RESUMO
The septation initiation network (SIN) is a conserved signal transduction network, which is important for cytokinesis in Schizosaccharomyces pombe. The SIN component Etd1p is required for association of some SIN proteins with the spindle pole body (SPB) during anaphase and for contractile ring formation. We show that tethering of Cdc7p or Sid1p to the SIN scaffold Cdc11p at the SPB, rescues etd1-Δ. Analysis of a suppressor of the mutant etd1-M9 revealed that SIN signalling is influenced by the carbon source of the cell. Growth on a non-fermentable carbon source glycerol reduces the requirement for SIN signalling but does not bypass it. The decreased need for SIN signalling is mediated largely by reduction of protein kinase A activity, and it is phenocopied by deletion of pka1 on glucose medium. We conclude that protein kinase A is an important regulator of the SIN, and that SIN signalling is regulated by the carbon source of the cell.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Schizosaccharomyces , Proteínas Quinases Dependentes de AMP Cíclico/genética , Schizosaccharomyces/genética , Citoesqueleto de Actina , Carbono , Transdução de SinaisRESUMO
Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS-STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS-STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS-STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS-STING signalling as a potential strategy to halt neurodegenerative processes during old age.
Assuntos
Envelhecimento , Encéfalo , Disfunção Cognitiva , Inflamação , Proteínas de Membrana , Doenças Neurodegenerativas , Nucleotidiltransferases , Animais , Humanos , Camundongos , Envelhecimento/metabolismo , Envelhecimento/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Efeito Espectador , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , DNA/imunologia , Inflamação/enzimologia , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Transtornos da Memória/enzimologia , Transtornos da Memória/metabolismo , Microglia/metabolismo , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/metabolismo , Nucleotidiltransferases/metabolismo , Especificidade de Órgãos , Transdução de Sinais , Hipocampo/metabolismo , Hipocampo/patologiaRESUMO
Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/genética , SARS-CoV-2/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Animais , Antivirais/farmacologia , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Interferons/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Especificidade da Espécie , Temperatura , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Despite its widespread use, RNA-seq is still too laborious and expensive to replace RT-qPCR as the default gene expression analysis method. We present a novel approach, BRB-seq, which uses early multiplexing to produce 3' cDNA libraries for dozens of samples, requiring just 2 hours of hands-on time. BRB-seq has a comparable performance to the standard TruSeq approach while showing greater tolerance for lower RNA quality and being up to 25 times cheaper. We anticipate that BRB-seq will transform basic laboratory practice given its capacity to generate genome-wide transcriptomic data at a similar cost as profiling four genes using RT-qPCR.
Assuntos
Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Análise de Sequência de RNA , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Subunit vaccines for prevention of congenital cytomegalovirus (CMV) infection based on glycoprotein B (gB) and pp65 are in clinical trials, but it is unclear whether simultaneous vaccination with both antigens enhances protection. We undertook evaluation of a novel bivalent vaccine based on nonreplicating lymphocytic choriomeningitis virus (rLCMV) vectors expressing a cytoplasmic tail-deleted gB [gB(dCt)] and full-length pp65 from human CMV in mice. Immunization with the gB(dCt) vector alone elicited a comparable gB-binding antibody response and a superior neutralizing response to that elicited by adjuvanted subunit gB. Immunization with the pp65 vector alone elicited robust T cell responses. Comparable immunogenicity of the combined gB(dCt) and pp65 vectors with the individual monovalent formulations was demonstrated. To demonstrate proof of principle for a bivalent rLCMV-based HCMV vaccine, the congenital guinea pig cytomegalovirus (GPCMV) infection model was used to compare rLCMV vectors encoding homologs of pp65 (GP83) and gB(dCt), alone and in combination versus Freund's adjuvanted recombinant gB. Both vectors elicited significant immune responses, and no loss of gB immunogenicity was noted with the bivalent formulation. Combined vaccination with rLCMV-vectored GPCMV gB(dCt) and pp65 (GP83) conferred better protection against maternal viremia than subunit or either monovalent rLCMV vaccine. The bivalent vaccine also was significantly more effective in reducing pup mortality than the monovalent vaccines. In summary, bivalent vaccines with rLCMV vectors expressing gB and pp65 elicited potent humoral and cellular responses and conferred protection in the GPCMV model. Further clinical trials of LCMV-vectored HCMV vaccines are warranted.
