RESUMO
Macrophages (MΦ) are increasingly recognized as HIV-1 target cells involved in the pathogenesis and persistence of infection. Paradoxically, in vitro infection assays suggest that virus isolates are mostly T-cell-tropic and rarely MΦ-tropic. The latter are assumed to emerge under CD4+ T-cell paucity in tissues such as the brain or at late stage when the CD4 T-cell count declines. However, assays to qualify HIV-1 tropism use cell-free viral particles and may not fully reflect the conditions of in vivo MΦ infection through cell-to-cell viral transfer. Here, we investigated the capacity of viruses expressing primary envelope glycoproteins (Envs) with CCR5 and/or CXCR4 usage from different stages of infection, including transmitted/founder Envs, to infect MΦ by a cell-free mode and through cell-to-cell transfer from infected CD4+ T cells. The results show that most viruses were unable to enter MΦ as cell-free particles, in agreement with the current view that non-M-tropic viruses inefficiently use CD4 and/or CCR5 or CXCR4 entry receptors on MΦ. In contrast, all viruses could be effectively cell-to-cell transferred to MΦ from infected CD4+ T cells. We further showed that viral transfer proceeded through Env-dependent cell-cell fusion of infected T cells with MΦ targets, leading to the formation of productively infected multinucleated giant cells. Compared to cell-free infection, infected T-cell/MΦ contacts showed enhanced interactions of R5 M- and non-M-tropic Envs with CD4 and CCR5, resulting in a reduced dependence on receptor expression levels on MΦ for viral entry. Altogether, our results show that virus cell-to-cell transfer overcomes the entry block of isolates initially defined as non-macrophage-tropic, indicating that HIV-1 has a more prevalent tropism for MΦ than initially suggested. This sheds light into the role of this route of virus cell-to-cell transfer to MΦ in CD4+ T cell rich tissues for HIV-1 transmission, dissemination and formation of tissue viral reservoirs.
Assuntos
Infecções por HIV , HIV-1 , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos , Infecções por HIV/metabolismo , HIV-1/metabolismo , Humanos , Macrófagos/metabolismo , Receptores CCR5/metabolismo , Internalização do VírusRESUMO
HIV-1-associated nephropathy (HIVAN) is a severe complication of HIV-1 infection. To gain insight into the pathogenesis of kidney disease in the setting of HIV, a transgenic (Tg) mouse model [CD4C/HIV-negative regulator factor (Nef)] was used in which HIV-1 nef expression is under control of regulatory sequences (CD4C) of the human CD4 gene, thus allowing expression in target cells of the virus. These Tg mice develop a collapsing focal segmental glomerulosclerosis associated with microcystic dilatation, similar to human HIVAN. To identify kidney cells permissive to the CD4C promoter, CD4C reporter Tg lines were used. They showed preferential expression in glomeruli, mainly in mesangial cells. Breeding CD4C/HIV Tg mice on 10 different mouse backgrounds showed that HIVAN was modulated by host genetic factors. Studies of gene-deficient Tg mice revealed that the presence of B and T cells and that of several genes was dispensable for the development of HIVAN: those involved in apoptosis (Trp53, Tnfsf10, Tnf, Tnfrsf1b, and Bax), in immune cell recruitment (Ccl3, Ccl2, Ccr2, Ccr5, and Cx3cr1), in nitric oxide (NO) formation (Nos3 and Nos2), or in cell signaling (Fyn, Lck, and Hck/Fgr). However, deletion of Src partially and that of Hck/Lyn largely abrogated its development. These data suggest that Nef expression in mesangial cells through hematopoietic cell kinase (Hck)/Lck/Yes novel tyrosine kinase (Lyn) represents important cellular and molecular events for the development of HIVAN in these Tg mice.
