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CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5
Abstract
For efficient entry into target cells, primary macrophage-tropic and laboratory-adapted human immunodeficiency viruses type 1 (HIV-1) require particular chemokine receptors, CCR-5 and CXCR-4, respectively, as well as the primary receptor CD4 (refs 1-6). Here we show that a complex of gp120, the exterior envelope glycoprotein, of macrophage-tropic primary HIV-1 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta (refs 7, 8). The apparent affinity of the interaction between gp120 and CCR-5 was dramatically lower in the absence of soluble CD4. Additionally, in the absence of gp120, an interaction between a two-domain CD4 fragment and CCR-5 was observed. A gp120 fragment retaining the CD4-binding site and overlapping epitopes was able to interact with CCR-5 only if the V3 loop, which can specify HIV-1 tropism and chemokine receptor choice, was also present on the molecule. Neutralizing antibodies directed against either CD4-induced or V3 epitopes on gp120 blocked the interaction of gp12O-CD4 complexes with CCR-5. These results suggest that HIV-1 attachment to CD4 creates a high-affinity binding site for CCR-5, leading to membrane fusion and virus entry.
... A step in the entry process intermediate between gp120-CD4 attachment and gp41 conformational changes involves a coreceptor for gp120 (21,31,37,85,98). Thus, after gp120 has bound to CD4, it changes conformation to enable it to bind to a coreceptor from the G-protein-coupled receptor superfamily (21,31,37,85,98,107,116). The most physiologically relevant coreceptors are the chemokine receptors CCR5 or CXCR4, the former used by HIV-1 strains that usually dominate early in infection and the latter used by viruses that sometimes emerge several years later or that are detectable only transiently (21,31,85,99,122). ...
... Alternatively, the Env complex on the fully AD101-resistant virus may have evolved to recognize a specific CCR5 isoform or conformation present only on human PBMC (or on primary cells in general) and absent from L1.2-CCR5 cells. The latter are CCR5 transfectants of a murine pre-B-cell lymphoma line (116), so that post-translational modifications of CCR5 may be subtly different from what happens in primary human cells. Again, there is precedent for chemokine receptors, including CCR5, existing in multiple isoforms and conformations (6,10,11,18,36,63,64,67,70). ...
We have described previously the generation of an escape variant of human immunodeficiency virus type 1 (HIV-1), under the selection pressure of AD101, a small molecule inhibitor that binds the CCR5 coreceptor (A. Trkola, S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P. Pugach, S. X. L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S. McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.
... The proteolytic cleavage site between gp120 and gp41 was altered, with two serine residues substituted for Arg 508 and Arg 511. In the HIV-1 JR-FL Env(2) glycoprotein, the amino acid sequence LVPRGS-(His) 6 was added to the C terminus of the cytoplasmic tail. For Env(2) expression, the env coding sequences were cloned immediately downstream of the tetracycline (Tet)-responsive element (TRE). ...
... After labeling, Env(2) trimers were purified using Zeba spin desalting columns (ThermoFisher) to remove free dyes. Finally, prior to imaging, fluorescence-labeled HIV-1 JR-FL Env(2) carrying the (His) 6 epitope tag was incubated with biotin-conjugated anti-(His) 6 tag antibody (HIS.H8; Invitrogen) at 4°C for 2 h. ...
The functional human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] is produced by cleavage of a conformationally flexible gp160 precursor. Gp160 cleavage or the binding of BMS-806, an entry inhibitor, stabilizes the pre-triggered, “closed” (State-1) conformation recognized by rarely elicited broadly neutralizing antibodies. Poorly neutralizing antibodies (pNAbs) elicited at high titers during natural infection recognize more “open” Env conformations (States 2 and 3) induced by binding the receptor, CD4. We found that BMS-806 treatment and crosslinking decreased the exposure of pNAb epitopes on cell-surface gp160; however, after detergent solubilization, crosslinked and BMS-806-treated gp160 sampled non-State-1 conformations that could be recognized by pNAbs. Cryo-electron microscopy of the purified BMS-806-bound gp160 revealed two hitherto unknown asymmetric trimer conformations, providing insights into the allosteric coupling between trimer opening and structural variation in the gp41 HR1 N region. The individual protomer structures in the asymmetric gp160 trimers resemble those of other genetically modified or antibody-bound cleaved HIV-1 Env trimers, which have been suggested to assume State-2-like conformations. Asymmetry of the uncleaved Env potentially exposes surfaces of the trimer to pNAbs. To evaluate the effect of stabilizing a State-1-like conformation of the membrane Env precursor, we treated cells expressing wild-type HIV-1 Env with BMS-806. BMS-806 treatment decreased both gp160 cleavage and the addition of complex glycans, implying that gp160 conformational flexibility contributes to the efficiency of these processes. Selective pressure to maintain flexibility in the precursor of functional Env allows the uncleaved Env to sample asymmetric conformations that potentially skew host antibody responses toward pNAbs.
IMPORTANCE
The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The functional Env trimer is produced by cleavage of the gp160 precursor in the infected cell. We found that the HIV-1 Env precursor is highly plastic, allowing it to assume different asymmetric shapes. This conformational plasticity is potentially important for Env cleavage and proper modification by sugars. Having a flexible, asymmetric Env precursor that can misdirect host antibody responses without compromising virus infectivity would be an advantage to a persistent virus like HIV-1.
... The interaction between a host CD4 receptor and a gp120 trimer on the external surface of the viral membrane induces a conformational shift in the envelope protein. This exposes further binding sites, allowing gp120 to bind to the viral co-receptors CXCR4 or CCR5 [71,72], inducing a further conformational shift which causes the trimeric gp41 to "spring out" and insert itself into the host-cell membrane [73,74]. This initiates membrane fusion, merging the host and viral membranes and enabling the viral capsid, which contains the viral RNA genome and viral proteins such as reverse transcriptase and integrase, to enter the cell [69,70]. ...
... While this provides more precise insight into specific co-receptor driven signaling processes, it is important to note that the concentrations of gp120 used in many of the following studies are unlikely to represent levels in cART-treated individuals. Further, the gp120 driving these processes in the CNS of cART-treated PLWH are likely to present on the virion surface in complex with gp41 and have a different structure than free gp120 [70,364], and may require interaction with CD4 [71,74], even if the downstream signaling effects are driven solely by the co-receptors. However, while some studies suggest there may be some differences between the responses of shed gp120 and virus associated gp120 [364], many indicate that monomeric gp120 and virus-associated gp120 largely initiate the same signaling cascades [213,264,285,365,366,[368][369][370]. ...
The HIV co-receptors, CCR5 and CXCR4, are necessary for HIV entry into target cells, interacting with the HIV envelope protein, gp120, to initiate several signaling cascades thought to be important to the entry process. Co-receptor signaling may also promote the development of neuroHIV by contributing to both persistent neuroinflammation and indirect neurotoxicity. But despite the critical importance of CXCR4 and CCR5 signaling to HIV pathogenesis, there is only one therapeutic (the CCR5 inhibitor Maraviroc) that targets these receptors. Moreover, our understanding of co-receptor signaling in the specific context of neuroHIV is relatively poor. Research into co-receptor signaling has largely stalled in the past decade, possibly owing to the complexity of the signaling cascades and functions mediated by these receptors. Examining the many signaling pathways triggered by co-receptor activation has been challenging due to the lack of specific molecular tools targeting many of the proteins involved in these pathways and the wide array of model systems used across these experiments. Studies examining the impact of co-receptor signaling on HIV neuropathogenesis often show activation of multiple overlapping pathways by similar stimuli, leading to contradictory data on the effects of co-receptor activation. To address this, we will broadly review HIV infection and neuropathogenesis, examine different co-receptor mediated signaling pathways and functions, then discuss the HIV mediated signaling and the differences between activation induced by HIV and cognate ligands. We will assess the specific effects of co-receptor activation on neuropathogenesis, focusing on neuroinflammation. We will also explore how the use of substances of abuse, which are highly prevalent in people living with HIV, can exacerbate the neuropathogenic effects of co-receptor signaling. Finally, we will discuss the current state of therapeutics targeting co-receptors, highlighting challenges the field has faced and areas in which research into co-receptor signaling would yield the most therapeutic benefit in the context of HIV infection. This discussion will provide a comprehensive overview of what is known and what remains to be explored in regard to co-receptor signaling and HIV infection, and will emphasize the potential value of HIV co-receptors as a target for future therapeutic development.
... As mentioned above, the concept of targeting cytokines or cytokine pathways to control specific diseases or processes is not new. After the role of receptors in viral entry was established, these pathways have even been targeted for infectious diseases such as HIV (CXCR4 and CCR5) [32][33][34]. Although most primary solid or intraepithelial tumors can be treated with several types of chemotherapy and radiation, adaptive resistance of tumor cells and recurrence of cancer have hampered durable success of these therapies. ...
Precision cancer medicine primarily aims to identify individual patient genomic variations and exploit vulnerabilities in cancer cells to select suitable patients for specific drugs. These genomic features are commonly determined by gene sequencing prior to therapy, to identify individuals who would be most responsive. This precision approach in cancer therapeutics remains a powerful tool that benefits a smaller pool of patients, sparing others from unnecessary treatments. A limitation of this approach is that proteins, not genes, are the ultimate effectors of biological functions, and therefore the targets of therapeutics. An additional dimension in precision medicine that considers an individual’s cytokine response to cancer therapeutics is proposed. Cytokine responses to therapy are multifactorial and vary among individuals. Thus, precision is dictated by the nature and magnitude of cytokine responses in the tumor microenvironment exposed to therapy. This review highlights cytokine responses as modules for precision medicine in cancer therapy, including potential challenges. For solid tumors, both detectability of cytokines in tissue fluids and their being amenable to routine sensitive analyses could address the difficulty of specimen collection for diagnosis and monitoring. Therefore, in precision cancer medicine, cytokines offer rational targets that can be utilized to enhance the efficacy of cancer therapy.
... As mentioned above, the concept of targeting cytokines or cytokine pathways to control specific diseases or processes is not new. After the role of receptors in viral entry was established, these pathways have even been targeted for infectious diseases such as HIV (CXCR4 and CCR5) [32][33][34]. Although most primary solid or intraepithelial tumors can be treated with several types of chemotherapy and radiation, adaptive resistance of tumor cells and recurrence of cancer have hampered durable success of these therapies. ...
Precision cancer medicine primarily aims to identify individual patient genomic variations and exploit vulnerabilities in cancer cells to select suitable patients for specific drugs. These genomic features are commonly determined by gene sequencing prior to therapy, to identify individuals who would be most responsive. This precision approach in cancer therapeutics remains a powerful tool that benefits a smaller pool of patients, sparing others from unnecessary treatments. A limitation of this approach is that proteins, not genes, are the ultimate effectors of biological functions, and therefore the targets of therapeutics. An additional dimension in precision medicine that considers an individual’s cytokine response to cancer therapeutics is proposed. Cytokine responses to therapy are multifactorial and vary among individuals. Thus, precision is dictated by the nature and magnitude of cytokine responses in the tumor microenvironment exposed to therapy. This review highlights cytokine responses as modules for precision medicine in cancer therapy, including potential challenges. For solid tumors, both detectability of cytokines in tissue fluids and their being amenable to routine sensitive analyses could address the difficulty of specimen collection for diagnosis and monitoring. Therefore, in precision cancer medicine, cytokines offer rational targets that can be utilized to enhance the efficacy of cancer therapy.
... All four of these individuals received bone marrow transplants of cells harboring a 32-bp dele-tion in the human C-C chemokine receptor type 5 gene (CCR5 D32), which prevents use of CCR5 as a co-receptor for HIV-1 entry into CD4 + T cells. [11][12][13][14][15][16][17] Strategies to block HIV-1 entry by ex vivo genetic modification of autologous T cells or CD34 + hematopoietic stem and progenitor cells (HSPCs) have been investigated using ribozymes, RNA interference, and inactivating the CCR5 or CXC chemokine receptor type 4 (CXCR4) co-receptor genes in HSPCs or CD4 + T cells (reviewed in Sheykhhasan et al. 18 ). Fusion inhibitor peptides, such as the T20 peptide approved for clinical use, 19 interfere with the fusion of the viral and host membranes by binding to the gp41 envelope helical trimeric heptad repeats (HRs) and blocking formation of a 6-helix bundle that enables fusion (reviewed in Wilen and co-workers 20,21 ). ...
Gene therapy strategies that effectively inhibit HIV-1 replication are needed to reduce the requirement for lifelong antiviral therapy and potentially achieve a functional cure. We previously designed self-activating lentiviral vectors that efficiently delivered and expressed a Vif-resistant mutant of APOBEC3G (A3G-D128K) to T cells, which potently inhibited HIV-1 replication and spread with no detectable virus. Here, we developed vectors that express A3G-D128K, membrane-associated fusion inhibitor peptide mC46, and O6-methylguanine-DNA-methyltransferase (MGMT) selectable marker for in vivo selection of transduced CD34+ hematopoietic stem and progenitor cells. MGMT-selected T cell lines MT4, CEM, and PM1 expressing A3G-D128K (with or without mC46) potently inhibited NL4-3 infection up to 45 days post infection with no detectable viral replication. Expression of mC46 was sufficient to block infection >80% in a single-cycle assay. Importantly, expression of mC46 provided a selective advantage to the A3G-D128K-modified T cells in the presence of replication competent virus. This combinational approach to first block HIV-1 entry with mC46, and then block any breakthrough infection with A3G-D128K, could provide an effective gene therapy treatment and a potential functional cure for HIV-1 infection.
... Other membrane proteins involved in virus entry are also of importance in understanding disease pathogenesis. The chemokine receptors CCR5 and CXCR4 have been reported as co-receptors involved in human immunodeficiency virus infection (9)(10)(11), and HLA class II receptors function as coreceptors in Epstein-Barr virus infection (12). Researchers working on vaccine and antiviral agent development are interested in the role of viral co-receptors (cofactors), in addition to primary receptors (13). ...
Coronavirus disease (COVID-19) represents a real threat to the global population, and understanding the biological features of the causative virus, i.e., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is imperative for mitigating this threat. Analyses of proteins such as primary receptors and co-receptors (co-factors), which are involved in the entry of SARS-CoV-2 into host cells, will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates present in proximity to the attachment sites of SARS-CoV-2 spike proteins, using proximity labeling and proteomic analysis. The identified proteins represent key candidate factors that may be required for viral entry. We found SARS-CoV-2 host protein DPP4, cell adhesion protein Cadherin-17, and glycoprotein CD133 co-localized with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells and thus showed potential as candidate factors. Additionally, our analysis of the experimental infection of HEK293T cells with a SARS-CoV-2 pseudovirus indicated a 2-fold enhanced infectivity in the CD133-ACE2-coexpressing HEK293T cells compared to that in HEK293T cells expressing ACE-2 alone. The information and resources regarding these co-receptor labeling and analysis techniques could be utilized for the development of antiviral agents against SARS-CoV-2 and other emerging viruses.
... It is worth noting that the V3 loop presents a complete stability in length and glycosylation sites. This V3 loop is key for viral tropism [175][176][177][178][179] and for the correct CD4 Env binding as revealed with anti-V3 neutralizing antibodies that abrogate Env-CD4 interaction, as the authors and others have reported [180,181]. Furthermore, during chronic infection, higher PNG density has been observed in the V1-V5 region of the gp120 subunit of the HIV-1 Env complex compared to the PNG density observed during the early acute phase of the infection [182]. ...
In the absence of antiviral therapy, HIV-1 infection progresses to a wide spectrum of clinical manifestations that are the result of an entangled contribution of host, immune and viral factors. The contribution of these factors is not completely established. Several investigations have described the involvement of the immune system in the viral control. In addition, distinct HLA-B alleles, HLA-B27, -B57-58, were associated with infection control. The combination of these elements and antiviral host restriction factors results in different clinical outcomes. The role of the viral proteins in HIV-1 infection has been, however, less investigated. We will review contributions dedicated to the pathogenesis of HIV-1 infection focusing on studies identifying the function of the viral envelope glycoprotein (Env) in the clinical progression because of its essential role in the initial events of the virus life-cycle. Some analysis showed that inefficient viral Envs were dominant in non-progressor individuals. These poorly-functional viral proteins resulted in lower cellular activation, viral replication and minor viral loads. This limited viral antigenic production allows a better immune response and a lower immune exhaustion. Thus, the properties of HIV-1 Env are significant in the clinical outcome of the HIV-1 infection and AIDS pathogenesis.
... La gp120 se lie au récepteur CD4 exprimé par les cellules immunitaires (LT CD4 + , monocytes/macrophages, et DC) par leur domaine de liaison au CD4 (Klatzmann and Gluckman, 1986;Klatzmann et al., 1984;Kwong et al., 1998;Lin et al., 2000;McDougal et al., 1986;Wu et al., 1996). Cette liaison induit un changement de conformation du trimère irréversible (Chen, 2019;Sattentau et al., 1993) et expose des sites jusqu'alors cachés tels que la boucle variable V3, essentiels à la fixation du corécepteur ( Figure 3) Freed et al., 1990). ...
Aucun vaccin contre le VIH-1 n’existe malgré plus de 30 ans de recherche. L’étude des mécanismes de défense développés par des patients contrôlant l’infection est importante pour définir des stratégies de vaccination efficaces et endiguer la pandémie. L’induction d’anticorps neutralisants (AcN) à large spectre est une piste attrayante pour contrôler l’infection VIH-1. Cependant, l’induction d’AcN nécessite de stimuler le système immunitaire avec un antigène conservé et d’induire une maturation des lymphocytes B spécifique. Les capacités de recombinaison, le fort taux de mutation et la conformation de l’unique protéine de surface Env sont responsables de la difficulté à trouver des antigènes cibles pour la vaccination. L’amélioration de la compréhension des mécanismes cellulaires et moléculaires des adjuvants permettront d’améliorer les stratégies vaccinales. Le vaccin candidat contre le VIH-1 composé du peptide W614A-3S-KLH formulé avec un adjuvant Squalene induit une réponse humorale neutralisant un large éventail de souches du VIH-1. La formulation de ce même peptide avec un adjuvant Alum induit des anticorps incapables de neutraliser différentes souches de VIH-1. Cette dichotomie entre deux adjuvant a permis d’étudier les réponses adaptatives et innées qui conduisent à la production d’AcN. La formulation Squalene a favorisé la réaction du centre germinatif et induit une maturation différente des LB par rapport à l’Alum et elle a induit un recrutement précoce et intense de neutrophiles dans les ganglions lymphatiques. Ces neutrophiles sont activés différemment en fonction de l’adjuvant utilisé mais leur rôle n’était pas déterminant pour l’induction d’une réponse humorale.
... In contrast with the more significant changes detected in the V2 and V5 loops, it is important to point to the stability in length and glycosylation of the V3 loop. This structure is key for viral tropism (Cocchi et al., 1996;Wu et al., 1996;Bieniasz et al., 1997;Speck et al., 1997;Isaka et al., 1999) and for the correct CD4 Env binding as revealed with anti-V3 neutralizing antibodies that abrogate Env-CD4 interaction (Trkola et al., 1996;Valenzuela et al., 1997). ...
The understanding of HIV-1 pathogenesis and clinical progression is incomplete due to the variable contribution of host, immune, and viral factors. The involvement of viral factors has been investigated in extreme clinical phenotypes from rapid progressors to long-term non-progressors (LTNPs). Among HIV-1 proteins, the envelope glycoprotein complex (Env) has been concentrated on in many studies for its important role in the immune response and in the first steps of viral replication. In this study, we analyzed the contribution of 41 Envs from 24 patients with different clinical progression rates and viral loads (VLs), LTNP-Elite Controllers (LTNP-ECs); Viremic LTNPs (vLTNPs), and non-controller individuals contemporary to LTNPs or recent, named Old and Modern progressors. We studied the Env expression, the fusion and cell-to-cell transfer capacities, as well as viral infectivity. The sequence and phylogenetic analysis of Envs were also performed. In every functional characteristic, the Envs from subjects with viral control (LTNP-ECs and vLTNPs) showed significant lower performance compared to those from the progressor individuals (Old and Modern). Regarding sequence analysis, the variable loops of the gp120 subunit of the Env (i.e., V2, V4, and mainly V5) of the progressor individuals showed longer and more glycosylated sequences than controller subjects. Therefore, HIV-1 Envs from virus of patients presenting viremic control and the non-progressor clinical phenotype showed poor viral functions and shorter sequences, whereas functional Envs were associated with virus of patients lacking virological control and with progressor clinical phenotypes. These correlations support the role of Env genotypic and phenotypic characteristics in the in vivo HIV-1 infection and pathogenesis.
... State 3 represents a more open three-CD4-bound Env trimer conformational state (43,(45)(46)(47) (Fig. 1). CD4 binding induces several structural changes such as movement of the V1/V2 loops from the trimer apex to the periphery (43,(46)(47)(48)(49)(50)(51)(52), reordering of the gp120 beta sheet bridging domain (46,50,(52)(53)(54), CoRBS formation (46,47,(55)(56)(57)(58), and gp41 HR1 coiled-coil assembly at the trimer axis (46,47,52,(59)(60)(61). HIV-1 Env trimers that favor State 3 can mediate infection of target cells with low or no CD4 expression (44,53,(62)(63)(64)(65)(66)(67)(68)(69)(70). ...
The HIV Env glycoprotein is the surface glycoprotein responsible for viral entry into CD4+ immune cells. During infection, Env also serves as a primary target for antibody responses, which are robust but unable to control virus replication. Immune evasion by HIV-1 Env appears to employ complex mechanisms to regulate what antigenic states are presented to the immune system. Immunodominant features appear to be distinct from epitopes that interfere with Env functions in mediating infection. Further, cell-cell transmission studies indicate that vulnerable conformational states are additionally hidden from recognition on infected cells, even though the presence of Env at the cell surface is required for viral infection through the virological synapse. Cell-cell infection studies support that Env on infected cells is presented in distinct conformations from that on virus particles. Here we review data regarding the regulation of conformational states of Env and assess how regulated sorting of Env within the infected cell may underlie mechanisms to distinguish Env on the surface of virus particles versus Env on the surface of infected cells. These mechanisms may allow infected cells to avoid opsonization, providing cell-to-cell infection by HIV with a selective advantage during evolution within an infected individual. Understanding how distinct Env conformations are presented on cells versus viruses may be essential to designing effective vaccine approaches and therapeutic strategies to clear infected cell reservoirs.
... For successful entry of the host cell, the HIV-1 particle binds first to CD4 and opens the CCR binding domain in the gp120 variable loop; then, binding to CCR5 forms an integration complex, which mediates fusion of the virion into the cell (67). To determine whether the mutations in CCR5 prevented infection, TZM-bls were exposed to HIV-1 BaL and infectability was assessed by Luciferase Assay (Figure 5D). ...
Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.
... After some false starts such as mistaking complement receptor 3 for HIV-l's coreceptor (Stoiber et al., 1997), it was shown that a chemokine receptor is the coreceptor on T-cells and APCs (Dragic et al., 1996;Deng et al., 1996;Choe et al., 1996;Doranz et al., 1996;Rottman et al., 1997;Jones etal., 1998;Albright etal., 1999, reviewed in D'Souza andHarden, 1996;Weiss, 1996;Ross et al., 1999). ...
Human immunodeficiency virus (HIV) infects about 40 million people worldwide. HIV is the causative agent of acquired immunodeficiency syndrome (AIDS). AIDS is characterised by a progressive decline in protective immunity that leads to opportunistic infection and eventually death. Although HIV-1 causes a decline in CD4' T-cell number and this undoubtedly contributes to the general immune deficit of AIDS, CD4+ T-cell loss does not completely explain the pathogenesis of AIDS. Death and anergy of uninfected T-cells is observed in AIDS, as are deficits in innate and specific immunity. Antigen presenting cells(APCs, including macrophages and dendritic cells, DCs, which are infectable by M-tropic strains of HIV-1 via the CCR-5 chemokine receptor) play a key role in orchestrating innate and adaptive immune responses and controlling T-cell activities including activation, anergy, deletion, tolerisation and memory by the provision of appropriate signals. APC dysregulation results in deficits of innate and adaptive immune responses. It is known that HIV-1 can cause APC dysregulation; this thesis examines some mechanisms by which this might occur. The HIV-1 envelope glycoprotein gpl20 mediates HIV-1 infection by binding to target cells via CD4 and CCR-5 and is focussed on throughout this work. Because gpl20 is found on the surface of HIV-1 and dissolved in the serum of HIV-1 infected patients, it has the ability to disrupt the function of both infected and uninfected APCs. Data in this thesis demonstrate that gp 120 causes a decline of cell-surface CD4 from human macrophages in vitro. A mechanism for this loss is proposed based on observations that it is significantly more substantial when CCR-5-binding gpl20, derived from M-tropicHIV-1 is used as opposed to CXCR-4-binding gpl20. CD4 loss is absent from macrophages that fail to express surface CCR-5 due to homozygosity for the naturally occurring ccr5A32mutation. It appears that CD4 loss by this novel CCR-5-dependent mechanism requires cross-linking of CCR-5, CD4 and gp 120 at the cell surface leading to receptor-mediated endocytosis of this protein complex. Confocal microscopy was used to visualise these endocytosed proteins inside macrophages and RT-PCR was used to investigate transcriptional regulation of CD4 and CCR-5 recovery. Endocytosis of the protein complex may change antigen presentation efficiencies. Possible implications for protective- and auto¬immunity are discussed. This thesis also presents evidence that pre-treatment with gp!20 leads to reduction in an APC's ability to stimulate antigen-specific proliferation of a T-cell line. Because this effect is not dependent on the tropism of the HIV-1 strain from which the gpl20 is derived, an alternative mechanism to CD4-loss was sought. The hypothesis that APC dysfunction is due to HIV-1 subversion of physiological mechanisms involving prostaglandin and the Notch signalling pathway, leading to inappropriate tolerance induction, was examined. Treatment of macrophages and DCs with gpl20 caused the transcriptional up-regulation of genes involved in the Notch pathway including Notch ligands, the presence of which on an APC has previously been shown to abrogate T-cell activation by the induction of an anergic phenotype. Preventing HIV-1 infection of APCs and the subsequent dysregulation of immune responses is a therapeutic goal. Branched, synthetic peptides based on discontinuous epitopes of gpl20and previously demonstrated to disrupt binding to CD4 and CCR-5 are shown to protect macrophages from infection with M-tropic HIV-1Bal- Possible refinements to peptide structure and their utility as anti-HIV-1 therapeutics or vaccines are discussed
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... RTCs are packaged into a proteinaceous capsid, which in turn is surrounded by the matrix and the viral envelope ( Figure 3D). 132 The glycoproteins that are anchored in the envelope bind to the host cell receptor at the plasma membrane to initiate the HIV-1 cell entry through membrane fusion, 133 resulting in the cytoplasmic release of the viral capsid. 134 It was recently shown that cytoplasmic HIV-1 capsids are subsequently imported into the nucleus via nuclear pore complexes. ...
The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments, and their accessory proteins. Motor proteins, which comprise the dynein, kinesin, and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as the rabies disease and the acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, i.e., rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins. This article is protected by copyright. All rights reserved. Microtubules, actin filaments and intermediate filaments are central elements of the cell's cytoskeleton. Motor proteins such as dyneins, kinesins and myosins allow directional intracellular transport of cargoes along these cytoskeleton elements. Viruses can exploit the cytoskeleton to promote different steps of their life cycle by hijacking distinct motor proteins. This review describes a few examples of the mechanisms that some human viruses have developed to subvert specific dyneins, kinesins and myosins.
... On CD4 1 cells, numerous cell surface molecules have been identified that may promote HIV infection via an interaction with gp120/gp41, including DC-SIGN, the mannose receptor, langerin, CXCR6, and CCR8 (103), none of which are known to be expressed by cervical epithelial cells (15,104). Whereas HIV uses the canonical coreceptor CXCR4 or CCR5 for entry into CD4 1 cells, whether FRT epithelial cells express CXCR4 or CCR5 and, if expressed, the extent to which HIV has affinity for these receptors in the absence of CD4 are controversial (105). CXCR4 and CCR5 (as well as CR3 [18]) are not expressed by the ectocervical and endocervical epithelial cell lines Ect1 and End1, respectively (72). ...
In women, the lower female reproductive tract is the primary site for HIV infection. How HIV traverses the epithelium to infect CD4 T cells in the submucosa is ill-defined.
... Binding of CD4 to viral gp120 causes repositioning of variable loops, including V1-V3 and, thereby, exposing co-receptor binding sites (Trkola et al. 1996;Wu et al. 1996). The α-chemokine receptor, CXCR4, and β-chemokine receptor CCR5, are the two key secondary cellular receptors identified for T cell line-tropic and macrophage-tropic HIV-1 isolates; they belong to the members of the G protein-coupled receptor superfamily (GPCRs) (Allen et al. 2007). ...
The innate immune system is comprised of both cellular and humoral players that recognise and eradicate invading pathogens. Therefore, the interplay between retroviruses and innate immunity has emerged as an important component of viral pathogenesis. HIV-1 infection in humans that results in hematologic abnormalities and immune suppression is well represented by changes in the CD4/CD8 T cell ratio and consequent cell death causing CD4 lymphopenia. The innate immune responses by mucosal barriers such as complement, DCs, macrophages, and NK cells as well as cytokine/chemokine profiles attain great importance in acute HIV-1 infection, and thus, prevent mucosal capture and transmission of HIV-1. Conversely, HIV-1 has evolved to overcome innate immune responses through RNA-mediated rapid mutations, pathogen-associated molecular patterns (PAMPs) modification, down-regulation of NK cell activity and complement receptors, resulting in increased secretion of inflammatory factors. Consequently, epithelial tissues lining up female reproductive tract express innate immune sensors including anti-microbial peptides responsible for forming primary barriers and have displayed an effective potent anti-HIV activity during phase I/II clinical trials.
... To initiate the viral entry process, the outer gp120 protomer of the trimer binds the receptor CD4 on the host cell surface. Following CD4 binding, Env undergoes conformational changes that lead to the formation of the coreceptor binding site (CoRBS) and engagement of CCR5 or CXCR4, the two known HIV-1 coreceptors (2)(3)(4)(5)(6)(7)(8)(9). After coreceptor binding, additional structural rearrangements within Env occur that lead to the formation of a sixhelix bundle from the helical heptad repeat HR1 and HR2 segments of the gp41 ectodomain to drive fusion of viral and target cell membranes (10,11). ...
Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity.
... Gp120 is part of the Env protein which consists of a trimer of Gp41-Gp120 heterodimers (Wyatt & Sodroski, 1998;Tran et al., 2012). First, Gp120 binds to the CD4 receptor and then to the co-receptor CXCR4 or, in other cases, to CCR5, depending on the sequence of the third variable region (V3) of Gp120 (Lapham et al., 1996;Wu et al., 1996). The binding of the viral glycoprotein Gp120 to CXCR4 is the event triggering the fusion between the virus and the plasma membrane of the cell, further assisted by Gp41, which becomes exposed due to conformational rearrangements once Gp120 binds to CD4 (Wilen et al., 2012). ...
CXCR4 is a chemokine receptor that belongs to the superfamily of G protein-coupled receptors. The natural ligand of CXCR4 is the chemokine CXCL12. The interaction between the receptor and its ligand activates numerous signaling pathways, controlling cell migration, survival, proliferation and chemotaxis. The CXCL12/CXCR4 axis is essential in embryogenesis, hematopoiesis, in the regulation of the immune system and in tissue repair after wounding. Several pathological conditions are linked to deregulated CXCR4 signaling, such as WHIM syndrome, autoimmune diseases and cancer, where CXCR4 upregulation promotes tumor growth and metastasis. Although the central role of CXCR4 in physiology and disease has been extensively investigated during the last twenty-five years, efforts to develop pharmaceutical compounds targeting CXCR4 have up to date limited success. Thus, there is a great interest in the exploration of alternative ways to regulate the expression and signaling of CXCR4.
In this study, we identified CMTM4, a member of the CKLF-like MARVEL Transmembrane domain containing protein family with relatively unknown function, as a novel interactor of CXCR4. CMTM4 has been mainly described as a tumor suppressor, and its expression is downregulated in many types of cancer by promoter methylation. We found that CMTM4, but not the closely related family member CMTM6, regulates the trafficking and protein stability of CXCR4 in transfected HEK 293T cells. To investigate how CMTM4 regulates CXCR4 signaling, we created a tetracycline-inducible CMTM4.F overexpression model in HeLa cells, a cervical cancer cell line that expresses endogenous CXCR4. We showed that CMTM4 co-immunoprecipitates and co-localizes with CXCR4 intracellularly and on the cell surface. Although CMTM4 does not affect the cell surface expression of CXCR4 or the ligand-independent endocytosis of the receptor, we found that it reduces the CXCL12-induced internalization of CXCR4. Additionally, we could show that CMTM4 expression reduces the activation of AKT in steady-state conditions and after ligand stimulation without affecting the activation of ERK1/2. The CXCL12-mediated chemotaxis and proliferation of CMTM4-expressing HeLa cells was also significantly attenuated while cell viability was not affected.
Overall, our findings identified CMTM4 as a novel interactor of CXCR4 and regulator of CXCL12/CXCR4 signaling that could potentially serve as a novel pharmacological approach to control CXCR4-mediated tumor growth and metastasis.
... Four accessory proteins, Vif, Vpr, Vpu, and Nef, counteract antiviral activity and bind to several host cell factors during infection and viral production ensuring viral pathogenesis (reviewed in Anderson and Hope, 2004;reviewed in Abraham, et al., 2012;reviewed in Sauter, et al., 2016;reviewed in Fabryova, et al., 2019). interaction of Env with a cellular co-receptor molecule (β-chemokine receptor CCR5 or α-chemokine receptor CXCR4 (reviewed in Berger, 1997;McDougal, et al., 1986;Kwong, et al., 1998;reviewed in Capon, et al., 1991;Wu, et al., 1996). These binding events of Env lead to a dramatic structural shift within gp41, which inserts a hydrophobic fusion protein into the host cell plasma membrane, and a subsequent formation of a six-helix bundle at the terminal regions of gp41 (Lu, et al., 1997;Swanstrom, et al., 2012;reviewed in Sattentau, et al., 1988) causes a close proximity of viral and cellular membranes (Liu, et al., 2008), followed by membrane fusion (Chan, et al., 1997). ...
Human Immunodeficiency Virus-1 (HIV-1) assembles and buds as non-infectious particles at the plasma membrane of host cells. During assembly, it forms an irregular hexameric ordered lattice consisting of the structural polyproteins Gag and GagProPol. To facilitate infectivity, the intrinsic protease (PR) of HIV-1 drives the maturation by processing these polyproteins at 5 and 9 cleavage sites (CS), respectively, after self-cleavage. This proteolytic part is tightly regulated temporally and sequentially, to ensure a correct morphological rearrangement by a specific release of subdomains inside the viral particle. Gag comprises the matrix (MA), capsid (CA), and nucleocapsid protein (NC) as well as two spacer peptides (SP1 and SP2) and the C-terminal p6-domain. After maturation, CA encapsulates a condensed complex of NC and the RNA genome copies as the conical core. Already subtle dynamical or structural changes can prevent a successful maturation and therefore impair viral infectivity. The concomitant start of maturation and assembly/budding processes so far prevented a precise time-course analysis of maturation in connection with structural and cofactor interactions, and the determination of pH during maturation. Previous proteolytic cleavage studies with in vitro translated Gag and viral particles already led to the categorization of the CSs regarding their processing rates: rapid (SP1-NC), intermediate (MA-CA, SP2-p6), and slow (CA-SP1, NC-SP2). However, these dynamics differ from the processing of synthetic CS peptides. That is why this work aimed to analyze the impact of Gag assembly, which is usually induced by the binding of nucleic acid, and other factors as specific mutations upon the dynamics of maturation. In previous studies, only the final products of maturation were analyzed in viral particles regarding morphology and processing results. Other time-course analyses excluded the verification of the Gag multimerization or the influence of nucleic acid as present in virus-producing cells. Thus, I wanted to compare the processing of Gag in an assembled structure or non-assembled state and additionally introduced specific cleavage site mutants and maturation altering compounds into my system. In order to tackle these open questions for Gag processing dynamics, an in vitro based processing approach for the analyses of proteolytic maturation was chosen, including non-assembled Gag and in vitro assembled ΔMACANCSP2, a truncated Gag variant. Therefore, I produced recombinant Gag with a C-terminal His-tag (Gag-His) in E. coli and optimized the protocol to yield high purity and no nucleic acid contamination, to avoid preliminary assembly. A given protocol to assemble ΔMACANCSP2 was optimized, which increased assembly efficiency and stability to endure the inconvenient conditions of the following processing experiment. Additionally, this newly created protocol could achieve assembly of ΔMACANCSP2 in the absence of any nucleic acid into curved filaments instead of spherical particles. As these filamentous structures are a novelty, further structural analysis of them could give more insight into the assembling properties of Gag in the future. Gag-His featured, independent of its intrinsic homodimerization, an altered order of processing in contrast to the assembled ΔMACANCSP2. The initial cleavage occurred at MA-CA and CA-SP1, followed by SP1-NC and SP2-p6, and at last, NC-SP2. In comparison, the processing of assembled ΔMACANCSP2 reproduced the same processing order shown in the literature, which was only marginally affected by the application of longer NA than 68 nt. The processing of assembled protein was finished up to six times faster than for the non-assembled, while the absence of or very shot (5 nucleotides) NA during the processing of assembled ΔMACANCSP2 caused a mixture of both results. While MA-CA and CA-SP1 are processed like assembled ΔMACANCSP2 with nucleic acid, SP1-NC and NC-SP2 are processed significantly slower than in the case of non-assembled Gag-His. These results suggest that the cleavage events of non-assembled Gag is dependent on the amino acid sequence of the CSs, and assembly causes for MA-CA and CA-SP1 a maturation restriction. Changing the pH of the processing procedure had a severe impact on the processing of CA-SP1 and NC-SP2 in an assembled or non-assembled protein. While the processing was fastest at pH 6.0, the optimum for PR activity, the processing was strongly reduced at pH 6.5 and even more at pH 7.0. The remaining three CSs were only marginally affected. Consequently, CA-SP1 and NC-SP2 might comprise pH-dependent structural domains or interactions, and a theoretical pH shift during maturation of viral particles could enable fast processing. The introduction of mutations known to inhibit proteolytic cleavage or a maturation inhibitor showed that the processing of each site, but CA-SP1, is independent of the cleavage of the other CSs. By inhibiting the processing at SP1-NC, the cleavage of CA-SP1 got delayed, which was observed in the presence and absence of nucleic acids. Interestingly, the inhibition of MA-CA cleavage led to the processing of a new cryptic CS, which was determined to be at the N-terminal region of CA. In summary of this work, assembly of Gag in the presence of nucleic acid accelerates maturation notably, whereas the single cleavage events are independent of each other and only temporally ordered. While assembly delays the processing at MA-CA and CA-SP1, the presence of nucleic acid is the actual key player to shorten the maturation, but it is not essential for Gag assembly.
... Preparing stable and homogenous samples of purified CCR5 or CXCR4 has been technically challenging, and various assays have, therefore, been employed to measure the binding affinity for the Env-coreceptor interactions in the presence of soluble CD4 (<10 nM for CCR5; 200-500 nM for CXCR4; [84][85][86][87]). The first structure of a full-length monomeric gp120 in complex with a soluble 4D-CD4 and an unmodified human CCR5 was determined by cryo-EM [83] (Figure 2B), revealing details of the interactions between gp120 and CCR5, largely consistent with the predictions based on previous mutational data [88,89]. ...
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.
... The relative molecular weight of LBPs and sulfated LBPs was determined by HPSEC of the Waters 2696 gel permeation chromatograph (US) [25] . A series of standard dextrans (1.0 mg) with average molecular weight of 1000 5900 11800 22800 47300 112000 212000 404000 788000 Da were dissolved in distilled water with 1.0 mg/mL concentration in turn, which were injected to Ultrahydrogel columns (7.8 cm × 300 cm).The puri ed water was used as mobile phase at 0.6mL/min ow rate, the injection concentration was 0.30%, and the injection volume was 50 µL with the column temperature at 50°C. and CCR5 (R5), via the V3 loop of gp120 [26,27] . HIV usingR5 as a coreceptor (R5-HIV) has been isolated from patients in the all stages. ...
BACKGROUND: Lycium barbarum polysaccharides (LBPs) belong to a very important class of biological macromolecules from Lycium barbarum berries in nature, and have received much attention due to their various biological activities. Since sulfated polysaccharides have antiviral activity in vitro, there is no published research on the sulfated modification of LBPs.
RESULTS: The objective of this study was to investigate the feasibility of sulfated modification of LBPs and their potential application in inhibiting HIV-1. The LBPs with different molecular weight were prepared by fractional precipitation from crude LBP through aqueous extraction, ethanol precipitation and deproteinization. The purified LBPs (G1, G2 and G3) were sulfated by chlorosulfonic acid pyridine method to give rise to the sulfated LBPs (G1S1-G1S4, G2S1-G2S4 and G3S1-G3S4) with different degrees of substitution. The anti-HIV-1activities were evaluated by TD50 of the cytotoxicity and IC50 of inhibitory activity using the CCK-8 and Magi test with Azidothymidine (AZT) as positive control. The results showed that LBPs and sulfated LBPs showed non-toxicity with the TD50 >100 μg/mL. Compared with the LBPs, the inhibition of anti-HIV-1 activity of sulfated LBPs was increased significantly with the IC50 value from 0.0924-0.1206 μg/mL to 0.0206-0.0722 μg/mL. The G1S4 (Mw=2.13×10⁴ Da and DS=1.12) showed excellent anti-HIV-1 activity with the IC50 value near to that of AZT (0.0200μg/mL), which would make it possible to become a candidate compound with anti-HIV-1 activity.
CONCLUSION: The outcome of the study indicated that sulfated modification of LBPs was feasible and sulfated LBPs had good potential as an anti-HIV drug.
... The entry of the human immunodeficiency virus type 1 (HIV-1) into target cells involves sequential binding of its surface envelope glycoprotein to cellular primary receptor CD4 and the chemokine receptor CCR5 or CXCR4. 1,[2][3][4] CCR5 is the major coreceptor used by HIV-1 and is expressed in CD4 + T cells and myeloid cells that are depleted during HIV-1 infection. [5][6][7][8][9] A natural 32 base-pair deletion in CCR5 (CCR5D32) leads to a premature stop codon and a nonfunctional gene product; therefore, CCR5 is not expressed on the cell surface. ...
CCR5 is a coreceptor of human immunodeficiency virus type 1 (HIV-1). Transplantation of hematopoietic stem cells homozygous for a 32-bp deletion in CCR5 resulted in a loss of detectable HIV-1 in two patients, suggesting that genetic strategies to knockout CCR5 expression would be a promising gene therapy approach for HIV-1-infected patients. In this study, we targeted CCR5 by CRISPR-Cas9 with a single-guide (sgRNA) and observed 35% indel frequency. When we expressed hCas9 and two gRNAs, the Surveyor assay showed that Cas9-mediated cleavage was increased by 10% with two sgRNAs. Genotype analysis on individual clones showed 11 of 13 carried biallelic mutations, where 4 clones had frameshift (FS) mutations. Taken together, these results indicate that the efficiency of biallelic FS mutations and the knockout of the CCR5 necessary to prevent viral replication were significantly increased with two sgRNAs. These studies demonstrate the knockout of CCR5 and the potential for translational development.
... 19 can influence the function of various cell types, including T and B lymphocytes, monocytes, endothelial cells, neurons, and astrocytes, which it does by binding to the CD4 receptor or the chemokine receptors, CCR5 and CXCR4. 23,24 Among its reported effects, HIV-1 gp120 has been shown to increase intracellular calcium levels, induce formation of inositol triphosphate, inhibit antigen-driven T-cell activation, alter cytokine production, induce apoptosis, and stimulate polyclonal B-cell activation. 25 HIV-1 gp120 has also been shown to stimulate the replication of human cytomegalovirus in infected monocytes. ...
Context.—Human herpesvirus 8 (HHV-8) is the presumed etiologic agent of Kaposi sarcoma (KS), the most common neoplasm in patients with acquired immunodeficiency syndrome. Current evidence indicates HHV-8 is necessary, but not sufficient, for KS development without the involvement of other cofactors. One potentially important cofactor is human immunodeficiency virus type 1 (HIV-1). Although HIV-1 is not essential for development of KS, studies have shown factors released from HIV-1–infected cells, including HIV-1 proteins and cytokines, promote the growth of KS cells in vitro. Recently, studies have shown that coculture of HIV-1–infected T cells with HHV-8–infected primary effusion lymphoma cell lines results in HHV-8 reactivation. This response was due, in part, to cytokines. However, only a portion of induced HHV-8 replication could be accounted for by cytokine stimulation, indicating that other factors, including HIV-1–associated proteins, may also be involved.
Objective.—To investigate a possible role for HIV-1 gp120 in HHV-8 reactivation.
Design.—Using an in vitro model system, we examined the effect of recombinant HIV-1 gp120 protein on HHV-8 replication in latently infected primary effusion lymphoma cell lines.
Main Outcome Measures.—Reactivation of HHV-8 was analyzed using Northern blot analysis and quantitative polymerase chain reaction for ORF26 messenger RNA expression, a gene encoding for the HHV-8 minor capsid protein produced only during reactivation. The results were extended and confirmed using a luciferase reporter construct driven by the HHV-8 ORF50 promoter, the first promoter activated during HHV-8 replication.
Results.—No evidence of enhanced HHV-8 replication was found following treatment with HIV-1 gp120. In addition, HIV-1 gp120 was unable to act synergistically with interferon-γ or hepatocyte growth factor/scatter factor to enhance reactivation of the virus in infected primary effusion lymphoma cell lines.
Conclusions.—HIV-1 gp120 does not appear to be responsible for the reactivation of HHV-8 demonstrated in our previous studies. Further studies are necessary to determine if other HIV-associated proteins, particularly Tat, gp160, and/or gp41, which are also released from infected cells, may be important in inducing HHV-8 reactivation.
... The lipofectamine method was also less toxic for the cells, allowing up to 3 high titre harvests from transfected cells. Macrophage-tropic strains o f HIV-1 infect human macrophages, predominantly via the co-receptor CCR5 (266). It has been shown previously that the susceptibility of the cells to these strains increases during monocyte to macrophage differentiation (190). ...
Viral vectors based on lentiviruses exhibit distinct advantages over other retroviral vectors because of their ability to transduce non-dividing cells. The use of lentiviral vectors for gene delivery of antigens and immuno-modulatory factors to human antigen presenting cells will allow initiation and modulation of clinically relevant immune responses. To this end we have explored the use of vectors based on the human immunodeficiency virus type 1 (HIV-1) to transduce human macrophages and dendritic cells. Human peripheral blood monocytes were susceptible to HIV vector transduction only after maturation into macrophages following 5 days culture. This maturation-dependence of infection was observed with vectors carrying HIV-1 accessory proteins and with transgene expression driven by 4 different promoters. Analysis of reverse transcription in freshly isolated monocytes and differentiated macrophages infected with HIV-based vectors showed that levels of viral DNA synthesis were equivalent. However nuclear viral DNA could only be detected in differentiated macrophages. Moreover, wild-type HIV-1 virions carrying the envelope of VSV-G also exhibited this pattern of differentiation-dependent transduction. Taken together these results demonstrate a differentiation-regulated restriction to HIV-1 in primary human monocytes. However, monocytes differentiated into dendritic cells in the presence of the cytokines IL-4 and GMCSF were susceptible to transduction at all stages of culture. Infection in freshly isolated monocytes could be rescued by subsequent dendritic cell differentiation, an effect that was shown to be due to culture of the cells in foetal calf serum. These results are of importance in the design of protocols for the infection of human antigen presenting cells by lentiviral vectors. They also provide evidence for a post-entry block to HIV-1 infection in freshly isolated human monocytes.
... Interactions of gp120 with the cell surface receptor CD4 6 and one of the chemokine receptors CCR5 or CXCR4 [7][8][9] initiates a series of conformational rearrangements during which gp120 is shed, thereby exposing gp41 which adopts an extended pre-fusion state 2,10 . The primary structure of gp41 is schematically illustrated in Fig. 1. ...
During the first steps of HIV infection the Env subunit gp41 is thought to establish contact between the membranes and to be the main driver of fusion. Here we investigated in liquid crystalline membranes the structure and cholesterol recognition of constructs made of a gp41 external region carrying a cholesterol recognition amino acid consensus (CRAC) motif and a hydrophobic membrane anchoring sequence. CD- und ATR-FTIR spectroscopies indicate that the constructs adopt a high degree of helical secondary structure in membrane environments. Furthermore, ¹⁵N and ²H solid-state NMR spectra of gp41 polypeptides reconstituted into uniaxially oriented bilayers agree with the CRAC domain being an extension of the transmembrane helix. Upon addition of cholesterol the CRAC NMR spectra remain largely unaffected when being associated with the native gp41 transmembrane sequence but its topology changes when anchored in the membrane by a hydrophobic model sequence. The ²H solid-state NMR spectra of deuterated cholesterol are indicative of a stronger influence of the model sequence on this lipid when compared to the native gp41 sequence. These observations are suggestive of a strong coupling between the transmembrane and the membrane proximal region of gp41 possibly enforced by oligomerization of the transmembrane helical region.
... Other membrane proteins involved in virus entry should also be noted. The chemokine receptors CCR5 and CXCR4 have been reported as co-receptors involved in human immunodeficiency virus infection (9)(10)(11), and HLA class II receptors function as co-receptors in Epstein-Barr virus infection (12). The fields of vaccine and antiviral development are of interest in regard to viral co-receptors (co-factors) in addition to primary receptors (13). ...
COVID-19 represents a real threat to the global population, and understanding the biological features of the causative virus (SARS-CoV-2) is imperative to aid in mitigating this threat. Analyses of proteins such as primary receptors and co-receptors (co-factors) that are involved in SARS-CoV-2 entry into host cells will provide important clues to help control the virus. Here, we identified host cell membrane protein candidates that were present in proximity to the attachment sites of SARS-CoV-2 spike proteins through the use of proximity labeling and proteomics analysis. The identified proteins represent candidate key factors that may be required for viral entry. Our results indicated that a number of membrane proteins, including DPP4, Cadherin-17, and CD133, were identified to co-localize with cell membrane-bound SARS-CoV-2 spike proteins in Caco-2 cells that were used to expand the SARS-CoV-2 virion. We anticipate that the information regarding these protein candidates will be utilized for the future development of vaccines and antiviral agents against SARS-CoV-2.
The Env trimers on the HIV-1 mediate virus entry into host cells. Env is synthesized in infected cells, modified by complex sugars, and cleaved to form a mature, functional Env, which is incorporated into virus particles. Env elicits antibodies in infected individuals, some of which can neutralize the virus. We found that antibodies co-expressed in the virus-producing cell can disrupt Env transit to the proper compartment for cleavage and sugar modification and, in some cases, block incorporation into viruses. These studies provide insights into the processes by which Env becomes functional in the virus-producing cell and may assist attempts to interfere with these events to inhibit HIV-1 infection.
CD4-mimetic compounds (CD4mcs) bind the human immunodeficiency virus (HIV-1) gp120 exterior envelope glycoprotein (Env) and compete for binding to CD4, the host receptor. CD4mcs prematurely trigger conformational changes in Env similar to those induced by CD4, leading to transient activation of infectivity followed by irreversible virus inactivation. Natural HIV-1 variants exhibit a wide range of susceptibilities to CD4mc inhibition, only a small fraction of which can be explained by variation in the gp120 Phe-43 cavity/vestibule where CD4mcs bind. Here, we study Envs from the resistant HIV-1 BG505 and the more sensitive HIV-1 191955_A4 clade A strains. The major determinant of the relative sensitivity of the HIV-1 191955_A4 Env to CD4mcs mapped to a single residue change (F317Y) in the tip of the gp120 V3 variable loop. In the Envs of several HIV-1 strains, replacement of the more prevalent Phe 317 with a tyrosine residue increased virus sensitivity to multiple CD4mcs. Tryptophan substitutions at residues 317 and 316 resulted in increases and decreases, respectively, in sensitivity to CD4mcs. Some of the gp120 V3 changes increased virus sensitivity to inactivation by both CD4mc and cold exposure, phenotypes indicative of increased Env triggerability. Infection of CD4-negative cells expressing the CCR5 coreceptor by these Env variants was triggered more efficiently by CD4mcs. For the panel of studied HIV-1 Envs, resistance to the CD4mcs was associated with decreased ability to support virus entry. These studies illustrate how variation in gp120 outside the CD4mc binding site can influence the sensitivity of natural HIV-1 strains to inhibition by these compounds.
IMPORTANCE
CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.
Cytokines are small-molecular-weight, proteinaceous, immunomodulating molecules that are crucial for controlling the growth and activity of blood and other immune cells. They are peptide molecules that cannot cross the lipid bilayer of a cell to enter the cytoplasm but are important for cell signaling. Cytokines bind to specific cell surface receptors and send intracellular signals. They are involved in autocrine, paracrine, and endocrine signaling. Normally, cytokines affect cell activation, division, apoptosis, or movement, but their biological effect depends on the type of cytokine and the cell involved. These essential chemical mediators function as biomarkers for a wide range of illnesses and control immunological and inflammatory responses through intricate networks. They can be discussed in terms of their pro- and anti-inflammatory activities as well as on the basis of their clinical significance. They take part in the immune response and serve as significant immune system communication network mediators. The development, proliferation, and response of immune cells are dynamically controlled by cytokines, which play a significant role in determining health. Multiple biological activities can result from a single cytokine's ability to act on various cell types and can be released by numerous kinds of cells. Cytokines have two common characteristics, redundancy and promiscuity, which are particularly notable in chemokines that are a specific type of cytokine, and can direct immune cell movement toward their target. There are different types of chemokines, like interferons, interleukins, tumor necrosis factor (TNFs), and growth factors. Chemokines, sometimes also referred to as chemotactic cytokines, are a broad family of small, secreted proteins that communicate by binding to the cell surfaces that have heptahelical chemokine receptors (CKRs). They are mostly well-known for their capacity to encourage the movement of cells, particularly that of leukocytes (white blood cells (WBCs)). Since they are involved in all inflammatory and immunological responses, whether beneficial or harmful, chemokines are essential for homeostasis of the immune system and development. This chapter provides a general review of the cytokine network, chemokines, and chemokine receptor families, highlights the chemokine network's numerous physical interactions, and covers the fundamental principles of chemokine function while providing specific examples.
HIV-1 envelope glycoproteins (Envs) mediate viral entry and are the sole target of neutralizing antibodies. Envs of most primary HIV-1 strains exist in a closed conformation and occasionally sample more open Env states. Thus, current knowledge guides immunogen design to mimic the closed Env conformation as the preferred target for eliciting broadly neutralizing antibodies (bnAbs) to block HIV-1 entry. Here we show that Env-preferred conformations of 6 out of 13 (46%) transmitted/founder (T/F) strains tested are incompletely closed. As a result, entry of these T/Fs into target cells is sensitive to antibodies that recognize internal epitopes exposed on open Env conformations. A cryo-electron microscopy structure of unliganded, incompletely closed T/F Envs (1059-SOSIP) at 3.6 Å resolution exhibits an asymmetric configuration of Env protomers with increased sampling of states with incompletely closed trimer apex. An additional structure of the 1059-SOSIP complexed with the CD4-induced antibody 17b without the requirement for prior sCD4 binding, together with double electron-electron resonance spectroscopy, provided further evidence for enriched occupancy of more open conformations. 1059 Env conformational flexibility was associated with resistance to bnAbs that preferentially neutralize the closed Env conformation. To follow the fate of incompletely closed Env in patients, we reconstructed de novo the post-transmission evolutionary pathway of a second T/F Env (CH040), which is sensitive to the V3-targeting antibody 19b and highly resistant to most bnAbs. Evolved viruses exhibited increased resistance to cold, soluble CD4 and 19b, all of which correlate with the evolution of a more closed Env state. Lastly, we show a correlation between efficient neutralization of multiple Env conformations and increased antiviral breadth of CD4-binding site (CD4bs) bnAbs. In particular, N6 CD4bs bnAb, which uniquely recognizes different Env conformations, efficiently neutralizes 50% of the HIV-1 strains that were resistant to VRC01 and transmitted during the first-in-humans antibody-mediated prevention trial (HVTN 704). VRC01-resistant Envs are incompletely closed based on their sensitivity to cold and on partial sensitivity to antibodies targeting internal epitopes, which are typically occluded in tightly closed Envs. Most VRC01-resistant Envs retain the VRC01 epitope according to VRC01 binding to their gp120 subunit at concentrations that have no significant effect on virus entry, and they exhibit cross resistance to other CD4bs bnAbs that preferentially neutralize the closed Env conformation. Our findings refine current knowledge of Env conformational states and provide guidance for developing new strategies for bnAb immunotherapy and Env-based immunogen design.
The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry into host cells by binding receptors, CD4 and CCR5/CXCR4, and fusing the viral and cell membranes. In infected cells, cleavage of the gp160 Env precursor yields the mature Env trimer, with gp120 exterior and gp41 transmembrane Env subunits. Env cleavage stabilizes the State-1 conformation, which is the major target for broadly neutralizing antibodies, and decreases the spontaneous sampling of more open Env conformations that expose epitopes for poorly neutralizing antibodies. During HIV-1 entry into cells, CD4 binding drives the metastable Env from a pretriggered (State-1) conformation into more “open,” lower-energy states. Here, we report that changes in two dissimilar elements of the HIV-1 Env trimer, namely particular gp120 glycans and the gp41 fusion peptide-proximal region (FPPR), can independently modulate the stability of State 1. Individual deletion of several gp120 glycans destabilized State 1, whereas removal of a V1 glycan resulted in phenotypes indicative of a more stable pretriggered Env conformation. Likewise, some alterations of the gp41 FPPR decreased the level of spontaneous shedding of gp120 from the Env trimer and stabilized the pretriggered State-1 Env conformation. State-1-stabilizing changes were additive and could suppress the phenotypes associated with State-1-destabilizing alterations in Env. Our results support a model in which multiple protein and carbohydrate elements of the HIV-1 Env trimer additively contribute to the stability of the pretriggered (State-1) conformation. The Env modifications identified in this study will assist efforts to characterize the structure and immunogenicity of the metastable State-1 conformation.
IMPORTANCE
The elicitation of antibodies that neutralize multiple strains of HIV-1 is an elusive goal that has frustrated the development of an effective vaccine. The pretriggered shape of the HIV-1 envelope glycoprotein (Env) spike on the virus surface is the major target for such broadly neutralizing antibodies. The “closed” pretriggered Env shape resists the binding of most antibodies but is unstable and often assumes “open” shapes that elicit ineffective antibodies. We identified particular changes in both the protein and the sugar components of the Env trimer that stabilize the pretriggered shape. Combinations of these changes were even more effective at stabilizing the pretriggered Env than the individual changes. Stabilizing changes in Env could counteract the effect of Env changes that destabilize the pretriggered shape. Locking Env in its pretriggered shape will assist efforts to understand the Env spike on the virus and to incorporate this shape into vaccines.
The mature human immunodeficiency virus (HIV) envelope glycoprotein (Env) trimer, which consists of noncovalently associated gp120 exterior and gp41 transmembrane subunits, mediates virus entry into cells. The pretriggered (State-1) Env conformation is the major target for broadly neutralizing antibodies (bNAbs), whereas receptor-induced downstream Env conformations elicit immunodominant, poorly neutralizing antibody (pNAb) responses. To examine the contribution of membrane anchorage to the maintenance of the metastable pretriggered Env conformation, we compared wild-type and State-1-stabilized Envs solubilized in detergents or in styrene-maleic acid (SMA) copolymers. SMA directly incorporates membrane lipids and resident membrane proteins into lipid nanoparticles (styrene-maleic acid lipid particles [SMALPs]). The integrity of the Env trimer in SMALPs was maintained at both 4°C and room temperature. In contrast, Envs solubilized in Cymal-5, a nonionic detergent, were unstable at room temperature, although their stability was improved at 4°C and/or after incubation with the entry inhibitor BMS-806. Envs solubilized in ionic detergents were relatively unstable at either temperature. Comparison of Envs solubilized in Cymal-5 and SMA at 4°C revealed subtle differences in bNAb binding to the gp41 membrane-proximal external region, consistent with these distinct modes of Env solubilization. Otherwise, the antigenicity of the Cymal-5- and SMA-solubilized Envs was remarkably similar, both in the absence and in the presence of BMS-806. However, both solubilized Envs were recognized differently from the mature membrane Env by specific bNAbs and pNAbs. Thus, detergent-based and detergent-free solubilization at 4°C alters the pretriggered membrane Env conformation in consistent ways, suggesting that Env assumes default conformations when its association with the membrane is disrupted. IMPORTANCE The human immunodeficiency virus (HIV) envelope glycoproteins (Envs) in the viral membrane mediate virus entry into the host cell and are targeted by neutralizing antibodies elicited by natural infection or vaccines. Detailed studies of membrane proteins rely on purification procedures that allow the proteins to maintain their natural conformation. In this study, we show that a styrene-maleic acid (SMA) copolymer can extract HIV-1 Env from a membrane without the use of detergents. The Env in SMA is more stable at room temperature than Env in detergents. The purified Env in SMA maintains many but not all of the characteristics expected of the natural membrane Env. Our results underscore the importance of the membrane environment to the native conformation of HIV-1 Env. Purification methods that bypass the need for detergents could be useful tools for future studies of HIV-1 Env structure and its interaction with receptors and antibodies.
