrane. Subsequent to fusion peptide insertion, additional conformational changes are thought to take place in HA that eventually lead to membrane fusion (reviewed by2Carr C.M Kim P.S Science. 1994; 266: 234-236Crossref PubMed Scopus (50) Google Scholar). The human immunodeficiency virus type 1 envelope (HIV-1 Env) has a number of proposed structural features that are very similar to HA and suggest a common mechanism of entry. However, most retroviruses, including HIV-1, enter cells in a pH-independent manner, and thus low pH cannot be the signal for the requisite conformational changes. It has been proposed that, for HIV-1, receptor recognition may serve as the trigger that initiates these conformational changes. Therefore, analysis of the virus–receptor interaction is likely to be critical for understanding entry of HIV-1. CD4 has been identified as a receptor for the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) as well as for simian immunodeficiency virus. Although CD4 binds tightly to the HIV-1 envelope glycoprotein gp120, expression of CD4 is not sufficient to allow HIV-1 entry into nonhuman cells, and a cofactor that functions in conjunction with CD4 has been postulated (10Maddon P.J Dalgleish A.G McDougal J.S Clapham P.R Weiss R.A Axel R Cell. 1986; 47: 333-348Abstract Full Text PDF PubMed Scopus (1496) Google Scholar). However, a recent breakthrough has caused an upheaval in the field of HIV-1 entry that is likely also to have profound implications on studies of HIV-1 pathogenesis, development of novel therapeutics and small animal models, and analysis of natural resistance to infection. This breakthrough is the identification of several members of the chemokine receptor family as coreceptors required for HIV-1 infection. With all the players sufficient for HIV-1 entry now identified, studies on the mechanism of entry by this virus can now proceed. The HIV-1 envelope (Env) glycoproteins gp120 (SU) and gp41 (TM) mediate HIV-1 infection of target cells. Like many other viral glycoproteins, they direct binding to the cell and are critical for the fusion of viral and host membranes. HIV-1 requires a host protein, CD4, on target cells for efficient infection. CD4 binds tightly (KD 1–2 nM) to the viral envelope surface glycoprotein, gp120. In addition, CD4 can induce conformational changes in the viral glycoproteins, as measured by antibody epitope changes and gp120 shedding from the virus (reviewed by14Sattentau Q.J Moore J.P Philos. Trans. R. Soc. London (Biol.). 1993; 342: 59-66Crossref PubMed Scopus (83) Google Scholar), but viral binding and these conformational changes are not sufficient for HIV-1 infection of cells. In nonhuman cells expressing CD4, HIV-1 binds, but virus–host membrane fusion does not occur. This species tropism of HIV-1 suggests that an accessory factor may be required, in addition to CD4, to elicit the correct alterations in Env that lead to membrane fusion. In addition to the species-specific restriction to HIV-1 host range outlined above, there are also restrictions on HIV-1 infection of human cells (reviewed by11Miedema F Meyaard L Koot M Klein M.R Toos M.T.L Groenink M Fouchier R.A.M Van't Wout A.B Tersmette M Schellekens P.T.A Schuitemaker H Immunol. Rev. 1994; 140: 35-72Crossref PubMed Scopus (139) Google Scholar). Most commonly used laboratory strains of HIV-1 readily infect cultured T cell lines and primary T lymphocytes but not primary monocytes or macrophages. These strains are termed T-tropic. Some primary isolates (viruses not extensively passaged in culture) exhibit a similar replication pattern in T cell lines and macrophages and are also defined as T-tropic. In contrast, most primary HIV-1 isolates grow poorly in established T cell lines but will replicate efficiently in primary lymphocytes, as well as in monocytes and macrophages, and are referred to as macrophage- or M-tropic. Importantly, the macrophage-tropic strains more closely resemble viruses present early and throughout the course of HIV-1 infection. It appears that the macrophage-tropic viruses are primarily responsible for transmission of HIV-1. The viral determinant of T- and M-tropism maps to the gp120 subunit of the HIV-1 