Assuntos
Nefropatia Associada a AIDS , Infecções por HIV , Camundongos , Humanos , Animais , Proteínas Tirosina Quinases/metabolismo , Nefropatia Associada a AIDS/genética , Nefropatia Associada a AIDS/patologia , Camundongos Transgênicos , Infecções por HIV/complicações , Tirosina , Quinases da Família src , Proteínas Proto-Oncogênicas c-hckRESUMO
HIV-1 is dependent on the host cell for providing the metabolic resources for completion of its viral replication cycle. Thus, HIV-1 replicates efficiently only in activated CD4+ T cells. Barriers preventing HIV-1 replication in resting CD4+ T cells include a block that limits reverse transcription and also the lack of activity of several inducible transcription factors, such as NF-κB and NFAT. Because FOXO1 is a master regulator of T cell functions, we studied the effect of its inhibition on T cell/HIV-1 interactions. By using AS1842856, a FOXO1 pharmacologic inhibitor, we observe that FOXO1 inhibition induces a metabolic activation of T cells with a G0/G1 transition in the absence of any stimulatory signal. One parallel outcome of this change is the inhibition of the activity of the HIV restriction factor SAMHD1 and the activation of the NFAT pathway. FOXO1 inhibition by AS1842856 makes resting T cells permissive to HIV-1 infection. In addition, we found that FOXO1 inhibition by either AS1842856 treatment or upon FOXO1 knockdown induces the reactivation of HIV-1 latent proviruses in T cells. We conclude that FOXO1 has a central role in the HIV-1/T cell interaction and that inhibiting FOXO1 with drugs such as AS1842856 may be a new therapeutic shock-and-kill strategy to eliminate the HIV-1 reservoir in human T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteína Forkhead Box O1/antagonistas & inibidores , Regulação da Expressão Gênica , Infecções por HIV/virologia , HIV-1/imunologia , Ativação Viral/imunologia , Replicação Viral , Animais , Linfócitos T CD4-Positivos/virologia , Ciclo Celular , Proteína Forkhead Box O1/genética , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Macaca fascicularis , Masculino , Latência ViralRESUMO
Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.
Assuntos
Reabsorção Óssea/etiologia , Infecções por HIV/complicações , HIV-1/fisiologia , Osteoclastos/virologia , Actinas/metabolismo , Animais , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Osso e Ossos/metabolismo , Adesão Celular , Feminino , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Camundongos , Osteoclastos/citologia , Osteoclastos/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Cell-cell fusion between eukaryotic cells is a general process involved in many physiological and pathological conditions, including infections by bacteria, parasites, and viruses. As obligate intracellular pathogens, viruses use intracellular machineries and pathways for efficient replication in their host target cells. Interestingly, certain viruses, and, more especially, enveloped viruses belonging to different viral families and including human pathogens, can mediate cell-cell fusion between infected cells and neighboring non-infected cells. Depending of the cellular environment and tissue organization, this virus-mediated cell-cell fusion leads to the merge of membrane and cytoplasm contents and formation of multinucleated cells, also called syncytia, that can express high amount of viral antigens in tissues and organs of infected hosts. This ability of some viruses to trigger cell-cell fusion between infected cells as virus-donor cells and surrounding non-infected target cells is mainly related to virus-encoded fusion proteins, known as viral fusogens displaying high fusogenic properties, and expressed at the cell surface of the virus-donor cells. Virus-induced cell-cell fusion is then mediated by interactions of these viral fusion proteins with surface molecules or receptors involved in virus entry and expressed on neighboring non-infected cells. Thus, the goal of this review is to give an overview of the different animal virus families, with a more special focus on human pathogens, that can trigger cell-cell fusion.
Assuntos
Fusão Celular , Membrana Celular/metabolismo , Fusão de Membrana , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Vírus/metabolismo , Animais , Humanos , Vírus/isolamento & purificaçãoRESUMO
HIV-1-infected macrophages participate in virus dissemination and establishment of virus reservoirs in host tissues, but the mechanisms for virus cell-to-cell transfer to macrophages remain unknown. Here, we reveal the mechanisms for cell-to-cell transfer from infected T cells to macrophages and virus spreading between macrophages. We show that contacts between infected T lymphocytes and macrophages lead to cell fusion for the fast and massive transfer of CCR5-tropic viruses to macrophages. Through the merge of viral material between T cells and macrophages, these newly formed lymphocyte-macrophage fused cells acquire the ability to fuse with neighboring noninfected macrophages. Together, these two-step envelope-dependent cell fusion processes lead to the formation of highly virus-productive multinucleated giant cells reminiscent of the infected multinucleated giant macrophages detected in HIV-1-infected patients and simian immunodeficiency virus-infected macaques. These mechanisms represent an original mode of virus transmission for viral spreading and a new model for the formation of macrophage virus reservoirs during infection.IMPORTANCE We reveal a very efficient mechanism involved in cell-to-cell transfer from infected T cells to macrophages and subsequent virus spreading between macrophages by a two-step cell fusion process. Infected T cells first establish contacts and fuse with macrophage targets. The newly formed lymphocyte-macrophage fused cells then acquire the ability to fuse with surrounding uninfected macrophages, leading to the formation of infected multinucleated giant cells that can survive for a long time, as evidenced in vivo in lymphoid organs and the central nervous system. This route of infection may be a major determinant for virus dissemination and the formation of macrophage virus reservoirs in host tissues during HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/citologia , Células Gigantes/virologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Macrófagos/citologia , Animais , Linfócitos T CD4-Positivos/virologia , Fusão Celular , Linhagem Celular , Células Gigantes/citologia , Células HEK293 , HIV-1/patogenicidade , Humanos , Células Jurkat , Macaca mulatta , Macrófagos/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Vírus da Imunodeficiência Símia/fisiologiaRESUMO
The variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from llamas that have been immunized with a trimeric gp140 bound to a CD4 mimic have been recently isolated (here referred to as VHH JM2 and VHH JM4, respectively). JM2 binds the CD4-binding site of gp120 and neutralizes HIV-1 strains from subtypes B, C, and G. JM4 binds gp120 and neutralizes HIV-1 strains from subtypes A, B, C, A/E, and G in a CD4-dependent manner. In the present study, we constructed glycosylphosphatidylinositol (GPI)-anchored VHH JM2 and JM4 along with an E4 control and transduced them into human CD4+ cell lines and primary CD4 T cells. We report that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. Expression of GPI-VHH JM4, but not GPI-VHH E4 and JM2, on the surface of transduced TZM.bl cells potently neutralizes multiple subtypes of HIV-1 isolates, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble single domain antibody (sdAb) JM4-resistant viruses. Moreover, transduction of CEMss-CCR5 cells with GPI-VHH JM4, but not with GPI-VHH E4, confers resistance to both cell-free and T cell-T cell transmission of HIV-1 and HIV-1 envelope-mediated fusion. Finally, GPI-VHH JM4-transduced human primary CD4 T cells efficiently resist both cell-free and T cell-T cell transmission of HIV-1. Thus, we conclude that VHH JM4, when targeted to the lipid rafts of the plasma membrane, efficiently neutralizes HIV-1 infection via both cell-free and T cell-T cell transmission. Our findings should have important implications for GPI-anchored antibody-based therapy against HIV-1. IMPORTANCE: Lipid rafts are specialized dynamic microdomains of the plasma membrane and have been shown to be gateways for HIV-1 budding as well as entry into T cells and macrophages. In nature, many glycosylphosphatidylinositol (GPI)-anchored proteins localize in the lipid rafts. In the present study, we developed GPI-anchored variable regions (VHHs) of two heavy chain-only antibodies, JM2 and JM4, from immunized llamas. We show that by genetically linking the VHHs with a GPI attachment signal, VHHs are targeted to the lipid rafts of the plasma membranes. GPI-VHH JM4, but not GPI-VHH JM2, in transduced CD4+ cell lines and human primary CD4 T cells not only efficiently blocks diverse HIV-1 strains, including tier 2 or 3 strains, transmitted founders, quasispecies, and soluble sdAb JM4-resistant strains, but also efficiently interferes T cell-T cell transmissions of HIV-1 and HIV-1 envelope-mediated fusion. Our findings should have important implications in GPI-anchored antibody-based therapy against HIV-1.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Camelídeos Americanos/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Camelídeos Americanos/genética , Linhagem Celular , Sistema Livre de Células , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Microdomínios da Membrana/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
Macrophages are motile leukocytes, targeted by HIV-1, thought to play a critical role in host dissemination of the virus. However, whether infection impacts their migration capacity remains unknown. We show that 2-dimensional migration and the 3-dimensional (3D) amoeboid migration mode of HIV-1-infected human monocyte-derived macrophages were inhibited, whereas the 3D mesenchymal migration was enhanced. The viral protein Nef was necessary and sufficient for all HIV-1-mediated effects on migration. In Nef transgenic mice, tissue infiltration of macrophages was increased in a tumor model and in several tissues at steady state, suggesting a dominant role for mesenchymal migration in vivo. The mesenchymal motility involves matrix proteolysis and podosomes, cell structures constitutive of monocyte-derived cells. Focusing on the mechanisms used by HIV-1 Nef to control the mesenchymal migration, we show that the stability, size, and proteolytic function of podosomes are increased via the phagocyte-specific kinase Hck and Wiskott-Aldrich syndrome protein (WASP), 2 major regulators of podosomes. In conclusion, HIV-1 reprograms macrophage migration, which likely explains macrophage accumulation in several patient tissues, which is a key step for virus spreading and pathogenesis. Moreover, Nef points out podosomes and the Hck/WASP signaling pathway as good candidates to control tissue infiltration of macrophages, a detrimental phenomenon in several diseases.