The HIV-1 envelope (Env) glycoprotein is conformationally dynamic and mediates membrane fusion required for cell entry. Single-molecule fluorescence resonance energy transfer (smFRET) of Env using peptide tags has provided mechanistic insights into the dynamics of Env conformations. Nevertheless, using peptide tags risks potential effects on structural integrity. Here, we aim to establish minimally invasive smFRET systems of Env on the virus by combining genetic code expansion and bioorthogonal click chemistry. Amber stop-codon suppression allows site-specifically incorporating noncanonical/unnatural amino acids (ncAAs) at introduced amber sites into proteins. However, ncAA incorporation into Env (or other HIV-1 proteins) in the virus context has been challenging due to low copies of Env on virions and incomplete amber suppression in mammalian cells. Here, we developed an intact amber-free virus system that overcomes impediments from preexisting ambers in HIV-1. Using this system, we successfully incorporated dual ncAAs at amber-introduced sites into Env on intact virions. Dual-ncAA incorporated Env retained similar neutralization sensitivities to neutralizing antibodies as wildtype. smFRET of click-labeled Env on intact amber-free virions recapitulated conformational profiles of Env. The amber-free HIV-1 infectious system also permits in-virus protein bioorthogonal labeling, compatible with various advanced microscopic studies of virus entry, trafficking, and egress in living cells.
Amber-free HIV-1 infectious systems actualized minimal invasive Env tagging for smFRET, versatile for in-virus bioorthogonal click labeling in advanced microscopic studies of virus-host interactions.
Human immunodeficiency virus (HIV-1) entry into cells involves triggering of the viral envelope glycoprotein (Env) trimer ([gp120/gp41]3) by the primary receptor, CD4, and coreceptors, CCR5 or CXCR4. The pretriggered (State-1) conformation of the mature (cleaved) Env is targeted by broadly neutralizing antibodies (bNAbs), which are inefficiently elicited compared with poorly neutralizing antibodies (pNAbs). Here, we characterize variants of the moderately triggerable HIV-1AD8 Env on virions produced by an infectious molecular proviral clone; such virions contain more cleaved Env than pseudotyped viruses. We identified three types of cleaved wild-type AD8 Env trimers on virions: (i) State-1-like trimers preferentially recognized by bNAbs and exhibiting strong subunit association; (ii) trimers recognized by pNAbs directed against the gp120 coreceptor-binding region and exhibiting weak, detergent-sensitive subunit association; and (iii) a minor gp41-only population. The first Env population was enriched and the other Env populations reduced by introducing State-1-stabilizing changes in the AD8 Env or by treatment of the virions with crosslinker or the State-1-preferring entry inhibitor, BMS-806. These stabilized AD8 Envs were also more resistant to gp120 shedding induced by a CD4-mimetic compound or by incubation on ice. Conversely, a State-1-destabilized, CD4-independent AD8 Env variant exhibited weaker bNAb recognition and stronger pNAb recognition. Similar relationships between Env triggerability and antigenicity/shedding propensity on virions were observed for other HIV-1 strains. State-1 Envs on virions can be significantly enriched by minimizing the adventitious incorporation of uncleaved Env; stabilizing the pretriggered conformation by Env modification, crosslinking or BMS-806 treatment; strengthening Env subunit interactions; and using CD4-negative producer cells. IMPORTANCE Efforts to develop an effective HIV-1 vaccine have been frustrated by the inability to elicit broad neutralizing antibodies that recognize multiple virus strains. Such antibodies can bind a particular shape of the HIV-1 envelope glycoprotein trimer, as it exists on a viral membrane but before engaging receptors on the host cell. Here, we establish simple yet powerful assays to characterize the envelope glycoproteins in a natural context on virus particles. We find that, depending on the HIV-1 strain, some envelope glycoproteins change shape and fall apart, creating decoys that can potentially divert the host immune response. We identify requirements to keep the relevant envelope glycoprotein target for broad neutralizing antibodies intact on virus-like particles. These studies suggest strategies that should facilitate efforts to produce and use virus-like particles as vaccine immunogens.
This study investigated the feasibility of sulfate modification of Lycium barbarum polysaccharides (LBPs) and their potential anti‐HIV‐1 application. LBPs with different molecular weights were prepared by fractional precipitation from crude LBP via aqueous extraction, ethanol precipitation, and deproteinization. The purified LBPs (G1, G2, and G3) were sulfated using the chlorosulfonic acid pyridine method to obtain sulfated LBPs (G1S1–G1S4, G2S1–G2S4, and G3S1–G3S4) with different sulfur degrees of substitution. The anti‐HIV‐1 activity in vitro was evaluated via the TD50 from the cytotoxicity test and the IC50 inhibitory activity. The cytotoxicity and inhibitory activity in vitro were carried out using the CCK‐8 assay and MAGI test with Azidothymidine (AZT) as a positive control. The results show that the LBPs and the sulfated derivatives were not toxic towards CCK‐8 cells with TD50>100 μg/mL and they had strong anti‐HIV‐1 activity with IC50 values of 0.02–0.12 μg/mL. Compared with the LBPs, the anti‐HIV‐1 activity of the sulfated LBPs increased with IC50 values from 0.0924–0.1206 μg/mL to 0.0206–0.0722 μg/mL. Sulfate modification of LBPs can increase their anti‐HIV pharmacological activity, the G1S4 (Mw=2.13×10⁴ Da and DS=1.12) showed relatively good in vitro anti‐HIV‐1 activity with an IC50 value near that of AZT (0.0200 μg/mL). The results suggest further detailed in vivo studies are warranted for these promising candidates with anti‐HIV‐1 activity.
CD4-mimetic compounds (CD4mcs) and conformational blockers like BMS-806 and BMS-529 (temsavir) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and conformational blockers inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor CD4 and is highly conserved among HIV-1 strains.
Depuis sa découverte en 1983, Le VIH-1 constitue un problème de santé publique majeur. A ce jour, malgré de nombreux traitements efficaces, les personnes infectées ne peuvent pas guérir, et le virus n’est toujours pas éradiqué. Pour prévenir l’émergence de souches résistances aux traitement ou plus agressives dans la progression de la maladie, il est essentiel de comprendre les interactions du virus avec la cellule qu’il infecte. L’entrée virale est pour cela une étape clé du cycle viral, avec la reconnaissance par le glycoprotéine virale gp120 du corécepteur, majoritairement CCR5. Il est désormais établi que le VIH-1 exploite la diversité des populations de récepteurs à la surface des cellules. Les travaux présentés dans cette thèse caractérisent, à l’échelle atomique, les différences de conformations correspondant aux populations ciblées par quatre virus différents. Nous avons utilisé la dynamique moléculaire pour simuler la dynamique du corécepteur CCR5 lié aux variantes de la gp120. L’analyse des trajectoires a conduit au développement d’une nouvelle méthode, ATOLL, qui permet de prédire les propriétés fonctionnelles du ligand, ici les variantes de la gp120, à partir de la position de extrémités intracellulaires des domaines transmembranaire d’un récepteur couplé à une protéine G, ici le CCR5. Nous avons aussi réussi à distinguer les populations de CCR5 liées aux gp120 par des motifs d’interactions intermoléculaires spécifiques. Dans le but d’exploiter ces motifs pour l’identification par criblage virtuel des petites molécules capables d’imiter une variante donnée de la gp120, nous avons développé LID, une méthode de score de docking capable de prendre en compte les multiples structures issues des simulations par dynamique moléculaire comme référence pour la sélection de poses.
Binding to the receptor, CD4, drives the pretriggered, "closed"(state-1) conformation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer into more "open"conformations (states 2 and 3). Broadly neutralizing antibodies, which are elicited inefficiently, mostly recognize the state-1 Env conformation, whereas the more commonly elicited poorly neutralizing antibodies recognize states 2/3. HIV-1 Env metastability has created challenges for defining the state-1 structure and developing immunogens mimicking this labile conformation. The availability of functional state-1 Envs that can be efficiently cross-linked at lysine and/or acidic amino acid residues might assist these endeavors. To that end, we modified HIV-1AD8 Env, which exhibits an intermediate level of triggerability by CD4. We introduced lysine/acidic residues at positions that exhibit such polymorphisms in natural HIV-1 strains. Env changes that were tolerated with respect to gp120-gp41 processing, subunit association, and virus entry were further combined. Two common polymorphisms, Q114E and Q567K, as well as a known variant, A582T, additively rendered pseudoviruses resistant to cold, soluble CD4, and a CD4-mimetic compound, phenotypes indicative of stabilization of the pretriggered state-1 Env conformation. Combining these changes resulted in two lysine-rich HIV-1AD8 Env variants (E.2 and AE.2) with neutralization- and cold-resistant phenotypes comparable to those of natural, less triggerable tier 2/3 HIV-1 isolates. Compared with these and the parental Envs, the E.2 and AE.2 Envs were cleaved more efficiently and exhibited stronger gp120-trimer association in detergent lysates. These highly cross-linkable Envs enriched in a pretriggered conformation should assist characterization of the structure and immunogenicity of this labile state.
The pretriggered shape of the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) is a major target for antibodies that can neutralize many strains of the virus. An effective HIV-1 vaccine may need to raise these types of antibodies, but this goal has proven difficult.
The envelope glycoprotein (Env) trimers on the surface of human immunodeficiency virus type 1 (HIV-1) mediate the entry of the virus into host cells and serve as targets for neutralizing antibodies. The cleaved, functional Env is incorporated into virus particles from the surface of the infected cell. We found that an uncleaved form of Env is transported to the cell surface by an unconventional route, but this nonfunctional Env is mostly excluded from the virus. Thus, only one of the pathways by which Env is transported to the surface of infected cells results in efficient incorporation into virus particles, potentially allowing the uncleaved Env to act as a decoy to the host immune system without compromising virus infectivity.
The chapter provides an overview of chemokine receptor classication, genomic location, structure, expression, binding, function, viral mimics, therapeutic implications, and potential decoy receptors. It discusses a hypothesis that the chemokine system, like the IL-1 pathway, has endogenous decoy receptors in addition to the Duffy antigen. Understanding the multifaceted roles of chemokines and their receptors in vivo has been facilitated by the development of transgenic mice and targeted deletion/mutagenesis of chemokines and their receptors. Chemokines are subdivided into classes based on the relative positions of their N-terminal cysteine residues. Chemokine receptors have a structure similar to that of other G protein-coupled receptors. The structure of chemokine receptors is characteristic of receptors, such as rhodopsin and the muscarinic acetylcholine receptor, which are coupled with guanine nucleotide binding proteins (G proteins). Chemokine receptor expression varies with cell type and receptor. Some receptors are restricted to certain cell types while others are expressed on many cell types. Chemokine receptors play a major role in two infectious diseases that cause significant morbidity and mortality worldwide: human immunodeciency virus (HIV) and malaria. One of the accepted models for chemokine/chemokine receptor interaction is the two-step binding and signaling model. In this model, the chemokine binds to the initial binding site, and the ensuing conformational change allows the pharmacophore to interact with the transmembrane helices to trigger signal transduction.