envelope protein. Alterations in the third variable region of gp120 (the V3 loop) specify M- versus T-tropism. Definition of viruses with distinct tropisms in human cells suggested that cell type–specific cofactors might be required in addition to CD4 for HIV-1 entry. It was unclear for quite some time whether these cofactors were the same factor(s) that determined the species specificity of HIV-1 infection. As discussed below, this question has now been answered. Two separate sets of observations demonstrate that chemokines are potent modulators of HIV-1 infection. First, CD8+ T lymphocytes have been shown to release protein factors that inhibit HIV-1 replication (4Cocchi F DeVico A.L Garzino-Demo A Arya S.K Gallo R.C Lusso P Science. 1995; 270: 1811-1815Crossref PubMed Scopus (2590) Google Scholar). Purification and sequencing of these proteins reveal that they are the chemokines macrophage–inflammatory protein-1α and -1β (MIP-1α and MIP-1β) and RANTES (for regulated on activation normal T cell expressed and secreted). These proteins are members of the CC family of chemokines (reviewed by9Horuk R Trends Pharmacol.Sci. 1994; 15: 159-165Google Scholar, 12Murphy P.M Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1114) Google Scholar). A second observation implicating chemokines as inhibitors of HIV-1 entry comes from studies by Koup and coworkers of individuals who have been exposed to HIV-1 multiple times but remain uninfected (13Paxton W.A Martin S.R Tse D O'Brien T.R Skurnick J VanDevanter N.L Padian N Braun J.F Kotler D.P Wolinsky S.M Koup R.A Nature Med. 1996; 2: 412-417Crossref PubMed Scopus (585) Google Scholar). CD4-positive lymphocytes from these individuals are relatively resistant to infection by HIV-1 in culture, yet the cells have normal levels of surface CD4. This resistance is restricted to the M-tropic strains of HIV-1 and is associated with the secretion of high levels of RANTES, MIP-1α, and MIP-1β by the CD4+ T lymphocytes from these individuals. The nature of the chemokine effect on HIV-1 infection is clarified somewhat when the coreceptors for HIV-1 entry are identified (see below). Over ten years elapsed after the identification of CD4 as a HIV-1 receptor before a coreceptor for HIV-1 entry was identified. This molecule has been quite elusive despite significant efforts aimed at identifying it. There have been a number of false starts along the way. However, in a recently published series of elegant experiments utilizing a sensitive fusion-assay system, Berger and colleagues have identified a protein they call fusin that when coexpressed with CD4 in murine NIH 3T3 cells allows fusion with cells bearing HIV-1 envelope proteins (5Deng H Liu R Ellmeier W Choe S Unutmaz D Burkhart M Di Marzio P Marmon S Sutton R.E Hill C.M Davis C.B Peiper S.C Schall T.J Littman D.R Landau N.R Nature. 1996; 381: 661-667Crossref PubMed Scopus (3166) Google Scholar; Figure 1). Furthermore, expression of CD4 and fusin in a number of different nonhuman cells renders them susceptible to infection with HIV-1. Antibodies directed against fusin blocks Env-mediated fusion and infection in human cells normally permissive for HIV-1. From these results, it is clear that fusin is a bona fide coreceptor for HIV-1. The specific requirement for human fusin and human CD4 explains the species tropism displayed by HIV. When analyzed with Env's from various viral types, fusin has been shown specifically to mediate T-tropic viral fusion and entry but not to function as a coreceptor for the macrophage-tropic viral Env's. Fusin mRNA is expressed in numerous cell types permissive for T-tropic HIV-1 viruses, including peripheral blood mononuclear cells, Jurkat T cells, HeLa cells, and H9 cells. Therefore, fusin also fulfills these criteria for the T cell–tropic cofactor, but the cofactor for macrophage-tropic viruses remains to be identified. The sequence of fusin reveals that it is a member of the superfamily of G protein–coupled receptors containing seven transmembrane segments and that it is most closely related (37% identity) to the receptor for interleukin-8, a member of the CXC family of chemokines. Fusin had been previously identified by several other