Assuntos
HIV-1/patogenicidade , Macrófagos/fisiologia , Macrófagos/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Animais , Linhagem Celular Tumoral , Estruturas da Membrana Celular/patologia , Estruturas da Membrana Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Reprogramação Celular/fisiologia , Infecções por HIV/patologia , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-hck/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
ANKHD1 is highly expressed in human acute leukemia cells and potentially regulates multiple cellular functions through its ankyrin-repeat domains. In order to identify interaction partners of the ANKHD1 protein and its role in leukemia cells, we performed a yeast two-hybrid system screen and identified SIVA, a cellular protein known to be involved in proapoptotic signaling pathways. The interaction between ANKHD1 and SIVA was confirmed by co-imunoprecipitation assays. Using human leukemia cell models and lentivirus-mediated shRNA approaches, we showed that ANKHD1 and SIVA proteins have opposing effects. While it is known that SIVA silencing promotes Stathmin 1 activation, increased cell migration and xenograft tumor growth, we showed that ANKHD1 silencing leads to Stathmin 1 inactivation, reduced cell migration and xenograft tumor growth, likely through the inhibition of SIVA/Stathmin 1 association. In addition, we observed that ANKHD1 knockdown decreases cell proliferation, without modulating apoptosis of leukemia cells, while SIVA has a proapoptotic function in U937 cells, but does not modulate proliferation in vitro. Results indicate that ANKHD1 binds to SIVA and has an important role in inducing leukemia cell proliferation and migration via the Stathmin 1 pathway. ANKHD1 may be an oncogene and participate in the leukemia cell phenotype.
Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Leucemia/patologia , Proteínas de Ligação a RNA/genética , Estatmina/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Inativação Gênica , Células HEK293 , Humanos , Células Jurkat , Leucemia/genética , Leucemia/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Estatmina/antagonistas & inibidores , Células U937RESUMO
BACKGROUND: Through incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhancement of virus infectivity in established CD4-positive cell lines via a nonenzymatic mechanism. RESULTS: We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV-1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte-derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2- and RPA32-depleted T lymphocytes. Reciprocally, viruses produced in UNG2-depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. CONCLUSIONS: Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV-1.
Assuntos
DNA Glicosilases/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteína de Replicação A/metabolismo , Transcrição Reversa , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Células Cultivadas , Humanos , Leucócitos Mononucleares/virologiaRESUMO
BACKGROUND: The HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies. RESULTS: We have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef-Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication. CONCLUSIONS: Together, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef.
Assuntos
Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , HIV-1/química , HIV-1/imunologia , Humanos , Ligação Proteica , Conformação ProteicaRESUMO
Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an â¼30 and â¼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , HIV-1/genética , HIV-1/patogenicidade , Proteínas Nucleares/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/deficiência , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Western Blotting , Eletroforese/métodos , Inativação Gênica , Células HEK293 , HIV-1/metabolismo , Humanos , Proteômica , RNA Interferente Pequeno , Tetraspanina 28/metabolismo , Ultracentrifugação , Vírion/metabolismo , alfa-Glucosidases/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.