Assuntos
Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Portadores de Fármacos , Vírus da Coriomeningite Linfocítica/genética , Fosfoproteínas/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Citomegalovirus/congênito , Vacinas contra Citomegalovirus/administração & dosagem , Modelos Animais de Doenças , Feminino , Cobaias , Camundongos Endogâmicos C57BL , Fosfoproteínas/genética , Linfócitos T/imunologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genéticaRESUMO
An important focus in vaccine research is the design of vaccine vectors with low seroprevalence and high immunogenicity. Replication-incompetent lymphocytic choriomeningitis virus (rLCMV) vectors do not elicit vector-neutralizing antibody responses, and homologous prime-boost regimens with rLCMV vectors induce boostable and protective T cell responses to model antigens in mice. However, cellular and humoral immune responses following homologous rLCMV vaccine regimens have not been rigorously evaluated in non-human primates (NHPs). To test whether rLCMV vectors constitute an effective vaccine platform in NHPs, we developed rLCMV vectors expressing SIVmac239 Env and Gag antigens and assessed their immunogenicity in mice and cynomolgus macaques. Immunization with rLCMV vaccine vectors expressing SIV Env and Gag was effective at generating SIV-specific T cell and antibody responses in both mice and NHPs. Epitope mapping using SIV Env in C57BL/6 mice demonstrated that rLCMV vectors induced sustained poly-functional responses to both dominant and subdominant epitopes. Our results suggest the potential of rLCMV vectors as vaccine candidates. Future SIV challenge experiments in rhesus macaques will be needed to assess immune protection by these vaccine vectors.
Assuntos
Antígenos Virais/imunologia , Portadores de Fármacos , Vírus da Coriomeningite Linfocítica/genética , Vacinas contra a SAIDS/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Macaca fascicularis , Camundongos Endogâmicos C57BL , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Vírus da Imunodeficiência Símia/genética , Linfócitos T/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Endogenous retroelements (EREs) account for about half of the mouse or human genome, and their potential as insertional mutagens and transcriptional perturbators is suppressed by early embryonic epigenetic silencing. Here, we asked how ERE control is maintained during the generation of induced pluripotent stem cells (iPSCs), as this procedure involves profound epigenetic remodeling. We found that all EREs tested were markedly up-regulated during the reprogramming of either mouse embryonic fibroblasts, human CD34(+) cells, or human primary hepatocytes. At the iPSC stage, EREs of some classes were repressed, whereas others remained highly expressed, yielding a pattern somewhat reminiscent of that recorded in embryonic stem cells. However, variability persisted between individual iPSC clones in the control of specific ERE integrants. Both during reprogramming and in iPS cells, the up-regulation of specific EREs significantly impacted on the transcription of nearby cellular genes. While transcription triggered by specific ERE integrants at highly precise developmental stages may be an essential step toward obtaining pluripotent cells, the broad and unspecific unleashing of the repetitive genome observed here may contribute to the inefficiency of the reprogramming process and to the phenotypic heterogeneity of iPSCs.
Assuntos
Retrovirus Endógenos/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Transcriptoma , Animais , Células Cultivadas , Reprogramação Celular , Inativação Gênica , Humanos , Camundongos , Regulação para CimaRESUMO
Ribosome-inactivating proteins (RIPs) are endowed with several medicinal properties, including antiviral activity. We demonstrate here that the recently identified type I RIP from Momordica balsamina also possesses antiviral activity, as determined by viral growth curve assays and single-round infection experiments. Importantly, this activity is at play even as doses where the RIP has no cytotoxic effect. In addition, balsamin inhibits HIV-1 replication not only in T cell lines but also in human primary CD4(+) T cells. This antiviral compound exerts its activity at a viral replicative step occurring later than reverse-transcription, most likely on viral protein translation, prior to viral budding and release. Finally, we demonstrate that balsamin antiviral activity is broad since it also impedes influenza virus replication. Altogether our results demonstrate that type I RIP can exert a potent anti-HIV-1 activity which paves the way for new therapeutic avenues for the treatment of viral infections.
