HIV-1 R5 Macrophage-Tropic Envelope Glycoprotein Trimers Bind CD4 with High Affinity, while the CD4 Binding Site on Non-macrophage-tropic, T-Tropic R5 Envelopes Is Occluded.

HIV-1 R5 variants exploit CCR5 as a coreceptor to infect both T cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic (mac-tropic) R5 viruses predominate in brain tissue and can be detected in cerebrospinal fluid but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by postbinding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. In this study, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and showed that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable. Our data indicated that the CD4 binding site (CD4bs) is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high-avidity interactions that overcome extremely low Env-CD4 affinities.IMPORTANCE HIV R5 variants bind to CD4 and CCR5 receptors on T cells and macrophages to initiate infection. Transmitted HIV variants infect T cells but not macrophages, and these viral strains persist in immune tissue even in late disease. Here we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibody binding to this site and neutralization of the virus, but it makes it difficult for virus-CD4 interactions to occur. Such viruses probably pass from T cell to T cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope-CD4 binding. Our data are highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein.

Efficiency of bridging-sheet recruitment explains HIV-1 R5 envelope glycoprotein sensitivity to soluble CD4 and macrophage tropism.

HIV-1 R5 viruses vary extensively in their capacity to infect macrophages. R5 viruses that confer efficient infection of macrophages are able to exploit low levels of CD4 for infection and predominate in brain tissue, where macrophages are a major target for infection. HIV-1 R5 founder viruses that are transmitted were reported to be non-macrophage-tropic. Here, we investigated the sensitivities of macrophage-tropic and non-macrophage-tropic R5 envelopes to neutralizing antibodies. We observed striking differences in the sensitivities of Env(+) pseudovirions to soluble CD4 (sCD4) and to neutralizing monoclonal antibodies (MAbs) that target the CD4 binding site. Macrophage-tropic R5 Envs were sensitive to sCD4, while non-macrophage-tropic Envs were significantly more resistant. In contrast, all Envs were sensitive to VRC01 regardless of tropism, while MAb b12 conferred an intermediate neutralization pattern where all the macrophage-tropic and about half of the non-macrophage-tropic Envs were sensitive. CD4, b12, and VRC01 share binding specificities on the outer domain of gp120. However, these antibodies differ in their ability to induce conformational changes on the trimeric envelope and in specificity for residues on the V1V2 loop stem and ß20-21 junction that are targets for CD4 in recruiting the bridging sheet. These distinct specificities of CD4, b12, and VRC01 likely explain the observed differences in Env sensitivity to inhibition by these reagents and provide an insight into the envelope mechanisms that control macrophage tropism. We present a model where the efficiency of bridging-sheet recruitment by CD4 is a major determinant of HIV-1 R5 envelope sensitivity to soluble CD4 and macrophage tropism.

Plugging gp120s cavity.

NBD-556 is a class of HIV-1 entry inhibitors that binds HIV-1 surface glycoprotein gp120 and induces conformational changes, which mimic those caused by CD4 binding. Here, Madani et al. (2008) show that NBD-556 binds to the highly conserved "Phe-43 cavity" on gp120.

The promise of CCR5 antagonists as new therapies for HIV-1.

The chemokine receptors CCR5 and CXCR4 were identified as HIV-1 co-receptors in 1996. Since then, a range of agents that bind these receptors and potently block HIV-1 infection have been described, including monoclonal antibodies, peptides and modified chemokines. However, small organic molecules that bind CCR5 are currently the most promising of the co-receptor antagonists for the potential treatment of HIV. These agents are now in advanced stages of clinical development and should soon augment current therapies, as well as being candidates for inclusion in microbicides. Unlike existing drugs that target HIV proteins (eg, reverse transcriptase and protease), co-receptor antagonists bind receptors encoded by the host. As a consequence, blockade of these receptors may result in immunosuppressive effects or other disorders. Furthermore, co-receptor inhibitors may also be more toxic than currently available HIV therapies, and it is not yet clear whether they will become candidates for first-line therapy. Nonetheless, safer, less toxic versions of such inhibitors may be achievable in the future. The use of CCR5 inhibitors as a second-line treatment increases the possibility that these reagents will select for more pathogenic CXCR4-using variants. The development of effective CXCR4 antagonists for dual treatment would be beneficial; however, whether long-term treatment with antagonists of the widely expressed CXCR4 receptor is feasible without toxicity is unknown. This review discusses the current status of CCR5 antagonists, their modes of action and their development for therapeutic use.

Variation in the biological properties of HIV-1 R5 envelopes: implications of envelope structure, transmission and pathogenesis.

HIV-1 R5 viruses predominantly use CCR5 as a coreceptor to infect CD4(+) T cells and macrophages. While R5 viruses generally infect CD4(+) T cells, research over the past few years has demonstrated that they vary extensively in their capacity to infect macrophages. Thus, R5 variants that are highly macrophage tropic have been detected in late disease and are prominent in brain tissue of subjects with neurological complications. Other R5 variants that are less sensitive to CCR5 antagonists and use CCR5 differently have also been identified in late disease. These latter variants have faster replication kinetics and may contribute to CD4 T-cell depletion. In addition, R5 viruses are highly variable in many other properties, including sensitivity to neutralizing antibodies and inhibitors that block HIV-1 entry into cells. Here, we review what is currently known about how HIV-1 R5 viruses vary in cell tropism and other properties, and discuss the implications of this variation on transmission, pathogenesis, therapy and vaccines.

Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes.

Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles.