RESUMO
To replicate in their hosts, viruses have to navigate the complexities of the mammalian cell, co-opting mechanisms of cellular physiology while defeating restriction factors that are dedicated to halting their progression. Primate lentiviruses devote a relatively large portion of their coding capacity to counteracting restriction factors by encoding accessory proteins dedicated to neutralizing the antiviral function of these intracellular inhibitors. Research into the roles of the accessory proteins has revealed the existence of previously undetected intrinsic defenses, provided insight into the evolution of primate lentiviruses as they adapt to new species and uncovered new targets for the development of therapeutics. This Review discusses the biology of the restriction factors APOBEC3, SAMHD1 and tetherin and the viral accessory proteins that counteract them.
Assuntos
Antígenos CD/metabolismo , Citosina Desaminase/metabolismo , Infecções por HIV/imunologia , HIV-1/fisiologia , Especificidade de Hospedeiro , Evasão da Resposta Imune , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Desaminases APOBEC , Animais , Evolução Biológica , Citidina Desaminase , Proteínas Ligadas por GPI/metabolismo , Infecções por HIV/virologia , Humanos , Terapia de Alvo Molecular , Proteína 1 com Domínio SAM e Domínio HDRESUMO
SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.
Assuntos
HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismo , Replicação Viral , Animais , Linhagem Celular , Humanos , Espaço Intracelular/metabolismo , Macaca mulatta , Macrófagos/imunologia , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteína 1 com Domínio SAM e Domínio HDRESUMO
V-set and immunoglobulin domain-containing 4 (VSIG4) is a complement receptor of the immunoglobulin superfamily that is specifically expressed on tissue resident macrophages, and its many reported functions and binding partners suggest a complex role in immune function. VSIG4 is reported to have a role in immune surveillance as well as in modulating diverse disease phenotypes such as infections, autoimmune conditions, and cancer. However, the mechanism(s) governing VSIG4's complex, context-dependent role in immune regulation remains elusive. Here, we identify cell surface and soluble glycosaminoglycans, specifically heparan sulfates, as novel binding partners of VSIG4. We demonstrate that genetic deletion of heparan sulfate synthesis enzymes or cleavage of cell-surface heparan sulfates reduced VSIG4 binding to the cell surface. Furthermore, binding studies demonstrate that VSIG4 interacts directly with heparan sulfates, with a preference for highly sulfated moieties and longer glycosaminoglycan chains. To assess the impact on VSIG4 biology, we show that heparan sulfates compete with known VSIG4 binding partners C3b and iC3b. Furthermore, mutagenesis studies indicate that this competition occurs through overlapping binding epitopes for heparan sulfates and complement on VSIG4. Together these data suggest a novel role for heparan sulfates in VSIG4-dependent immune modulation.
Assuntos
Glicosaminoglicanos , Heparitina Sulfato , Heparitina Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Membrana Celular/metabolismo , SulfatosRESUMO
The triphosphohydrolase SAMHD1 (sterile α motif and histidine-aspartate domain-containing protein 1) restricts HIV-1 replication in nondividing myeloid cells by depleting the dNTP pool, preventing reverse transcription. SAMHD1 is also reported to have ribonuclease activity that degrades the virus genomic RNA. Human SAMHD1 is regulated by phosphorylation of its carboxyl terminus at Thr-592, which abrogates its antiviral function yet has only a small effect on its phosphohydrolase activity. In the mouse, SAMHD1 is expressed as two isoforms (ISF1 and ISF2) that differ at the carboxyl terminus due to alternative splicing of the last coding exon. In this study we characterized the biochemical and antiviral properties of the two mouse isoforms of SAMHD1. Both are antiviral in nondividing cells. Mass spectrometry analysis showed that SAMHD1 is phosphorylated at several amino acid residues, one of which (Thr-634) is homologous to Thr-592. Phosphomimetic mutation at Thr-634 of ISF1 ablates its antiviral activity yet has little effect on phosphohydrolase activity in vitro dGTP caused ISF1 to tetramerize, activating its catalytic activity. In contrast, ISF2, which lacks the phosphorylation site, was significantly more active, tetramerized, and was active without added dGTP. Neither isoform nor human SAMHD1 had detectable RNase activity in vitro or affected HIV-1 genomic RNA stability in newly infected cells. These data support a model in which SAMHD1 catalytic activity is regulated through tetramer stabilization by the carboxyl-terminal tail, phosphorylation destabilizing the complexes and inactivating the enzyme. ISF2 may serve to reduce the dNTP pool to very low levels as a means of restricting virus replication.