Assuntos
Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Anti-HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Camelídeos Americanos , Reações Cruzadas , Anticorpos Anti-HIV/imunologia , Humanos , Imunização/métodos , Anticorpos de Domínio Único/imunologiaRESUMO
Although cells of the myeloid lineages, including tissue macrophages and conventional dendritic cells, were rapidly recognized, in addition to CD4+ T lymphocytes, as target cells of HIV-1, their specific roles in the pathophysiology of infection were initially largely neglected. However, numerous studies performed over the past decade, both in vitro in cell culture systems and in vivo in monkey and humanized mouse animal models, led to growing evidence that macrophages play important direct and indirect roles as HIV-1 target cells and in pathogenesis. It has been recently proposed that macrophages are likely involved in all stages of HIV-1 pathogenesis, including virus transmission and dissemination, but above all, in viral persistence through the establishment, together with latently infected CD4+ T cells, of virus reservoirs in many host tissues, the major obstacle to virus eradication in people living with HIV. Infected macrophages are indeed found, very often as multinucleated giant cells expressing viral antigens, in almost all lymphoid and non-lymphoid tissues of HIV-1-infected patients, where they can probably persist for long period of time. In addition, macrophages also likely participate, directly as HIV-1 targets or indirectly as key regulators of innate immunity and inflammation, in the chronic inflammation and associated clinical disorders observed in people living with HIV, even in patients receiving effective antiretroviral therapy. The main objective of this review is therefore to summarize the recent findings, and also to revisit older data, regarding the critical functions of tissue macrophages in the pathophysiology of HIV-1 infection, both as major HIV-1-infected target cells likely found in almost all tissues, as well as regulators of innate immunity and inflammation during the different stages of HIV-1 pathogenesis.
Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Vírus da Imunodeficiência Símia , Humanos , Animais , Camundongos , Macrófagos , HIV-1/fisiologia , Inflamação , Linfócitos T CD4-Positivos , Latência Viral , Replicação ViralRESUMO
The HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the accuracy of reverse transcription. This role of Vpr was related to the recruitment of the nuclear form of the uracil DNA glycosylase (UNG2) enzyme into virus particles, but several conflicting findings have been reported regarding the role of UNG2 encapsidation on viral infectivity. Here, we report that the catalytic activity of UNG2 was not required for influencing HIV-1 mutation, and this function of UNG2 was mapped within a 60-amino-acid domain located in the N-terminal region of the protein required for direct interaction with the p32 subunit of the replication protein A (RPA) complex. Importantly, enforced recruitment of overexpressed UNG2 into virions resulted in a net increase of virus infectivity, and this positive effect on infectivity was also independent of the UNG2 enzymatic activity. In contrast, virus infectivity and replication, as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of either endogenous UNG2 or RPA p32. Taken together, these results demonstrate that incorporation of UNG2 into virions has a positive impact on HIV-1 infectivity and replication and positively influences the reverse transcription process through a nonenzymatic mechanism involving the p32 subunit of the RPA complex.
Assuntos
DNA Glicosilases/metabolismo , Infecções por HIV/enzimologia , HIV-1/fisiologia , Vírion/fisiologia , Linhagem Celular , DNA Glicosilases/genética , Infecções por HIV/virologia , HIV-1/genética , Humanos , Ligação Proteica , Vírion/genética , Replicação Viral , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismoRESUMO
HIV-1 Nef is essential for AIDS pathogenesis, but this viral protein is not targeted by antiviral strategies. The functions of Nef are largely related to perturbations of intracellular trafficking and signaling pathways through leucine-based and polyproline motifs that are required for interactions with clathrin-associated adaptor protein complexes and SH3 domain-containing proteins, such as the phagocyte-specific kinase Hck. We previously described a single-domain antibody (sdAb) targeting Nef and inhibiting many, but not all, of its biological activities. We now report a further development of this anti-Nef strategy through the demonstration of the remarkable inhibitory activity of artificial Nef ligands, called Neffins, comprised of the anti-Nef sdAb fused to modified SH3 domains. The Neffins inhibited all key activities of Nef, including Nef-mediated CD4 and major histocompatibility complex class I (MHC-I) cell surface downregulation and enhancement of virus infectivity. When expressed in T lymphocytes, Neffins specifically inhibited the Nef-induced mislocalization of the Lck kinase, which contributes to the alteration of the formation of the immunological synapse. In macrophages, Neffins inhibited the Nef-induced formation of multinucleated giant cells and podosome rosettes, and it counteracted the inhibitory activity of Nef on phagocytosis. Since we show here that these effects of Nef on macrophage and T cell functions were both dependent on the leucine-based and polyproline motifs, we confirmed that Neffins disrupted interactions of Nef with both AP complexes and Hck. These results demonstrate that it is possible to inhibit all functions of Nef, both in T lymphocytes and macrophages, with a single ligand that represents an efficient tool to develop new antiviral strategies targeting Nef.