laboratories as an “orphan” chemokine receptor and given the names LCR-1, HUMSTSR, and LESTR. The natural ligand for fusin is presently unknown, but fusin does not bind RANTES, MIP-1α, or MIP-1β. The discovery of fusin, coupled with the observed effects of the CC chemokines RANTES, MIP-1α, and MIP-1β on infection by M-tropic HIV-1 viruses, strongly suggests that other chemokine receptor family members might display coreceptor function for the M-tropic viruses. Six receptors for the CC family of chemokines have been identified: CC-CKR1, CC-CKR2, CC-CKR3, CC-CKR4, CC-CKR5, and the Duffy blood group antigen. All these proteins are seven transmembrane–domain, G protein–coupled receptors (see reviews by9Horuk R Trends Pharmacol.Sci. 1994; 15: 159-165Google Scholar, 12Murphy P.M Annu. Rev. Immunol. 1994; 12: 593-633Crossref PubMed Scopus (1114) Google Scholar). In general, there is broad overlap in the ligands bound by the CC chemokine receptors, and many bind RANTES, MIP-1α, and MIP-1β. Characterization of the receptor expression patterns is incomplete. Using both HIV-1 infection assays and Env-mediated cell–cell fusion experiments, several groups tested the known receptors for the CC family of chemokines by transient transfection into cells and assessed HIV-1 coreceptor function. Nearly simultaneously, five groups reported that CC-CKR5 was the major coreceptor for the macrophage-tropic strains of HIV-1 (Figure 1). CC-CKR5 allows both membrane fusion and infection by numerous strains of M-tropic viruses (1Alkhatib G Combadiere C Broder C.C Feng Y Murphy P.M Berger E.A Science, in press. 1996; Google Scholar, 3Choe H Farzan M Sun Y Sullivan N Rollins B Ponath P.D Wu L Mackay C.R LaRosa G Newman W Gerard N Gerard C Sodroski J Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar, 5Deng H Liu R Ellmeier W Choe S Unutmaz D Burkhart M Di Marzio P Marmon S Sutton R.E Hill C.M Davis C.B Peiper S.C Schall T.J Littman D.R Landau N.R Nature. 1996; 381: 661-667Crossref PubMed Scopus (3166) Google Scholar, 6Doranz B.J Rucker J Yi Y Smyth R.J Samson M Peiper S Parmentier M Collman R.G Doms R.W Cell. 1996; 85: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (1674) Google Scholar, 7Dragic T Litwin V Allaway G.P Martin S.R Huang Y Nagashima K.A Cayanan C Maddon P.J Koup R.A Moore J.P Paxton W.A Nature. 1996; 381: 667-674Crossref PubMed Scopus (2791) Google Scholar). RANTES, MIP-1α, and MIP-1β, the chemokine ligands for this receptor that had previously been demonstrated to inhibit HIV-1 infection, have been shown to block HIV-1 Env-mediated membrane fusion directed by CC-CKR5 (1Alkhatib G Combadiere C Broder C.C Feng Y Murphy P.M Berger E.A Science, in press. 1996; Google Scholar, 7Dragic T Litwin V Allaway G.P Martin S.R Huang Y Nagashima K.A Cayanan C Maddon P.J Koup R.A Moore J.P Paxton W.A Nature. 1996; 381: 667-674Crossref PubMed Scopus (2791) Google Scholar). It appears that CC-CKR5 is the major M-tropic HIV-1 cofactor. However, as discussed below, there is some flexibility in the choice of CC-CKR used as coreceptor for at least some M-tropic envelopes. Although the macrophage-tropic viruses all appear to utilize CC-CKR5, there are viral isolate–specific differences in the use of other CC-CKRs for entry. For example, the M-tropic HIV-1 strains ADA and YU2 appear to use CC-CKR3 in addition to CC-CKR5 (3Choe H Farzan M Sun Y Sullivan N Rollins B Ponath P.D Wu L Mackay C.R LaRosa G Newman W Gerard N Gerard C Sodroski J Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar). A dual-tropic HIV-1, 89.6, is quite promiscuous in its choice of coreceptor using the CC chemokine receptors CC-CKR2b, CC-CKR3, and CC-CKR5, as well as fusin (6Doranz B.J Rucker J Yi Y Smyth R.J Samson M Peiper S Parmentier M Collman R.G Doms R.W Cell. 1996; 85: 1149-1158Abstract Full Text Full Text PDF PubMed Scopus (1674) Google ScholarChoe et al., 1996). When genetically distant strains of macrophage-tropic HIV-1 were examined for coreceptor use, all of these viruses were able to utilize CC-CKR5. However, the viruses from clades E and C did so at only approximately 10% the level of the other viruses. The inability of envelope proteins from these clades to utilize efficiently