Chemokine Receptors and HIV entry

Over the last few years, researchers have determined critical structural and functional aspects of HIV entry into host cells. This information is critical to accelerating the development of microbicides that could block HIV from attaching to and infecting target cells, particularly those on the mucosal surfaces. HIV is an envelope virus that must attach to target cells and undergo conformational changes before it can fuse to the cell membrane and discharge its genetic material into the cell cytoplasm. Since the discovery of HIV, it was clear that the CD4 molecule is the primary receptor for HIV, but it soon became apparent that HIV needed another molecule or coreceptor that in conjunction with CD4 enabled binding as well as membrane fusion and infection. Researchers began to suspect that co-receptors existed when they learned that various strains of HIV infect different CD4-bearing cells. Two major types of HIV strains have been identified: macrophagetropic and T-cell-tropic viruses. In vitro studies have shown that M-tropic viruses will infect macrophages and primary T cells but will not infect T cell lines, although these viruses can attach to these cells. M-tropic viruses are the ones typically transmitted between humans and are the most common viruses isolated from asymptomatic individuals. T-tropic viruses appear to evolve over the course of the disease as a consequence of mutations in the envelope protein. These HIV strains typically fail to infect macrophages. In early 1996, Edward Berger and his coworkers at the National Institutes of Health discovered the first HIV co-receptor: CXCR4, a seven transmembrane chemokine receptor involved in inflammation and hematopoiesis. CXCR4 was subsequently shown to be a co-receptor for T-tropic HIV strains. Other researchers subsequently discovered that M-tropic viruses use a related chemokine receptor known as CCR5 and do not infect primary T cells or other cells that do not bear this co-receptor. It is important to note that there are evolutionary, intermediate viruses that are dual-tropic which can infect cells that have either CXCR4 or CCR5. HIV-1 cell tropism therefore is largely due to the expression patterns of these two co-receptors. Studies conducted in vitro have shown that other related chemokine receptors can co-express with CD4 at high levels and support virus infections by a subset of laboratory viral strains. Only one HIV strain, however, has been shown in vivo to infect human T cells independently of CCR5 and CXCR4. This particular virus uses the STL23 receptor. It is important to study rare and laboratory strains because, when effective CCR5 antagonists are developed and deployed, there is the possibility that HIV could evolve by using these alternative receptors. When the viral envelope protein binds to CD4, it causes a structural alteration in the protein that enables it to interact with either CCR5 or CXCR4 co-receptors. This final binding event triggers the final conformational changes that lead to membrane fusion. The envelope protein known as the fusion peptide physically inserts itself into the target cell membrane and transiently becomes an integral dependent of two membranes, the biomembrane. In this process, the membrane lipids essentially mix and then fusion occurs, causing viral RNA to be discharged into the cytoplasm. The importance of CCR5 for transmission was shown by that 1 percent of Caucasians lack the CCR5 receptor due to a naturally occurring 32 base pair deletion in the reading frame. These individuals do not appear to suffer any ill affects and are highly resistant to HIV-infection, although they are not completely immune to infection. HIV-infected individuals who are heterozygous, having only one copy of this mutant gene, have a two- to four-year survival advantage over individuals who do not have this mutation. These individuals have significantly fewer CCR5 receptors on their cells. Studies using a quantitative fusion assay have shown that CCR5 receptor levels appear to correlate with infection levels. While cells with low CCR5 levels can be infected, cells with low levels of alternate receptors such as STL33 do not become infected. These findings suggest that developing means to reduce CCR5 expression has the potential of significantly reducing virus transmission and subsequent disease. HIV attachment is a variable process that is cell type dependent. A variety of factors can influence virus binding. For instance, HIV has an affinity for cells with high levels of the poly-anion heparan sulfate. Soluble heparan sulfate in a number of soluble poly-anions can block HIV infection in vitro. Heparan sulfate is a highly charged molecule that binds to other viruses as well, including herpes simplex, pseudorabies, and adeno-associated viruses. T-tropic viruses efficiently bind to these poly-anions, while M-tropic viruses do not readily bind to them. T-tropic viruses, which use the CXCR4 co-receptor, have V3 loops that present basic surfaces to cell membranes that likely give them a high affinity for poly-anions like heparan sulfate. In addition, the V3 loop on M-tropic viruses has a more neutral charge than the one on T-tropic viruses. There are many other dimensions to viral adhesion. Retroviruses can incorporate into their membranes a variety of cellular proteins. Researchers at the University Medical Center St. Radboud3 recently identified a new protein that appears to play a significant role in viral presentation and binding: DC-SIGN, a receptor that is expressed at very high levels on dendritic cells like those found in mucosal tissues. DC-SIGN is a C-type lectin that binds to sugars and therefore has an affinity for gp120, the heavily glycosylated envelope protein of HIV. This receptor enables dendritic cells to present virus to CD4+, co-receptor-positive T cells. When virus binds to this dendritic cell receptor, it can remain stable for up to four days or longer. If it migrates to a lymph node, it may present virus to different T cells that have CD4 and the appropriate co-receptor. Dan Littman and others have studied DC-SIGN in in vitro infection assays and found that viral transmission from dendritic cells to T cells is significantly decreased by inhibiting the binding of HIV to the DC-SIGN receptor. X-ray crystallography studies have delineated the structural complex of gp120 and CD4 and have shown how CD4 fits into a pocket in the gp120 protein. This pocket is a potential site for small molecule inhibitors. While such inhibitors have yet to be developed, researchers are pursuing a variety of approaches to block the interaction between the virus and this chemokine receptor. The chemokines, or the ligands to these receptors, can block HIV infection. This approach, however, is not practical since these proteins are relatively large and expensive to produce. A number of small molecule inhibitors have been identified that block viral binding to HIV co-receptors CXCR4 and CCR5. There is published data on two cationic peptide inhibitors that bind to CXCR4. One of these, LX40-4C, has passed through phase II clinical testing. Additional CXCR4-specific peptide inhibitors are under development: a bicyclam compound (AMD-3100) and two small peptide inhibitors T-22 and ALX40-4C. Although their structures are dissimilar, these agents are cationic with a basic charge, which increases their affinity for the positively charged CXCR4 receptors and thereby increases their potential for preventing T-tropic viruses from infecting their target cells. These small cationic compounds are specific to CXCR4 receptors and do not bind to CCR5, which has a more neutral charge. While CXCR4 inhibitors bind to this receptor through a charged interaction, CCR5 interactions are more hydrophobic. CCR5 has a hydrophobic pocket that fits close to the membrane surface. TAK-779 fits into this pocket and may alter receptor conformation and thereby block virus infection. Research advances have provided significant structural information on how HIV binds to and infects its human target cells. This information has created opportunities for designing inhibitors that could block virus entry. Since M-tropic viruses that utilize the CCR5 co-receptor are the virus strains associated with primary transmission, developing inhibitors to prevent HIV from binding to the CCR5 co-receptor is one of the most promising opportunities for developing an effective microbicide to prevent HIV transmission.