Roles of Virion-Incorporated CD162 (PSGL-1), CD43, and CD44 in HIV-1 Infection of T Cells

Nascent HIV-1 particles incorporate the viral envelope glycoprotein and multiple host transmembrane proteins during assembly at the plasma membrane. At least some of these host transmembrane proteins on the surface of virions are reported as pro-viral factors that enhance virus attachment to target cells or facilitate trans-infection of CD4⁺ T cells via interactions with non-T cells. In addition to the pro-viral factors, anti-viral transmembrane proteins are incorporated into progeny virions. These virion-incorporated transmembrane proteins inhibit HIV-1 entry at the point of attachment and fusion. In infected polarized CD4⁺ T cells, HIV-1 Gag localizes to a rear-end protrusion known as the uropod. Regardless of cell polarization, Gag colocalizes with and promotes the virion incorporation of a subset of uropod-directed host transmembrane proteins, including CD162, CD43, and CD44. Until recently, the functions of these virion-incorporated proteins had not been clear. Here, we review the recent findings about the roles played by virion-incorporated CD162, CD43, and CD44 in HIV-1 spread to CD4⁺ T cells.     

Efficient HIV-1 transmission from macrophages to T cells across transient virological synapses

Macrophages are reservoirs of HIV-1 infection, proposed to transmit virus to CD4⁺ T cells, the primary target of the virus. Here we report that human monocyte-derived macrophages (MDMs) rapidly spread HIV-1 to autologous CD4⁺ T cells resulting in productive infection. Transmission takes place across transient adhesive contacts between T cells and MDMs, which have the features of a virological synapse including copolarization of CD4 on the T cell with HIV-1 Gag and Env on the macrophage. We propose that an infected MDM can infect at least one T cell every 6 hours. Since HIV-1–infected macrophages can survive for many weeks, these results highlight the central role played by macrophages in HIV-1 infection and pathogenesis.     

HIV-1 Latency in Monocytes/Macrophages

Human immunodeficiency virus type 1 (HIV-1) targets CD4⁺ T cells and cells of the monocyte/macrophage lineage. HIV pathogenesis is characterized by the depletion of T lymphocytes and by the presence of a population of cells in which latency has been established called the HIV-1 reservoir. Highly active antiretroviral therapy (HAART) has significantly improved the life of HIV-1 infected patients. However, complete eradication of HIV-1 from infected individuals is not possible without targeting latent sources of infection. HIV-1 establishes latent infection in resting CD4⁺ T cells and findings indicate that latency can also be established in the cells of monocyte/macrophage lineage. Monocyte/macrophage lineage includes among others, monocytes, macrophages and brain resident macrophages. These cells are relatively more resistant to apoptosis induced by HIV-1, thus are important stable hideouts of the virus. Much effort has been made in the direction of eliminating HIV-1 resting CD4⁺ T-cell reservoirs. However, it is impossible to achieve a cure for HIV-1 without considering these neglected latent reservoirs, the cells of monocyte/macrophage lineage. In this review we will describe our current understanding of the mechanism of latency in monocyte/macrophage lineage and how such cells can be specifically eliminated from the infected host.     

Molecular architecture of the uncleaved HIV-1 envelope glycoprotein trimer

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, a membrane-fusing machine, mediates virus entry into host cells and is the sole virus-specific target for neutralizing antibodies. Binding the receptors, CD4 and CCR5/CXCR4, triggers Env conformational changes from the metastable unliganded state to the fusion-active state. We used cryo-electron microscopy to obtain a 6-Å structure of the membrane-bound, heavily glycosylated HIV-1 Env trimer in its uncleaved and unliganded state. The spatial organization of secondary structure elements reveals that the unliganded conformations of both glycoprotein (gp)120 and gp41 subunits differ from those induced by receptor binding. The gp120 trimer association domains, which contribute to interprotomer contacts in the unliganded Env trimer, undergo rearrangement upon CD4 binding. In the unliganded Env, intersubunit interactions maintain the gp41 ectodomain helical bundles in a “spring-loaded” conformation distinct from the extended helical coils of the fusion-active state. Quaternary structure regulates the virus-neutralizing potency of antibodies targeting the conserved CD4-binding site on gp120. The Env trimer architecture provides mechanistic insights into the metastability of the unliganded state, receptor-induced conformational changes, and quaternary structure-based strategies for immune evasion.     