Assuntos
Infecções por HIV/enzimologia , HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Multimerização Proteica , RNA Viral/metabolismo , Replicação Viral/fisiologia , Substituição de Aminoácidos , Animais , Infecções por HIV/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação de Sentido Incorreto , Fosforilação , RNA Viral/genética , Proteína 1 com Domínio SAM e Domínio HD , Células U937RESUMO
BACKGROUND: Monocytes, the primary myeloid cell-type in peripheral blood, are resistant to HIV-1 infection as a result of the lentiviral restriction factor SAMHD1. Toll-like receptors recognize microbial pathogen components, inducing the expression of antiviral host proteins and proinflammatory cytokines. TLR agonists that mimic microbial ligands have been found to have activity against HIV-1 in macrophages. The induction of restriction factors in monocytes by TLR agonist activation has not been well studied. To analyze restriction factor induction by TLR activation in monocytes, we used the imidazoquinoline TLR7/8 agonist R848 and infected with HIV-1 reporter virus that contained packaged viral accessory protein Vpx, which allows the virus to escape SAMHD1-mediated restriction. RESULTS: R848 prevented the replication of Vpx-containing HIV-1 and HIV-2 in peripheral blood mononuclear cells and monocytes. The block was post-entry but prior to reverse transcription of the viral genomic RNA. The restriction was associated with destabilization of the genomic RNA molecules of the in-coming virus particle. R848 treatment of activated T cells did not protect them from infection but treated monocytes produced high levels of proinflammatory cytokines, including type-I IFN that protected bystander activated T cells from infection. CONCLUSION: The activation of TLR7/8 induces two independent restrictions to HIV-1 replication in monocytes: a cell-intrinsic block that acts post-entry to prevent reverse transcription; and a cell-extrinsic block, in which monocytes produce high levels of proinflammatory cytokines (primarily type-I IFN) that protects bystander monocytes and T lymphocytes. The cell-intrinsic block may result from the induction of a novel restriction factor, which can be termed Lv5 and acts by destabilizing the in-coming viral genomic RNA, either by the induction of a host ribonuclease or by disrupting the viral capsid. TLR agonists are being developed for therapeutic use to diminish the size of the latent provirus reservoir in HIV-1 infected individuals. Such drugs may both induce latent provirus expression and restrict virus replication during treatment.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Imidazóis/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/virologia , Linhagem Celular , Células HEK293 , HIV-1/fisiologia , Humanos , Interferon Tipo I/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Ativação Linfocitária/efeitos dos fármacos , Monócitos/imunologia , Proteínas Monoméricas de Ligação ao GTP/genética , RNA Viral , Transcrição Reversa , Proteína 1 com Domínio SAM e Domínio HD , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Ketoconazole binds to and antagonizes pregnane X receptor (PXR) activation. RESULTS: Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. CONCLUSION: Ketoconazole genetically interacts with specific PXR surface residues. SIGNIFICANCE: A yeast-based genetic method to discover novel nuclear receptor interactions with ligands that associate with surface binding sites is suggested. The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple "gain-of-function" mutants that were "resistant" to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications.
Assuntos
Receptores de Esteroides/química , Saccharomyces cerevisiae/efeitos dos fármacos , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Antifúngicos/farmacologia , Sítios de Ligação , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Farmacorresistência Fúngica , Humanos , Cetoconazol/farmacologia , Simulação de Acoplamento Molecular , Mutagênese , Proteína Oncogênica pp60(v-src)/biossíntese , Proteína Oncogênica pp60(v-src)/genética , Receptor de Pregnano X , Ligação Proteica , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/fisiologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Rifampina/farmacologia , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional , Técnicas do Sistema de Duplo-Híbrido , XenobióticosRESUMO
Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.