any of the potential coreceptors examined (CC-CKR1–5 or fusin) suggests that the clade E Env's studied are either inherently less efficient than the clade B envelopes or that they may primarily utilize an alternative coreceptor. Taken together, the results showing diverse receptor utilization by the dual-tropic virus and the inefficient utilization of the tested CKRs by the E and C clades suggest that the list of chemokine receptors implicated as HIV-1 cofactors is likely to grow. Furthermore, it also hints that the pattern of coreceptor utilization for various HIV-1 strains will surely be quite complex (i.e., there is not likely to be one coreceptor for all HIV-1 strains). The region of HIV-1 envelope responsible for determining coreceptor utilization was examined by Sodroski and coworkers using chimeric envelope proteins (3Choe H Farzan M Sun Y Sullivan N Rollins B Ponath P.D Wu L Mackay C.R LaRosa G Newman W Gerard N Gerard C Sodroski J Cell. 1996; 85: 1135-1148Abstract Full Text Full Text PDF PubMed Scopus (2062) Google Scholar). Substitution of the V3 loop from two M-tropic strains into a T-tropic (HXB2) envelope gene altered the coreceptor choice such that CC-CKR5 was now used by these envelopes. Although a very limited number of chimeras were used in this analysis, the results suggest that, as expected from numerous previous studies mapping T and M cell tropism, the V3 domain of Env determines coreceptor use. However, analysis of a much larger panel of chimeric Env's will be required to confirm this conclusion. The implications of the discovery of the HIV-1 coreceptors are numerous and exciting. First, with these coreceptors in hand, studies on the mechanism of entry of HIV-1 will be greatly facilitated. For entry to occur, the virus must bind to the cell and then undergo a series of conformational changes postulated to be triggered by the receptor. As mentioned above, CD4 can induce some conformational changes in HIV-1 Env, but these are insufficient for entry. Possible roles for the coreceptors in promoting fusion include inducing further changes in Env either by direct interaction or indirectly by association with CD4, binding Env to orient the protein correctly with respect to the membrane to allow fusion, or possibly inducing chemokine signaling (involving Ca2+ influx), which may be required to depolarize or somehow destabilize the membrane so that fusion can occur. The ways in which chemokines interfere with fusion and infection can also be addressed. Do HIV-1 Env and chemokines directly compete for receptor, or is the coreceptor down-regulated in response to chemokine overexpression, making it unavailable for the virus? Is there genetic polymorphism in the CC-CKR receptors that could account for the relative insensitivity of some individuals to HIV-1 infection? If the polymorphic receptors function less efficiently for both HIV-1 entry and chemokine stimulation, one might expect observed viral resistance and increased levels of chemokines. Also, the chemokine receptors and/or chemokine levels might determine the viral burden and thus the rate of disease progression in infected individuals. The discovery of chemokine receptors as cofactors may also lead to development of new therapeutic strategies aimed at blocking the viral access to the receptor using small molecule inhibitors instead of chemokines. The role of the coreceptors in HIV-1 pathogenesis can also be analyzed. Inappropriate signaling, mediated by direct interaction of HIV-1 envelope and the coreceptor, could contribute to pathogenesis of the virus. In addition, a number of chemokine receptor homologs have been identified in DNA viruses, including human cytomegalovirus and Herpes saimiri (a close relative of the Kaposi sarcoma–associated herpes virus). It is interesting to speculate that interaction of HIV-1 with these receptors could be involved in pathogenesis by these viruses that are associated with HIV-1 infection. Finally, identification of factors (CD4 and fusin or CC-CKR5) sufficient for HIV-1 entry may allow development of a small animal model for HIV-1 infection in which viral replication could be studied and therapeutic interventions might be tested.