HIV-1 Entry and Membrane Fusion Inhibitors

HIV-1 (human immunodeficiency virus type 1) infection begins with the attachment of the virion to a host cell by its envelope glycoprotein (Env), which subsequently induces fusion of viral and cell membranes to allow viral entry. Upon binding to primary receptor CD4 and coreceptor (e.g., chemokine receptor CCR5 or CXCR4), Env undergoes large conformational changes and unleashes its fusogenic potential to drive the membrane fusion. The structural biology of HIV-1 Env and its complexes with the cellular receptors not only has advanced our knowledge of the molecular mechanism of how HIV-1 enters the host cells but also provided a structural basis for the rational design of fusion inhibitors as potential antiviral therapeutics. In this review, we summarize our latest understanding of the HIV-1 membrane fusion process and discuss related therapeutic strategies to block viral entry.     

Quantifying Susceptibility of CD4+ Stem Memory T-Cells to Infection by Laboratory Adapted and Clinical HIV-1 Strains

CD4⁺ T cells are principal targets for human immunodeficiency virus type 1 (HIV-1) infection. CD4⁺ T cell subsets are heterogeneous cell populations, divided by functional and phenotypic differences into naïve and memory T cells. The memory CD4⁺ T cells are further segregated into central, effector and transitional memory cell subsets by functional, phenotypic and homeostatic characteristics. Defining the distribution of HIV-1 infection in different T cell subsets is important, as this can play a role in determining the size and composition of the viral reservoir. Both central memory and transitional memory CD4⁺ T cells have been described as long-lived viral reservoirs for HIV. Recently, the newly described stem memory T cell subset has also been implicated as a long-lived HIV reservoir. Using green fluorescent protein (GFP) reporter strains of HIV-1 and multi parameter flow cytometry, we developed an assay to simultaneously quantify the susceptibility of stem memory (TSCM), central memory, effector memory, transitional memory and naïve CD4⁺ T cell subsets, to HIV-1 infection in vitro. We show that TSCM are susceptible to infection with laboratory adapted and clinical HIV-1 strains. Our system facilitates the quantitation of HIV-1 infection in alternative T cell subsets by CCR5- and CXCR4-using viruses across different HIV-1 subtypes, and will be useful for studies of HIV-1 pathogenesis and viral reservoirs.     

Vaccine-induced plasma IgA specific for the C1 region of the HIV-1 envelope blocks binding and effector function of IgG