Assuntos
Didesoxinucleotídeos/metabolismo , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Linhagem Celular , Expressão Gênica , Genes Reporter , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Interações Hospedeiro-Patógeno , Humanos , Lamivudina/farmacologia , Luciferases/genética , Luciferases/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/virologia , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Monoméricas de Ligação ao GTP/genética , Organofosfonatos/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteína 1 com Domínio SAM e Domínio HD , Estavudina/farmacologia , Tenofovir , Replicação Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
BACKGROUND: SAMHD1 is a triphosphohydrolase that restricts the replication of HIV-1 and SIV in myeloid cells. In macrophages and dendritic cells, SAMHD1 restricts virus replication by diminishing the deoxynucleotide triphosphate pool to a level below that which supports lentiviral reverse transcription. HIV-2 and related SIVs encode the accessory protein Vpx to induce the proteasomal degradation of SAMHD1 following virus entry. While SAMHD1 has been shown to restrict HIV-1 and SIV, the breadth of its restriction is not known and whether other viruses have a means to counteract the restriction has not been determined. RESULTS: We show that SAMHD1 restricts a wide array of divergent retroviruses, including the alpha, beta and gamma classes. Murine leukemia virus was restricted by SAMHD1 in macrophages yet removal of SAMHD1 did not alleviate the block to infection because of an additional block to viral nuclear import. Prototype foamy virus (PFV) and Human T cell leukemia virus type I (HTLV-1) were the only retroviruses tested that were not restricted by SAMHD1. PFV reverse transcribes predominantly prior to entry and thus is unaffected by the dNTP level in the target cell. It is possible that HTLV-1 has a mechanism to render the virus resistant to SAMHD1-mediated restriction. CONCLUSION: The results suggest that SAMHD1 has broad anti-retroviral activity against which most viruses have not found an escape.
Assuntos
Macrófagos/virologia , Proteínas Monoméricas de Ligação ao GTP/farmacologia , Células Mieloides/virologia , Retroviridae/efeitos dos fármacos , Retroviridae/patogenicidade , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Células Dendríticas/metabolismo , Células Dendríticas/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Células Jurkat , Macrófagos/imunologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células Mieloides/metabolismo , Retroviridae/classificação , Retroviridae/fisiologia , Proteína 1 com Domínio SAM e Domínio HDRESUMO
Sterile alpha motif domain- and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase that restricts the replication of lentiviruses in myeloid cells by hydrolyzing the cellular deoxynucleotide triphosphates to a level below that which is required for reverse transcription. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) encode the accessory protein viral protein X (Vpx) that counteracts SAMHD1. Vpx recruits SAMHD1 to a cullin4A-RING E3 ubiquitin ligase (CRL4), which targets the enzyme for proteasomal degradation. Vpx and SAMHD1 both localize to the nucleus of the cell. We identified the nuclear localization sequence (NLS) of SAMHD1 as a conserved KRPR sequence at amino acid residues 11 to 14. SAMHD1 lacking a functional NLS localized to the cytoplasm but retained its triphosphohydrolase and antiviral activities. However, cytoplasmic SAMHD1 was resistant to Vpx-induced degradation, and its antiviral activity was not counteracted by Vpx. Cytoplasmic SAMHD1 interacted with Vpx and retained it in the cytoplasm. The inhibition of nuclear export with leptomycin B did not impair the ability of Vpx to degrade SAMHD1. These findings suggest that SAMHD1 is targeted by Vpx for ubiquitination and degradation in the nucleus.