Assuntos
Antivirais/metabolismo , HIV-1/metabolismo , Anticorpos de Cadeia Única/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sequência de Aminoácidos , Antígenos CD4/metabolismo , Linhagem Celular , Regulação para Baixo/imunologia , Ordem dos Genes , HIV-1/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Macrófagos/metabolismo , Dados de Sequência Molecular , Fagocitose/imunologia , Ligação Proteica/imunologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Fator de Transcrição AP-1/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Domínios de Homologia de src/genética , Domínios de Homologia de src/imunologiaRESUMO
The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.
Assuntos
Anticorpos de Cadeia Única/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Síndrome da Imunodeficiência Adquirida/terapia , Animais , Camelídeos Americanos/imunologia , Células Cultivadas , Embrião de Mamíferos , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Anticorpos Anti-HIV/farmacologia , Células HeLa , Humanos , Imunoterapia/métodos , Células Jurkat , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Semen is an important vector for sexual HIV-1 transmission. Although CXCR4-tropic (X4) HIV-1 may be present in semen, almost exclusively CCR5-tropic (R5) HIV-1 causes systemic infection after sexual intercourse. To identify factors that may limit sexual X4-HIV-1 transmission, we generated a seminal fluid-derived compound library and screened it for antiviral agents. We identified four adjacent fractions that blocked X4-HIV-1 but not R5-HIV-1 and found that they all contained spermine and spermidine, abundant polyamines in semen. We showed that spermine, which is present in semen at concentrations up to 14 mM, binds CXCR4 and selectively inhibits cell-free and cell-associated X4-HIV-1 infection of cell lines and primary target cells at micromolar concentrations. Our findings suggest that seminal spermine restricts sexual X4-HIV-1 transmission.
Assuntos
Infecções por HIV , HIV-1 , Humanos , Espermidina/farmacologia , Espermina/farmacologia , Infecções por HIV/tratamento farmacológico , Linhagem Celular , Receptores CXCR4RESUMO
Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4+ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.
Assuntos
Linfócitos T CD4-Positivos , Fusão Celular , Infecções por HIV , Macrófagos , Humanos , Linfócitos T CD4-Positivos/metabolismo , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Macrófagos/metabolismo , Macrófagos/virologia , Actomiosina/metabolismoRESUMO
Phagocytosis in macrophages is receptor mediated and relies on actin polymerization coordinated with the focal delivery of intracellular membranes that is necessary for optimal phagocytosis of large particles. Here we show that phagocytosis by various receptors was inhibited in primary human macrophages infected with wild-type HIV-1 but not with a nef-deleted virus. We observed no major perturbation of F-actin accumulation, but adaptor protein 1 (AP1)-positive endosome recruitment was inhibited in HIV-1-infected cells. Expression of negative factor (Nef) was sufficient to inhibit phagocytosis, and myristoylation as well as the LL and DD motifs involved in association of Nef with AP complexes were important for this inhibition. We observed that Nef interferes with AP1 in association with membranes and/or with a cleaved regulatory form of AP1. Finally, an alteration of the recruitment of vesicle-associated membrane protein (VAMP3)- and tumor necrosis factor-alpha (TNFalpha)-positive recycling endosomes regulated by AP1, but not of VAMP7-positive late endosomes, was observed in phagocytic cups of HIV-1-infected macrophages. We conclude that HIV-1 impairs optimal phagosome formation through Nef-dependent perturbation of the endosomal remodeling relying on AP1. We therefore identified a mechanism of macrophage function down-regulation in infected cells.