Analysis of correlates of risk of infection in the RV144 HIV-1 vaccine efficacy trial demonstrated that plasma IgG against the HIV-1 envelope (Env) variable region 1 and 2 inversely correlated with risk, whereas HIV-1 Env-specific plasma IgA responses directly correlated with risk. In the secondary analysis, antibody-dependent cellular cytotoxicity (ADCC) was another inverse correlate of risk, but only in the presence of low plasma IgA Env-specific antibodies. Thus, we investigated the hypothesis that IgA could attenuate the protective effect of IgG responses through competition for the same Env binding sites. We report that Env-specific plasma IgA/IgG ratios are higher in infected than in uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env glycoprotein 120 (gp120). An Env-specific monomeric IgA mAb isolated from an RV144 vaccinee also inhibited the ability of natural killer cells to kill HIV-1–infected CD4⁺ T cells coated with RV144-induced IgG antibodies. We show that monomeric Env-specific IgA, as part of postvaccination polyclonal antibody response, may modulate vaccine-induced immunity by diminishing ADCC effector function.The phase III RV144 ALVAC/AIDSVAX B/E HIV-1 vaccine efficacy trial in Thailand demonstrated 31.2% estimated vaccine efficacy through 42 mo of follow-up (1). Analysis of correlates of risk of infection indicated that envelope (Env)-specific plasma antibody responses were associated with a lower infection risk in vaccinees (2). Though plasma Env variable region 1 and 2 (V1/V2) IgG correlated with decreased infection risk, high levels of anti–HIV-1 Env plasma IgA correlated with increased infection risk (2). Interaction analyses demonstrated that, in the presence of low IgA Env antibodies, antibody-dependent cellular cytotoxicity (ADCC) responses inversely correlated with risk of infection, whereas in the presence of high IgA Env plasma antibodies, there was no correlation with risk of infection (2). Because there was no overall enhancement of infection risk in the trial (1), we hypothesized that Env IgA might block potentially protective effector functions of Env IgG antibodies.Antibody function depends, in part, on ability to bind to Fc receptors (FcR) on effector cells. The antibody isotype and subclass influences its affinity for different cellular FcRs (3, 4). IgG antibodies that mediate ADCC through natural killer (NK) cells bind to FcγRIIIa (CD16). In contrast, IgA antibodies do not bind to FcγRIIIa, but, rather, have high affinity for FcαRI (CD89) expressed by monocytes/macrophages and polymorphonuclear cells (PMN). This differential profile of FcR binding by IgG and IgA antibodies impacts the effector function capabilities of these antibody isotypes.Here, we examined plasma IgA and monomeric IgA monoclonal antibodies from RV144 vaccine recipients to test the hypothesis that some fraction of the vaccine-elicited IgA response could block IgG-mediated ADCC function. We found that a fraction of the HIV-1–specific IgA response was directed against conformational epitopes in the first constant (C1) region of glycoprotein 120 (gp120), a specificity of RV144 vaccine trial antibodies previously shown to mediate ADCC via NK cells (5, 6). Two IgA monoclonal antibodies, CH38 IgA₂ and CH29 IgA₂, were targeted to gp120 ADCC epitopes expressed on the surface of HIV-1–infected CD4 T cells. These IgA antibodies did not mediate ADCC via NK effector cells themselves, but rather blocked ADCC effector function on infected CD4⁺ T-cell targets by HIV-1 Env IgG. Thus, the RV144 vaccine-elicited polyclonal antibody response included IgA antibodies with specificities that blocked IgG-mediated ADCC.     

TRIM5α Restriction of HIV-1-N74D Viruses in Lymphocytes Is Caused by a Loss of Cyclophilin A Protection

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein CPSF6. In primary human CD4⁺ T cells, HIV-1-N74D viruses exhibit an infectivity defect when compared to wild-type. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4⁺ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D viruses is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5α_hu) restriction in CD4⁺ T cells, we found that depletion of TRIM5α_hu in CD4⁺ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5α_hu. Accordingly, TRIM5α_hu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In agreement with the notion that N74D capsids are defective in their ability to bind Cyp A, we found that HIV-1-N74D viruses were 20-fold less sensitive to TRIMCyp restriction when compared to wild-type viruses in OMK cells. Structural analysis revealed that N74D hexameric capsid protein in complex with PF74 is different from wild-type hexameric capsid protein in complex with PF74, which explains the defect of N74D capsids to interact with Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4⁺ T cells is due to a loss of Cyp A protection from TRIM5α_hu restriction activity.     

HIV-1 gp41 contains two sites for interaction with several proteins on the helper T-lymphoid cell line, H9.

OBJECTIVE: To characterize putative binding sites for HIV-1 gp41 to H9 cells. DESIGN: Based on accumulating evidence in the literature that HIV-1 can bind to cell surfaces independent of CD4, we attempted to clarify whether gp41, the transmembrane HIV-1 protein, contributes to CD4-independent binding. We therefore tested binding of gp41 to cells. METHODS: Using fluorescence-activated cell-sorter analysis, we examined the binding of recombinant gp160 (gp160) and soluble gp41 (sgp41; Env amino acids 539-684) to H9 cells, and located the putative binding site(s) of gp41 by inhibition using 16 HIV-1 Env peptides. Putative HIV-1 receptor proteins in H9 cell lysates were Western blotted and precipitated using sgp41. RESULTS: sgp41 bound to the CD4+ H9 cells and rgp160 bound to H9 cells independent of gp120-binding sites on CD4 molecules. Two gp41 peptides (Env amino acids 591-605 and 651-665) inhibited the binding of sgp41 to H9 cells. Four bands, of 37, 40, 55 and 97 kD, were blotted in H9 cell lysates, and three bands, 40, 97 and 108 kD, were observed in the precipitation analysis using lysates of 125I-surface-labelled H9 cells and sgp41 attached to sepharose beads. CONCLUSIONS: HIV-1 gp41 contains two putative binding sites to H9 cells. These sites may be located within Env amino acids 591-605 (ERYLKDQQLLGIWGC) and 651-665 (TLLEESQNQQEKNEQ). Using two different techniques, five proteins (37, 40, 55, 97 and 108 kD) were identified in H9 lysates as possible candidates for gp41 binding.     