Assuntos
HIV-1/efeitos dos fármacos , Momordica/metabolismo , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Farmacorresistência Viral/genética , HIV-1/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Células Jurkat , Mutação , Linfócitos T/patologia , Linfócitos T/virologiaRESUMO
BACKGROUND: Dendritic cells and their subsets, located at mucosal surfaces, are among the first immune cells to encounter disseminating pathogens. The cellular restriction factor BST-2/tetherin (also known as CD317 or HM1.24) potently restricts HIV-1 release by retaining viral particles at the cell surface in many cell types, including primary cells such as macrophages. However, BST-2/tetherin does not efficiently restrict HIV-1 infection in immature dendritic cells. RESULTS: We now report that BST-2/tetherin expression in myeloid (myDC) and monocyte-derived dendritic cells (DC) can be significantly up-regulated by IFN-α treatment and TLR-4 engagement with LPS. In contrast to HeLa or 293T cells, infectious HIV-1 release in immature DC and IFN-α-matured DC was only modestly affected in the absence of Vpu compared to wild-type viruses. Strikingly, immunofluorescence analysis revealed that BST-2/tetherin was excluded from HIV containing tetraspanin-enriched microdomains (TEMs) in both immature DC and IFN-α-matured DC. In contrast, in LPS-mediated mature DC, BST-2/tetherin exerted a significant restriction in transfer of HIV-1 infection to CD4+ T cells. Additionally, LPS, but not IFN-α stimulation of immature DC, leads to a dramatic redistribution of cellular restriction factors to the TEM as well as at the virological synapse between DC and CD4+ T cells. CONCLUSIONS: In conclusion, we demonstrate that TLR-4 engagement in immature DC significantly up-regulates the intrinsic antiviral activity of BST-2/tetherin, during cis-infection of CD4+ T cells across the DC/T cell virological synapse. Manipulating the function and potency of cellular restriction factors such as BST-2/tetherin to HIV-1 infection, has implications in the design of antiviral therapeutic strategies.
Assuntos
Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , HIV-1/imunologia , Sinapses Imunológicas/virologia , Receptor 4 Toll-Like/imunologia , Vírion/imunologia , Antígenos CD/genética , Linfócitos T CD4-Positivos/virologia , Diferenciação Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica/imunologia , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Interferon-alfa/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/virologia , Cultura Primária de Células , Transdução de Sinais , Tetraspaninas/genética , Tetraspaninas/imunologia , Receptor 4 Toll-Like/genética , Vírion/efeitos dos fármacos , Liberação de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Influenza virus infections lead to a burst of type I interferon (IFN) in the human respiratory tract, which most probably accounts for a rapid control of the virus. Although in mice, IFN-induced Mx1 factor mediates a major part of this response, the situation is less clear in humans. Interestingly, a recently identified IFN-induced cellular protein, tetherin (also known as CD317, BST-2, or HM1.24), exerts potent antiviral activity against a broad range of retroviruses, as well as several other enveloped viruses, by impeding the release of newly generated viral particles from the cell surface. Here we show that influenza virus belongs to the targets of this potent antiviral factor. Ectopic expression of tetherin strongly inhibited fully replicative influenza virus. In addition, depleting endogenous tetherin increased viral production of influenza virions, both in cells constitutively expressing tetherin and upon its induction by IFN. We further demonstrate, by biochemical and morphological means, that tetherin exerts its antiviral action by tethering newly budded viral particles, a mechanism similar to the one that operates against HIV-1. In addition, we determined that the magnitude of tetherin antiviral activity is comparable with or higher than the one of several previously identified anti-influenza cellular factors, such as MxA, ADAR1, ISG15, and viperin. Finally, we demonstrate that influenza virus reduces the impact of tetherin-mediated restriction on its replication by several mechanisms. First, the influenza virus NS1 protein impedes IFN-mediated tetherin induction. Second, influenza infection leads to a decrease of tetherin steady state levels, and the neuraminidase surface protein partly counteracts its activity. Overall, our study helps to delineate the intricate molecular battle taking place between influenza virus and its host cells.