HIV use the CD4 molecule as their primary cellular receptor. Residues in the N-terminal domain (D1) of CD4 are crucial to HIV attachment through the gp120 envelope component. However, other regions of CD4 appear to be required subsequently for virus- and cell-cell fusion. Little is understood of the post-binding steps which may differ between HIV variants. We report a novel anti-CD4 mAb that does not block CD4/gp120 binding, but that does efficiently block both viral infection and cell-cell syncytia formation, and define its contact site as residues in CD4 D2 using both mouse/human CD4 chimeras and CD4 substitution mutants. We also investigated the basis for its antiviral effect. Using the CD4 D2 specific mAb, we identify another conserved step in HIV infection, as evidenced by its ability to neutralize a broad range of primary isolates and T cell-line passaged strains. Monovalent forms of the mAb were used to determine if its activity was due to masking of the D2 epitope, to steric inhibition, or bivalency. Our data indicate that both binding site and bivalency of the mAb underlie its potency. The need for bivalency is not simply explained by affinity, because monovalent forms can displace the intact mAb and reverse its protective effect. These results provide evidence that binding of the D2-specific mAb prevents structural alterations necessary for membrane fusion.
The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C'' and FG. This structure is consistent with C'C'' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.
The envelope glycoprotein gp120 of primate immunodeficiency viruses initiates viral attachment to CD4+ cells by binding to the CD4 antigen on host cell surfaces. However, among different CD4+ cell types, different viruses display distinct host cell ranges and cytopathicities. Determinants for both of these biological properties have been mapped to the env gene. We have quantitatively compared the CD4 binding affinities of gp120 proteins from viruses exhibiting different host cell tropisms and cytopathicities. The viral proteins were produced by using a Drosophila cell expression system and were purified to greater than 90% homogeneity. Drosophila-produced gp120 from T-cell tropic human immunodeficiency virus type 1 (HIV-1) BH10 exhibits binding to soluble recombinant CD4 (sCD4) and syncytia inhibition potency identical to that of pure authentic viral gp120. Relative to the affinity of HIV-1 BH10 gp120 for sCD4, that of dual tropic HIV-1 Ba-L is 6-fold lower, that of restricted T-cell tropic simian immunodeficiency virus mac is 70-fold lower, and that of noncytopathic HIV-2 ST is greater than 280-fold lower. Thus, viruses that utilize CD4 for infection do so by using a remarkably wide range of envelope affinities. These differences in affinity may play a role in determining cell tropism and cytopathicity.
Insertion of four amino acids into various locations within the amino-terminal halves of the human immunodeficiency virus type 1 gp120 or gp41 envelope glycoprotein disrupts the noncovalent association of these two envelope subunits (M. Kowalski, J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. A. Haseltine, and J. Sodroski, Science 237:1351-1355, 1987). To localize the determinants on the gp120 envelope glycoprotein important for subunit association, amino acids conserved among primate immunodeficiency viruses were changed. Substitution mutations affecting either of two highly conserved regions located at the amino (residues 36 to 45) and carboxyl (residues 491 to 501) ends of the mature gp120 molecule resulted in nearly complete dissociation of the envelope glycoprotein subunits. Partial dissociation phenotypes were observed for some changes affecting residues in the third and fourth conserved gp120 regions. These results suggest that hydrophobic regions at both ends of the gp120 glycoprotein contribute to noncovalent association with the gp41 transmembrane glycoprotein.
The genome of the human immunodeficiency virus type 1 (HIV-1) is highly heterogeneous. Some of this genomic variability is reflected in the biologic and serologic differences observed among various strains of HIV-1. To map the viral determinants that correlate with pathogenicity of the virus, recombinant viruses were generated between biologically active molecular clones of HIV-1 strains that show differences in T-cell or macrophage tropism, cytopathogenicity, CD4 antigen modulation, and susceptibility to serum neutralization. The results of these studies indicate that the envelope region contains the major determinants of these viral features. Further studies with sequence exchanges within this region should help identify specific domains that contribute to HIV pathogenesis.
The complement anaphylatoxin peptide C5a is well known to activate human polymorphonuclear leukocytes through receptor-mediated processes. C5a has also been reported to activate eosinophils for both chemotaxis and hexose uptake. We characterized the receptor molecule for human C5a on human eosinophils and compared it with the receptor on human neutrophils. At 4 degrees C, uptake of 1 nM 125I-C5a reaches equilibrium within 10 min on both cell types. Binding of 125I-C5a occurs over a concentration range comparable to that which stimulates lysosomal enzyme release and hexose uptake in both cell types. Scatchard analyses of the data indicate the presence of two receptor populations on eosinophils; a high affinity receptor with 15,000-20,000 sites/cell and a Kd of 3.1 +/- 0.6 x 10(-11) M, and a low affinity receptor with approximately 375,000 sites/cell and a Kd of 1 x 10(-7) M. Parallel experiments with neutrophils indicate the presence of a single receptor population with approximately 90,000 sites/cell and a Kd of 4.8 +/- 0.1 x 10(-10)M. The eosinophil receptor molecule was further characterized by covalently cross-linking 125I-C5a to cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material. Autoradiography indicates the presence of a dominant C5a-eosinophil receptor complex with an apparent mass of 60-65 kDa. The corresponding neutrophil-C5a receptor complex has an apparent mass of 50-52 kDa as observed by others. When the cross-linked 125I-C5a-receptor complex was treated with cyanogen bromide, different patterns were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for neutrophils and eosinophils. Thus, human eosinophils have a receptor for C5a anaphylatoxin which appears to be distinct from the C5a receptor present on human neutrophils.
We have used virus neutralization and antibody-binding techniques to define the epitope for a human monoclonal antibody, designated 19b, within the V3 region of the gp120 surface glycoprotein of human immunodeficiency virus type 1. Unusually, the 19b epitope encompasses residues on both flanks of the V3 loop. However, 19b binding to gp120 is independent of sequences at the crown of the V3 loop, provided that they are compatible with the formation of a type II beta turn that is presumably necessary to juxtapose the antigenic residues on the V3 flanks. By comparing the V3 sequences of virus gp120s able and unable to bind 19b, we were able to define the canonical 19b epitope as -I----G--FY-T, where residues at the positions indicated by the gaps do not contribute directly to the 19b-binding site. A few conservative substitutions at the more critical residues are also compatible with 19b binding. Inspection of V3 sequences in the human immunodeficiency virus database indicated that the canonical 19b epitope is well conserved among isolates from the North American-European clade B and also among clade E isolates from Thailand and clade F isolates from Brazil. A minority of gp120s from clades A and C also possess the 19b epitope. Consistent with the theoretical predictions of its cross-clade reactivity, 19b was found to bind to gp120s from clades A, B, C, E, and F in immunoassays. However, 19b was not able to reduce the infectivity of primary viruses from clades A, E, and F that were predicted to possess the 19b epitope and only modestly reduced the infectivity of a clade C virus at low input virus concentrations. Cross-clade neutralization via V3-directed antibodies may, therefore, be difficult, even if the antibodies show broad reactivities in binding assays and the viruses theoretically possess the relevant binding site.
The binding of human immunodeficiency virus type 1 (HIV-1) to the cellular receptor CD4 has been suggested to induce conformational changes in the viral envelope glycoproteins that promote virus entry. Conserved, discontinuous epitopes on the HIV-1 gp120 glycoprotein recognized by the 17b, 48d, and A32 antibodies are preferentially exposed upon the binding of soluble CD4 (sCD4). The binding of the 17b and 48d antibodies to the gp120 glycoprotein can also be enhanced by the binding of the A32 antibody. Here we constructed HIV-1 gp120 mutants in which the variable segments of the V1/V2 and V3 structures were deleted, individually or in combination, while the 17b, 48d, and A32 epitopes were retained. The effects of the variable loop deletions on the function of the HIV-1 envelope glycoproteins and on the exposure of epitopes induced by sCD4 or A32 binding to the monomeric gp120 glycoprotein were examined. The variable-loop-deleted envelope glycoproteins were able to mediate virus entry, albeit at lower efficiencies than those of the wild-type glycoproteins. Thus, the V1/V2 and V3 variable sequences contribute to the efficiency of HIV-1 entry but are not absolutely required for the process. Neither the V1/V2 nor V3 loops were necessary for the increase in exposure of the 17b/48d epitopes induced by binding of the A32 monoclonal antibody. By contrast, induction of the 17b, 48d, and A32 epitopes by sCD4 binding apparently involves a movement of the V1/V2 loops, which in the absence of CD4 partially mask these epitopes on the native gp120 monomer. The results obtained with a mutant glycoprotein containing a deletion of the V1 loop alone indicated that the contribution of the V2 loop to these phenomena was more significant than that of the V1 sequences. These results suggest that the V1/V2 loops, which have been previously implicated in CD4-modulated, postattachment steps in HIV-1 entry, contribute to CD4-induced gp120 conformational changes detected by the 17b, 48d, and A32 antibodies.
To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection.
Interaction with the CD4 receptor enhances the exposure on the human immunodeficiency type 1 gp120 exterior envelope glycoprotein of conserved, conformation-dependent epitopes recognized by the 17b and 48d neutralizing monoclonal antibodies. The 17b and 48d antibodies compete with anti-CD4 binding antibodies such as 15e or 21h, which recognize discontinuous gp120 sequences near the CD4 binding region. To characterize the 17b and 48d epitopes, a panel of human immunodeficiency virus type 1 gp120 mutants was tested for recognition by these antibodies in the absence or presence of soluble CD4. Single amino acid changes in five discontinuous, conserved, and generally hydrophobic regions of the gp120 glycoprotein resulted in decreased recognition and neutralization by the 17b and 48d antibodies. Some of these regions overlap those previously shown to be important for binding of the 15e and 21h antibodies or for CD4 binding. These results suggest that discontinuous, conserved epitopes proximal to the binding sites for both CD4 and anti-CD4 binding antibodies become better exposed upon CD4 binding and can serve as targets for neutralizing antibodies.
Effective vaccines against the human immunodeficiency virus (HIV) must cope with the genetic variation of the viral envelope (gp120) to combat or prevent acquired immunodeficiency syndrome (AIDS). Here we describe novel epitopes that are accentuated when gp120 complexes with its receptor (CD4). The presentation of these epitopes results through conformational rearrangements in the CD4/gp120 complex. Monoclonal antibodies directed to these epitopes inhibit syncytium formation, thus indicating the potential use of these epitopes as subunit vaccines.
The structure, replicative properties, and sensitivity to neutralization by soluble CD4 and monoclonal antibodies were examined for molecularly cloned envelope glycoproteins derived from human immunodeficiency virus type 1 (HIV-1) viruses either isolated directly from patients or passaged in T-cell lines. Complementation of virus entry into peripheral blood mononuclear cell targets by primary patient envelope glycoproteins exhibited efficiencies ranging from that observed for the HXBc2 envelope glycoproteins, which are derived from a T-cell line-passaged virus, to approximately fivefold-lower values. The ability of the envelope glycoproteins to complement virus entry roughly correlated with sensitivity to neutralization by soluble CD4. Laboratory-adapted viruses were sensitive to neutralization by monoclonal antibodies directed against the CD4-binding site and the third variable (V3) loop of the gp120 glycoprotein. By comparison, viruses with envelope glycoproteins from primary patient isolates exhibited decreased sensitivity to neutralization by these monoclonal antibodies; for these viruses, neutralization sensitivity correlated with replicative ability. Subinhibitory concentrations of soluble CD4 and a CD4-binding site-directed antibody significantly enhanced the entry of viruses containing envelope glycoproteins from some primary patient isolates. The sensitivity of viruses containing the different envelope glycoproteins to neutralization by soluble CD4 or monoclonal antibodies could be predicted by assays dependent on the binding of the inhibitory molecule to the oligomeric envelope glycoprotein complex but less well by assays measuring binding to the monomeric gp120 glycoprotein. These results indicate that the intrinsic structure of the oligomeric envelope glycoprotein complex of primary HIV-1 isolates, while often less than optimal with respect to the mediation of early events in virus replication, allows a relative degree of resistance to neutralizing antibodies. The interplay of selective forces for higher virus replication efficiency and resistance to neutralizing antibodies could explain the temporal course described for the in vivo emergence of HIV-1 isolates with differing phenotypes.
Forty-six monoclonal antibodies (MAbs) able to bind to the native, monomeric gp120 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) LAI (HXBc2) strain were used to generate a competition matrix. The data suggest the existence of two faces of the gp120 glycoprotein. The binding sites for the viral receptor, CD4, and neutralizing MAbs appear to cluster on one face, which is presumably exposed on the assembled, oligomeric envelope glycoprotein complex. A second gp120 face, which is presumably inaccessible on the envelope glycoprotein complex, contains a number of epitopes for nonneutralizing antibodies. This analysis should be useful for understanding both the interaction of antibodies with the HIV-1 gp120 glycoprotein and neutralization of HIV-1.