NK cytotoxicity against CD4+ T cells during HIV-1 infection: a gp41 peptide induces the expression of an NKp44 ligand

HIV infection leads to a state of chronic immune activation and progressive deterioration in immune function, manifested most recognizably by the progressive depletion of CD4⁺ T cells. A substantial percentage of natural killer (NK) cells from patients with HIV infection are activated and express the natural cytotoxicity receptor (NCR) NKp44. Here we show that a cellular ligand for NKp44 (NKp44L) is expressed during HIV-1 infection and is correlated with both the progression of CD4⁺ T cell depletion and the increase of viral load. CD4⁺ T cells expressing this ligand are highly sensitive to the NK lysis activity mediated by NKp44⁺ NK cells. The expression of NKp44L is induced by the linear motif NH2-SWSNKS-COOH of the HIV-1 envelope gp41 protein. This highly conserved motif appears critical to the sharp increase in NK lysis of CD4⁺ T cells from HIV-infected patients. These studies strongly suggest that induction of NKp44L plays a key role in the lysis of CD4⁺ T cells by activated NK cells in HIV infection and consequently provide a framework for considering how HIV-1 may use NK cell immune surveillance to trigger CD4⁺ T cells. Understanding this mechanism may help to develop future therapeutic strategies and vaccines against HIV-1 infection.     

In vivo imaging of CD8+ T cell-mediated elimination of malaria liver stages

CD8⁺ T cells are specialized cells of the adaptive immune system capable of finding and eliminating pathogen-infected cells. To date it has not been possible to observe the destruction of any pathogen by CD8⁺ T cells in vivo. Here we demonstrate a technique for imaging the killing of liver-stage malaria parasites by CD8⁺ T cells bearing a transgenic T cell receptor specific for a parasite epitope. We report several features that have not been described by in vitro analysis of the process, chiefly the formation of large clusters of effector CD8⁺ T cells around infected hepatocytes. The formation of clusters requires antigen-specific CD8⁺ T cells and signaling by G protein-coupled receptors, although CD8⁺ T cells of unrelated specificity are also recruited to clusters. By combining mathematical modeling and data analysis, we suggest that formation of clusters is mainly driven by enhanced recruitment of T cells into larger clusters. We further show various death phenotypes of the parasite, which typically follow prolonged interactions between infected hepatocytes and CD8⁺ T cells. These findings stress the need for intravital imaging for dissecting the fine mechanisms of pathogen recognition and killing by CD8⁺ T cells.     

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate

The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4⁺ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4⁺ CCR-5⁺ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1_BX08 isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1_LAI strain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.     

A Replication-Competent HIV Clone Carrying GFP-Env Reveals Rapid Env Recycling at the HIV-1 T Cell Virological Synapse