Assuntos
Antígenos CD/fisiologia , Influenza Humana/metabolismo , Orthomyxoviridae/metabolismo , Animais , Antígenos CD/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cães , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/fisiologia , Regulação Viral da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Células HeLa , Humanos , Influenza Humana/virologia , Microscopia Eletrônica/métodos , Modelos Biológicos , Plasmídeos/metabolismo , Interferência de RNA , Subtilisina/metabolismoRESUMO
Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novel super-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellular restriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1 (HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherin that preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopy revealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin at the plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors. Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combined biochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini into assembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichment of the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides new insights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-host interactions.
Assuntos
Antígenos CD/metabolismo , Membrana Celular/metabolismo , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Vírion/metabolismo , Antígenos CD/química , Linhagem Celular , Membrana Celular/imunologia , Cor , Imunofluorescência/métodos , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão/métodos , Transfecção , Vírion/imunologiaRESUMO
DC are major targets of HIV-1 during the early events of infection. Yet, HIV-1 infects these cells only inefficiently in vitro as compared with CD4+T lymphocytes. Accordingly, we have previously identified a strong post-entry block to HIV-1 replication in MDDC as a result of the cellular restriction factor A3G. Furthermore, we have demonstrated that As2O3, a drug used to treat acute promyelocytic leukemia, can fully eliminate the potent post-entry restriction of HIV-1 infection in MDDC and in blood-derived MyDC by mechanisms that were unclear. We are now exploring the interplay between As2O3 and A3G-mediated restriction in primary DC subsets. Here, we report that As2O3 counteracts A3G-mediated restriction in MyDC but not in MDDC. RNAi of A3G in MyDC indicated that the As2O3-mediated increase of HIV-1 infection was largely dependent on the presence of the cellular restriction factor. This study reveals an unexpected interplay between As2O3 and A3G-mediated restriction to HIV-1 infection in primary human MyDC.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Citidina Desaminase/fisiologia , Células Dendríticas/virologia , HIV-1/efeitos dos fármacos , Células Mieloides/virologia , Óxidos/farmacologia , Desaminase APOBEC-3G , Elementos Alu , Trióxido de Arsênio , Células HEK293 , Humanos , Interferon-alfa/farmacologia , Retroelementos , Tropismo ViralRESUMO
Retroelements are important evolutionary forces but can be deleterious if left uncontrolled. Members of the human APOBEC3 family of cytidine deaminases can inhibit a wide range of endogenous, as well as exogenous, retroelements. These enzymes are structurally organized in one or two domains comprising a zinc-coordinating motif. APOBEC3G contains two such domains, only the C terminal of which is endowed with editing activity, while its N-terminal counterpart binds RNA, promotes homo-oligomerization, and is necessary for packaging into human immunodeficiency virus type 1 (HIV-1) virions. Here, we performed a large-scale mutagenesis-based analysis of the APOBEC3G N terminus, testing mutants for (i) inhibition of vif-defective HIV-1 infection and Alu retrotransposition, (ii) RNA binding, and (iii) oligomerization. Furthermore, in the absence of structural information on this domain, we used homology modeling to examine the positions of functionally important residues and of residues found to be under positive selection by phylogenetic analyses of primate APOBEC3G genes. Our results reveal the importance of a predicted RNA binding dimerization interface both for packaging into HIV-1 virions and inhibition of both HIV-1 infection and Alu transposition. We further found that the HIV-1-blocking activity of APOBEC3G N-terminal mutants defective for packaging can be almost entirely rescued if their virion incorporation is forced by fusion with Vpr, indicating that the corresponding region of APOBEC3G plays little role in other aspects of its action against this pathogen. Interestingly, residues forming the APOBEC3G dimer interface are highly conserved, contrasting with the rapid evolution of two neighboring surface-exposed amino acid patches, one targeted by the Vif protein of primate lentiviruses and the other of yet-undefined function.