A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional
complementary DNA (cDNA) cloning strategy. This protein, designated “fusin,” is a putative G protein-coupled receptor with
seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell
fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target cells.
Fusin messenger RNA levels correlated with HIV-1 permissiveness in diverse human cell types. Fusin acted preferentially for
T cell line-tropic isolates, in comparison to its activity with macrophage-tropic HIV-1 isolates.
Here, we show that the beta-chemokine receptor CKR-5 serves as a cofactor for M-tropic HIV viruses. Expression of CKR-5 with CD4 enables nonpermissive cells to form syncytia with cells expressing M-tropic, but not T-tropic, HIV-1 env proteins. Expression of CKR-5 and CD4 enables entry of a M-tropic, but not a T-tropic, virus strain. A dual-tropic primary HIV-1 isolate (89.6) utilizes both Fusin and CKR-5 as entry cofactors. Cells expressing the 89.6 env protein form syncytia with QT6 cells expressing CD4 and either Fusin or CKR-5. The beta-chemokine receptors CKR-3 and CKR-2b support HIV-1 89.6 env-mediated syncytia formation but do not support fusion by any of the T-tropic or M-tropic strains tested. Our results suggest that the T-tropic viruses characteristic of disease progression may evolve from purely M-tropic viruses prevalent early in virus infection through changes in the env protein that enable the virus to use multiple entry cofactors.
The chemokine eotaxin is unusual in that it appears to be a highly specific chemoattractant for eosinophils. Ligand-binding studies with radiolabeled eotaxin demonstrated a receptor on eosinophils distinct from the known chemokine receptors CKR-1 and -2. The distinct eotaxin binding site on human eosinophils also bound RANTES (regulated on activation T expressed and secreted) and monocyte chemotactic protein (MCP)3. We have now isolated a cDNA from eosinophils, termed CKR-3, with significant sequence similarity to other well characterized chemokine receptors. Cells transfected with CKR-3 cDNA bound radiolabeled eotaxin specifically and with high affinity, comparable to the binding affinity observed with eosinophils. This receptor also bound RANTES and MCP-3 with high affinity, but not other CC or CXC chemokines. Furthermore, receptor transfectants generated in a murine B cell lymphoma cell line migrated in transwell chemotaxis assays to eotaxin, RANTES, and MCP-3, but not to any other chemokines. A monoclonal antibody recognizing CKR-3 was used to show that eosinophils, but not other leukocyte types, expressed this receptor. This pattern of expression was confirmed by Northern blot with RNA from highly purified leukocyte subsets. The restricted expression of CKR-3 on eosinophils and the fidelity of eotaxin binding to CKR-3, provides a potential mechanism for the selective recruitment and migration of eosinophils within tissues.
Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.
Human immunodeficiency virus-type 1 (HIV-1) entry requires fusion cofactors on the CD4+ target cell. Fusin, a heterotrimeric GTP-binding protein (G protein)-coupled receptor, serves as a cofactor for T cell line-tropic
isolates. The chemokines RANTES, MIP-1α, and MIP-1β, which suppress infection by macrophage-tropic isolates, selectively inhibited
cell fusion mediated by the corresponding envelope glycoproteins (Envs). Recombinant CC CKR5, a G protein-coupled receptor
for these chemokines, rendered CD4-expressing nonhuman cells fusion-competent preferentially with macrophage-tropic Envs.
CC CKR5 messenger RNA was detected selectively in cell types susceptible to macrophage-tropic isolates. CC CKR5 is thus a
fusion cofactor for macrophage-tropic HIV-1 strains.
The CD4 molecule is a T cell surface glycoprotein that interacts with high affinity with the envelope glycoprotein of the human immunodeficiency virus, HIV, thus serving as a cellular receptor for this virus. To define the sites on CD4 essential for binding to gp120, we produced several truncated, soluble derivatives of CD4 and a series of 26 substitution mutants. Quantitative binding analyses with the truncated proteins demonstrate that the determinants for high affinity binding lie solely with the first 106 amino acids of CD4 (the V1 domain), a region having significant sequence homology to immunoglobulin variable regions. Analysis of the substitution mutants further defines a discrete binding site within this domain that overlaps a region structurally homologous to the second complementarity-determining region of antibody variable domains. Finally, we demonstrate that the inhibition of virus infection and virus-mediated cell fusion by soluble CD4 proteins depends on their association with gp120 at this binding site.
We have established a stable, continuous culture Drosophila Schneider 2 cell line that efficiently expresses a secreted, truncated form of the HIV envelope gp120 protein in a regulated manner. The Drosophila produced recombinant gp120 protein is highly glycosylated, is recognized by gp120-specific monoclonal antibodies, binds to the CD4 receptor and has the ability to inhibit syncytia formation between uninfected CD4+ cells and HIV infected cells. We conclude that this recombinant Drosophila envelope protein is an appropriate mimic of the authentic viral envelope protein. Thus, the Drosophila cell provides a continuous, stable culture system for the efficient expression of secreted forms of complex surface glycoproteins in quantities sufficient for detailed analyses.
A human mAb (HmAb) termed F105 was obtained by fusion of antibody-producing EBV-transformed cells with the HMMA2.11TG/O cell line. F105 is an IgG1 kappa antibody that binds to the surfaces of cells infected with all HIV-1 strains tested: MN, RF, IIIB, and SF2, but not uninfected cells. The HmAb immunoprecipitates GP120 from all four strains. F105 does not react with denatured GP120 on Western blots, but does react with viral lysates and purified GP120 dotted onto nitrocellulose filter paper under nondenaturing conditions. rGP120 from SF2 and soluble rCD4 inhibit antibody binding to infected cells in a dose-dependent manner. F105 inhibits the binding of free, infectious virions to uninfected HT-H9 cells with 50% of maximal (100%) inhibition at approximately 1 microgram/ml. F105 inhibits infection of HT-H9 cells by 100 tissue culture infective dose 50% units of MN and IIIB strains with 50% inhibition at concentrations of HmAb readily achievable in man. It appears that the F105 HmAb reacts with a conformationally defined epitope on HIV-1/GP120 that is exposed on the free virion and is important for binding to the cell surface by the virion. The epitope, which is immunogenic in humans, appears to be within, or topographically near, the CD4-binding site. F105 and the F105 epitope are potentially useful in therapy and in the design of peptide or anti-Id based vaccines; monitoring of the expression of the Id may prove useful in evaluating immune responses in infected individuals or vaccinated volunteers.
Cells of the monocyte-macrophage lineage are targets for human immunodeficiency virus-1 (HIV-1) infection in vivo. However, many laboratory strains of HIV-1 that efficiently infect transformed T cell lines replicate poorly in macrophages. A 20-amino acid sequence from the macrophage-tropic BaL isolate of HIV-1 was sufficient to confer macrophage tropism on HTLV-IIIB, a T cell line--tropic isolate. This small sequence element is in the V3 loop, the envelope domain that is the principal neutralizing determinant of HIV-1. Thus, the V3 loop not only serves as a target of the host immune response but is also pivotal in determining HIV-1 tissue tropism.
Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.
Deletions of the major variable regions (V1/V2, V3, and V4) of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein were created to study the role of these regions in function and antigenicity. Deletion of the V4 region disrupted processing of the envelope glycoprotein precursor. In contrast, the deletion of the V1/V2 and/or V3 regions yielded processed exterior envelope glycoproteins that retained the ability to interact with the gp41 transmembrane glycoprotein and the CD4 receptor. Shedding of the gp120 exterior glycoprotein by soluble CD4 was observed for the mutant with the V3 deletion but did not occur for the V1/V2-deleted mutant. None of the deletion mutants formed syncytia or supported virus entry. Importantly, the affinity of neutralizing antibodies directed against the CD4-binding region for the multimeric envelope glycoprotein complex was increased dramatically by the removal of both the V1/V2 and V3 structures. These results indicate that, in addition to playing essential roles in the induction of membrane fusion, the major variable regions mask conserved neutralization epitopes of the HIV-1 gp120 glycoprotein from antibodies. These results explain the temporal pattern associated with generation of HIV-1-neutralizing antibodies following infection and suggest stratagems for eliciting improved immune responses to conserved gp120 epitopes.
The cloning of several receptors activated by either CC or CXC chemokines and belonging to the G protein-coupled family of receptors has been reported recently. In the present work, we describe the cloning of a human gene, named ChemR13, encoding a new CC-chemokine receptor. The gene encodes a protein of 352 amino acids with a calculated molecular mass of 40 600 Da and displaying a single potential site for N-linked glycosylation. Using a set of overlapping lambda clones, the genomic organisation of the locus was investigated, demonstrating that the ChemR13 gene is physically linked, and in the same orientation, as the CC-CKR2 gene that encodes a receptor for the monocyte chemoattractant protein-1 (MCP-1). A distance of 17.5 kb separates the two coding regions, which share 75% identity in nucleic acid and amino acid sequences. Human ChemR13 was functionally expressed in a stably transfected CHO-K1 cell line. Physiological responses to chemokines were monitored using a microphysiometer. Macrophage inflammatory protein 1 alpha (MIP-1 alpha) was the most potent agonist. MIP-1 beta and RANTES were also active at physiological concentrations. The other CC-chemokines, MCP-1, MCP-2 and MCP-3, as well as CXC-chemokines (IL-8, GRO alpha) had no effect. ChemR13 receptor transcripts were detected by Northern blotting in the promyeloblastic cell line KG-1A, suggesting a potential role in the control of granulocytic lineage proliferation or differentiation. ChemR13 is thus a new member of the growing family of chemokine receptors that mediate the recruitment of cells involved in immune and inflammatory processes. Being the fifth functionally identified receptor in his class, this new CC-chemokine receptor (CC-CKR) is tentatively designated CC-CKR5.
The beta-chemokines MIP-1alpha, MIP-1beta and RANTES inhibit infection of CD4+ T cells by primary, non-syncytium-inducing (NSI) HIV-1 strains at the virus entry stage, and also block env-mediated cell-cell membrane fusion. CD4+ T cells from some HIV-1-exposed uninfected individuals cannot fuse with NSI HIV-1 strains and secrete high levels of beta-chemokines. Expression of the beta-chemokine receptor CC-CKR-5 in CD4+, non-permissive human and non-human cells renders them susceptible to infection by NSI strains, and allows env-mediated membrane fusion. CC-CKR-5 is a second receptor for NSI primary viruses.
Entry of HIV-1 into target cells requires cell-surface CD4 and additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T-cell lines was recently identified and named fusin. However, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains in vivo. The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the beta-chemokines RANTES, MIP-1alpha and MIP-1beta.
Chemokines affect leukocyte chemotactic and activation activities through specific G protein-coupled receptors. In an effort to map the closely linked CC chemokine receptor genes, we identified a novel chemokine receptor encoded 18 kilobase pairs downstream of the monocyte chemoattractant protein-1 (MCP-1) receptor (CCR2) gene on human chromosome 3p21. The deduced amino acid sequence of this novel receptor, designated CCR5, is most similar to CCR2B, sharing 71% identical residues. Transfected cells expressing the receptor bind RANTES (regulated on activation normal T cell expressed), MIP-1beta, and MIP-1alpha with high affinity and generate inositol phosphates in response to these chemokines. This same combination of chemokines has recently been shown to potently inhibit human immunodeficiency virus replication in human peripheral blood leukocytes (Cocchi, F., DeVico, A. L., Garzino-Demo, A., Arya, S. K., Gallo, R. C., and Lusso, P.(1995) Science 270, 1811-1815). CCR5 is expressed in lymphoid organs such as thymus and spleen, as well as in peripheral blood leukocytes, including macrophages and T cells, and is the first example of a human chemokine receptor that signals in response to MIP-1beta.
We examined the ability of chemokine receptors and related G protein-coupled receptors to facilitate infection by primary, clinical HIV-1 isolates. CCR5, when expressed along with CD4, the HIV-1 receptor, allowed cell lines resistant to most primary HIV-1 isolates to be infected. CCR3 facilitated infection by a more restricted subset of primary viruses, and binding of the CCR3 ligand, eotaxin, inhibited infection by these isolates. Utilization of CCR3 and CCR5 on the target cell depended upon the sequence of the third variable (V3) region of the HIV-1 gp120 exterior envelope glycoprotein. The ability of various members of the chemokine receptor family to support the early stages of HIV-1 infection helps to explain viral tropism and beta-chemokine inhibition of primary HIV-1 isolates.