HIV-1 infection is enhanced by cell–cell adhesions between infected and uninfected T cells called virological synapses (VS). VS are initiated by the interactions of cell-surface HIV-1 envelope glycoprotein (Env) and CD4 on target cells and act as sites of viral assembly and viral transfer between cells. To study the process that recruits and retains HIV-1 Env at the VS, a replication-competent HIV-1 clone carrying an Env-sfGFP fusion protein was designed to enable live tracking of Env within infected cells. Combined use of surface pulse-labeling of Env and fluorescence recovery after photobleaching (FRAP) studies, enabled the visualization of the targeted accumulation and sustained recycling of Env between endocytic compartments (EC) and the VS. We observed dynamic exchange of Env at the VS, while the viral structural protein, Gag, was largely immobile at the VS. The disparate exchange rates of Gag and Env at the synapse support that the trafficking and/or retention of a majority of Env towards the VS is not maintained by entrapment by a Gag lattice or immobilization by binding to CD4 on the target cell. A FRAP study of an Env endocytosis mutant showed that recycling is not required for accumulation at the VS, but is required for the rapid exchange of Env at the VS. We conclude that the mechanism of Env accumulation at the VS and incorporation into nascent particles involves continuous internalization and targeted secretion rather than irreversible interactions with the budding virus, but that this recycling is largely dispensable for VS formation and viral transfer across the VS.     

From the Cover: Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo

Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4⁺T cells. Some of these CD4⁺T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4⁺ T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1–infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4⁺T cells can be a reservoir of infectious HIV-1.HIV-1 infection can be controlled, but not cured, by combination antiretroviral therapy (1); cells infected with replication-competent HIV-1 persist despite many years of combination antiretroviral therapy (cART). Recently, we and others (2, 3) showed that there is frequent clonal expansion of HIV-1–infected cells in patients and that these clones can persist for more than 10 y. Most of the proviruses that persist in patients on cART are defective (4), and it is not known whether clonally expanded cells can harbor a replication-competent provirus. It has been suggested that CD4⁺ T-cell clones are not likely to contain replication-competent HIV-1 because expression of viral proteins is cytotoxic, and the host’s immune system would preferentially eliminate infected cells that express viral proteins (4). Only a small proportion of integrated proviruses are intact (4), and it has been proposed that clonally expanded CD4⁺ T cells contain only defective proviruses and that intact proviruses that make up the reservoir are found only in unexpanded cells (5). Previous studies have reported that populations of virus with identical sequences emerge in the plasma after several years of suppressive cART (6, 7), but it is not known whether the identical viruses found in the blood of patients are infectious. We report here the identification of a highly expanded clone of HIV-infected CD4⁺ T cells that produced infectious virus at a level that caused persistent plasma viremia. This result shows that cells containing replication-competent HIV-1 proviruses can clonally expand and persist in vivo, presenting a challenge for achieving a cure of HIV-1 infection.     

The integrin α4β7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1

Both activated and resting CD4⁺ T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin α₄β₇ (α₄β₇), the gut mucosal homing receptor. We find that α₄β₇^high CD4⁺ T cells are more susceptible to productive infection than are α₄β₇^low-neg CD4⁺ T cells in part because this cellular subset is enriched with metabolically active CD4⁺ T cells. α₄β₇^high CD4⁺ T cells are CCR5^high and CXCR4^low; on these cells, α₄β₇ appears in a complex with CD4. The specific affinity of gp120 for α₄β₇ provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.     

Antigenic fractions of Leishmania (Viannia) braziliensis: the immune response characterization of patients at the initial phase of disease

American cutaneous leishmaniasis (ACL) has different clinical manifestations and these manifestations are dependent on the immunological status of the host. As CD4⁺ and CD8⁺ T cells and their mediators play a fundamental role in the host response to Leishmania and there is also a search for antigenic molecules to be used as future vaccines and tools for prognostic tests, this study characterized ACL patients’ immune response after stimulation with soluble and insoluble fractions of L. (V.) braziliensis. We demonstrated a prevailing production of the Th2 cytokines, IL‐4 and IL‐10 and a specific production of IFN‐γ and TNF‐α in patients before treatment. There was also a predominance of CD4⁺ T cells and a small percentage CD8⁺ T cells. The insoluble antigenic fraction primarily stimulated CD4⁺ T cells, while the soluble antigenic fraction showed a mixed profile, with CD4⁺ T cells being the main responsible for Th2 cytokines and CD8⁺ T cells for Th1 cytokines. Therefore, our results showed that a down‐modulation of the Th1 type of response occurs in the initial phase of L. braziliensis disease, being the antigenic fractions capable of stimulating a specific immune response.