Assuntos
Citidina Desaminase/genética , Citidina Desaminase/imunologia , HIV-1/imunologia , Desaminase APOBEC-3G , Sequência de Aminoácidos , Animais , Sequência Conservada , Citidina Desaminase/metabolismo , Dimerização , Humanos , Lentivirus de Primatas , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Primatas , Ligação Proteica , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and beta-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a beta-TrCP2-dependent manner. Accordingly, in conditions where Vpu-beta-TrCP2-tetherin interplay was not operative, including cells stably knocked down for beta-TrCP2 expression or cells expressing a dominant negative form of beta-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its beta-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication.
Assuntos
Antígenos CD/metabolismo , HIV-1/patogenicidade , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático , Proteínas Ligadas por GPI , Células HeLa , Humanos , Imunoprecipitação , Interferon-alfa/metabolismo , Camundongos , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Vírion/metabolismoRESUMO
Human APOBEC3 proteins are editing enzymes that can interfere with the replication of exogenous retroviruses such as human immunodeficiency virus (HIV), hepadnaviruses such as hepatitis B virus (HBV), and with the retrotransposition of endogenous retroelements such as long-interspersed nuclear elements (LINE) and Alu. Here, we show that APOBEC3G, but not other APOBEC3 family members, binds 7SL RNA, the common ancestor of Alu RNAs that is specifically recruited into HIV virions. Our data further indicate that APOBEC3G recognizes 7SL RNA and Alu RNA by its common structure, the Alu domain, suggesting a mechanism for APOBEC3G- mediated inhibition of Alu retrotransposition. However, we also demonstrate that APOBEC3F and APOBEC3G are normally recruited into and inhibit the infectivity of DeltaVif HIV1 virions when 7SLRNA is prevented from accessing particles by RNA interference against SRP14 or by over expression of SRP19, both components of the signal recognition particle. We thus conclude that 7SL RNA is not an essential mediator of the virion packaging of these antiviral cytidine deaminases.
Assuntos
Elementos Alu/genética , Citidina Desaminase/metabolismo , RNA Citoplasmático Pequeno/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Desaminase APOBEC-3G , Elementos Alu/fisiologia , Sequência de Bases , Linhagem Celular , Citidina Desaminase/genética , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , RNA Citoplasmático Pequeno/química , Partícula de Reconhecimento de Sinal/química , Vírion/metabolismo , Montagem de VírusRESUMO
Interferon (IFN) has been part of the standard treatment of chronic hepatitis B infection for more than 2 decades, yet the mechanism of action of this antiviral remains poorly understood. It was recently observed that members of the human APOBEC family of cytidine deaminases endowed with anti-hepatitis B virus (HBV) activity are upregulated by type I and II IFNs. However, we demonstrated that, in tissue culture, these cellular enzymes are not essential effectors of the anti-HBV action of these cytokines. Here, we show that murine APOBEC3 (muA3) can also block HBV replication. While expressed at low levels in the mouse liver at baseline, muA3 is upregulated upon IFN induction. However, in HBV-transgenic muA3 knockout mice, IFN induction blocked HBV DNA production as efficiently as in control HBV-transgenic muA3-competent animals. We conclude that APOBEC3 is not an essential mediator of the IFN-mediated inhibition of HBV in vivo.
Assuntos
Citidina Desaminase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Vírus da Hepatite B , Hepatite B/virologia , Interferons/farmacologia , Replicação Viral/genética , Animais , Primers do DNA/genética , Genótipo , Hepatite B/tratamento farmacológico , Imunoprecipitação , Interferons/uso terapêutico , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação Viral/efeitos dos fármacosRESUMO
Dendritic cells are central to the early events of human immunodeficiency virus type 1 (HIV-1) transmission, but HIV-1 infects dendritic cells inefficiently in vitro compared to activated CD4(+) T cells. There is a strong postentry restriction of HIV-1 infection in dendritic cells, partly mediated by the cellular restriction factor APOBEC3G. Here, we reveal that arsenic trioxide markedly increases HIV infection of immature and mature dendritic cells as well as blood-derived myeloid dendritic cells in an APOBEC3G- and TRIM5alpha-independent way. Our data suggest the presence of powerful, arsenic-sensitive antiviral activities in primary human immune cells of the dendritic cell lineage.