Assuntos
Núcleo Celular/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos/genética , Proteínas Culina/metabolismo , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Sinais de Localização Nuclear/genética , Plasmídeos/genética , Proteína 1 com Domínio SAM e Domínio HD , UbiquitinaçãoRESUMO
A cell-based assay was developed to detect neutralizing anti-drug antibodies (NAbs) against odronextamab, a CD20xCD3 bispecific monoclonal antibody (mAb) under investigation for treatment of CD20+ B cell malignancies. In this assay, odronextamab bridges between two cell types, CD20-expressing HEK293 cells and CD3-expressing Jurkat T cells that generate a luciferase signal upon CD3 clustering. Patient samples containing NAbs directed to either arm of the bispecific drug block the odronextamab bridge formation between the cell lines thus preventing the generation of the luciferase signal. We determined that other anti-CD20 therapeutics also block bridge formation, resulting in false-positive results. In patient samples from odronextamab clinical trials, approximately 30% of baseline samples had a strong false-positive NAb signal that correlated with the presence of prior rituximab (anti-CD20) therapy. We determined that rituximab interference can be minimized by the addition of anti-rituximab antibodies in the NAb assay. Understanding and mitigating the impact of prior biologic exposure is increasingly important for implementing a successful bioanalytical strategy to support clinical drug development, especially in the immuno-oncology field. Odronextamab neutralizing antibody assay, interference, and mitigation. A Design of the odronextamab neutralizing antibody (NAb) assay where anti-CD20xCD3 drug bridges between CD20-expressing HEK293 cells and Jurkat T cells expressing an NFAT response element and luciferase reporter. True NAb prevents odronextamab from bridging between target and effector cells, thus preventing the expression of luciferase. B Interference with odronextamab from other anti-CD20 therapeutic antibodies (e.g., rituximab) from prior disease treatment generates a false-positive NAb result. Assay interference can be mitigated with an anti-idiotypic antibody against the interfering therapy.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais , Anticorpos Neutralizantes , Antígenos CD20 , Células HEK293 , Humanos , RituximabRESUMO
Dendritic cells are professional antigen-presenting cells of the immune system and are major producers of type-I interferon. Their role in HIV-1 infection is not well understood. They express CD4 and CCR5 yet appear to be resistant to infection. In culture, infection of the cells with HIV-1 is inhibited by the host cell restriction factor SAMHD1. Lentiviruses such as HIV-2/SIVmac counteract the restriction by encoding Vpx, a virion-packaged accessory protein that induces the proteasomal degradation of SAMHD1. In this study we investigated SAMHD1-mediated restriction in the two major dendritic cell subsets: plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC). The cells were highly resistant to HIV-1 and expressed high levels of SAMHD1. SAMHD1 amino acid residue T592, a target of CDK1 phosphorylation, was unphosphorylated, corresponding to the antiviral form of the enzyme. The resistance to infection was not counteracted by Vpx and SAMHD1 was not degraded in these cells. Treatment of pDCs with a cocktail of antibodies that blocked type-I interferon signaling partially restored the ability of Vpx to induce SAMHD1 degradation and caused the cells to become partially permissive to infection. pDCs and mDCs responded to HIV-1 virions by inducing an innate immune response but did not appear to sense newly produced Gag protein. The findings suggest that in vivo, dendritic cells serve as sentinels to alert the immune system to the virus but do not themselves become infected by virtue of high levels of SAMHD1.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Proteínas Monoméricas de Ligação ao GTP/imunologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Células Cultivadas , Humanos , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteína 1 com Domínio SAM e Domínio HDRESUMO
SAMHD1 restricts the replication of HIV-1 and other retroviruses in human myeloid and resting CD4(+) T cells and that is counteracted in SIV and HIV-2 by the Vpx accessory protein. The protein is a phosphohydrolase that lowers the concentration of deoxynucleoside triphosphates (dNTP), blocking reverse transcription of the viral RNA genome. Polymorphisms in the gene encoding SAMHD1 are associated with Aicardi-Goutières Syndrome, a neurological disorder characterized by increased type-I interferon production. SAMHD1 is conserved in mammals but its role in restricting virus replication and controlling interferon production in non-primate species is not well understood. We show that SAMHD1 is catalytically active and expressed at high levels in mouse spleen, lymph nodes, thymus and lung. siRNA knock-down of SAMHD1 in bone marrow-derived macrophages increased their susceptibility to HIV-1 infection. shRNA knock-down of SAMHD1 in the murine monocytic cell-line RAW264.7 increased its susceptibility to HIV-1 and murine leukemia virus and increased the levels of the dNTP pool. In addition, SAMHD1 knock-down in RAW264.7 cells induced the production of type-I interferon and several interferon-stimulated genes, modeling the situation in Aicardi-Goutières Syndrome. Our findings suggest that the role of SAMHD1 in restricting viruses is conserved in the mouse. The RAW264.7 cell-line serves as a useful tool to study the antiviral and innate immune response functions of SAMHD1.
Assuntos
HIV-1/fisiologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Células Mieloides/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Interferon beta/metabolismo , Lentivirus , Luciferases , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteína 1 com Domínio SAM e Domínio HDRESUMO
APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active E. coli-produced A3A. The enzyme binds to single-stranded DNA with a Kd of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.