Assuntos
Arsênio/toxicidade , Proteínas de Transporte/metabolismo , Citidina Desaminase/metabolismo , Células Dendríticas/virologia , Lentivirus/metabolismo , Desaminase APOBEC-3G , Antivirais/farmacologia , Fatores de Restrição Antivirais , Trióxido de Arsênio , Arsenicais , Linfócitos T CD4-Positivos/virologia , Linhagem da Célula , Regulação Viral da Expressão Gênica , HIV-1/metabolismo , Células HeLa , Humanos , Lentivirus/genética , Infecções por Lentivirus/metabolismo , Óxidos/toxicidade , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Zidovudina/farmacologiaRESUMO
Interferons (IFNs) play a major role in the control of hepatitis B virus (HBV), whether as endogenous cytokines limiting the spread of the virus during the acute phase of the infection or as drugs for the treatment of its chronic phase. However, the mechanism by which IFNs inhibit HBV replication has so far remained elusive. Here, we show that type I and II IFN treatment of human hepatocytes induces the production of APOBEC3G (A3G) and, to a lesser extent, that of APOBEC3F (A3F) and APOBEC3B (A3B) but not that of two other cytidine deaminases also endowed with anti-HBV activity, activation-induced cytidine deaminase (AID), and APOBEC1. Most importantly, we reveal that blocking A3B, A3F, and A3G by combining RNA interference and the virion infectivity factor (Vif) protein of human immunodeficiency virus does not abrogate the inhibitory effect of IFNs on HBV. We conclude that these cytidine deaminases are not essential effectors of IFN in its action against this pathogen.
Assuntos
Citidina Desaminase/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Interferon Tipo I/farmacologia , Interferon gama/farmacologia , Replicação Viral/efeitos dos fármacos , Desaminase APOBEC-1 , Desaminase APOBEC-3G , Linhagem Celular Tumoral , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , Citocinas/farmacologia , Citosina Desaminase/antagonistas & inibidores , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Hepatócitos/enzimologia , Humanos , Interferons , Antígenos de Histocompatibilidade Menor , Nucleosídeo Desaminases/antagonistas & inibidores , Nucleosídeo Desaminases/genética , Nucleosídeo Desaminases/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) has antiretroviral activity associated with the hypermutation of viral DNA through cytosine deamination. APOBEC3G has two cytosine deaminase (CDA) domains; the catalytically inactive amino-terminal domain of APOBEC3G (N-CDA) carries the Vif interaction domain. There is no 3-D structure of APOBEC3G solved by X-ray or nuclear magnetic resonance. METHODOLOGY/PRINCIPAL FINDINGS: We predicted the structure of human APOBEC3G based on the crystal structure of APOBEC2. To assess the model structure, we evaluated 48 mutants of APOBEC3G N-CDA that identify novel variants altering DeltaVif HIV-1 infectivity and packaging of APOBEC3G. Results indicated that the key residue D128 is exposed at the surface of the model, with a negative local electrostatic potential. Mutation D128K changes the sign of that local potential. In addition, two novel functionally relevant residues that result in defective APOBEC3G encapsidation, R122 and W127, cluster at the surface. CONCLUSIONS/SIGNIFICANCE: The structure model identifies a cluster of residues important for packaging of APOBEC3G into virions, and may serve to guide functional analysis of APOBEC3G.
Assuntos
Citidina Desaminase/química , Modelos Moleculares , Desaminase APOBEC-3G , Western Blotting , Cristalografia por Raios X , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
We describe a point mutation creating an additional ATG codon in the 5' untranslated region (UTR) of the HAMP gene, in a patient with juvenile hemochromatosis. By transient in vitro transfection studies, we provide evidence that the additional ATG is functional and prevents normal hepcidin production by inducing an aberrant translation initiation of the pre-hepcidin mRNA.