RESUMO
RNA-modulating factors not only regulate multiple steps of cellular RNA metabolism, but also emerge as key effectors of the immune response against invading viral pathogens including human immunodeficiency virus type-1 (HIV-1). However, the cellular RNA-binding proteins involved in the establishment and maintenance of latent HIV-1 reservoirs have not been extensively studied. Here, we screened a panel of 62 cellular RNA-binding proteins and identified NEDD4-binding protein 1 (N4BP1) as a potent interferon-inducible inhibitor of HIV-1 in primary T cells and macrophages. N4BP1 harbours a prototypical PilT N terminus-like RNase domain and inhibits HIV-1 replication by interacting with and degrading viral mRNA species. Following activation of CD4+ T cells, however, N4BP1 undergoes rapid cleavage at Arg 509 by the paracaspase named mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1). Mutational analyses and knockout studies revealed that MALT1-mediated inactivation of N4BP1 facilitates the reactivation of latent HIV-1 proviruses. Taken together, our findings demonstrate that the RNase N4BP1 is an efficient restriction factor of HIV-1 and suggest that inactivation of N4BP1 by induction of MALT1 activation might facilitate elimination of latent HIV-1 reservoirs.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ativação Viral/genética , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Infecções por HIV/metabolismo , Humanos , Interferon-alfa/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Latência ViralRESUMO
Zika virus (ZIKV) causes severe birth defects and can be transmitted via sexual intercourse. Semen from ZIKV-infected individuals contains high viral loads and may therefore serve as an important vector for virus transmission. Here we analyze the effect of semen on ZIKV infection of cells and tissues derived from the anogenital region. ZIKV replicates in all analyzed cell lines, primary cells, and endometrial or vaginal tissues. However, in the presence of semen, infection by ZIKV and other flaviviruses is potently inhibited. We show that semen prevents ZIKV attachment to target cells, and that an extracellular vesicle preparation from semen is responsible for this anti-ZIKV activity. Our findings suggest that ZIKV transmission is limited by semen. As such, semen appears to serve as a protector against sexual ZIKV transmission, despite the availability of highly susceptible cells in the anogenital tract and high viral loads in this bodily fluid.
Assuntos
Sêmen/imunologia , Doenças Virais Sexualmente Transmissíveis/transmissão , Ligação Viral , Infecção por Zika virus/transmissão , Zika virus/fisiologia , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , Vesículas Extracelulares/imunologia , Feminino , Fibroblastos , Genitália/citologia , Voluntários Saudáveis , Humanos , Concentração Inibidora 50 , Masculino , Cultura Primária de Células , RNA Viral/isolamento & purificação , Sêmen/citologia , Sêmen/virologia , Doenças Virais Sexualmente Transmissíveis/virologia , Células Vero , Carga Viral/imunologia , Replicação Viral/imunologia , Zika virus/isolamento & purificação , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologiaRESUMO
HIV-1 causes chronic inflammation and AIDS in humans, whereas related simian immunodeficiency viruses (SIVs) replicate efficiently in their natural hosts without causing disease. It is currently unknown to what extent virus-specific properties are responsible for these different clinical outcomes. Here, we incorporate two putative HIV-1 virulence determinants, i.e., a Vpu protein that antagonizes tetherin and blocks NF-κB activation and a Nef protein that fails to suppress T cell activation via downmodulation of CD3, into a non-pathogenic SIVagm strain and test their impact on viral replication and pathogenicity in African green monkeys. Despite sustained high-level viremia over more than 4 years, moderately increased immune activation and transcriptional signatures of inflammation, the HIV-1-like SIVagm does not cause immunodeficiency or any other disease. These data indicate that species-specific host factors rather than intrinsic viral virulence factors determine the pathogenicity of primate lentiviruses.
Assuntos
HIV-1/patogenicidade , Especificidade de Hospedeiro , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Lentivirus de Primatas/crescimento & desenvolvimento , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas Virais Reguladoras e Acessórias/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Sequência de Aminoácidos , Animais , Antígeno 2 do Estroma da Médula Óssea/genética , Antígeno 2 do Estroma da Médula Óssea/imunologia , Complexo CD3/genética , Complexo CD3/imunologia , Chlorocebus aethiops , Feminino , Regulação da Expressão Gênica , HIV-1/crescimento & desenvolvimento , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Lentivirus de Primatas/patogenicidade , Ativação Linfocitária , NF-kappa B/genética , NF-kappa B/imunologia , Alinhamento de Sequência , Transdução de Sinais , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Transcrição Gênica , Carga Viral , Proteínas Virais Reguladoras e Acessórias/genética , Virulência , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
In chronic myeloid leukemia (CML), the translocation t(9;22) results in the fusion protein BCR-ABL (breakpoint cluster region-abelson murine leukemia), a tyrosine kinase mediating oncogenic signaling which is successfully targeted by treatment with BCR-ABL inhibitors like imatinib. However, BCR-ABL inhibitors may also affect antitumor immunity. For instance, it was reported that imatinib impairs the function of dendritic cells (DCs) that play a central role in initiating and sustaining T cell responses. Meanwhile, second generation BCR-ABL inhibitors like nilotinib, which inhibits BCR-ABL with enhanced potency have become standard of treatment, at least in patients with BCR-ABL kinase domain mutations. In this study we analyzed the influence of therapeutic concentrations of nilotinib on human monocyte-derived DCs and compared its effects to imatinib. We found that both tyrosine kinase inhibitors (TKI) comparably and significantly impaired differentiation of monocytes to DCs as revealed by curtated downregulation of CD14 and reduced upregulation of CD1a and CD83. This was only partially restored after withdrawal of the TKI. Moreover, both TKI significantly reduced activation-induced IL-12p70 and C-C motif chemokine ligand (CCL) 3 secretion, while divergent TKI effects for CCL2 and CCL5 were observed. In contrast, only nilotinib significantly impaired the migratory capacity of DCs and their capacity to induce T-cell immune responses in MLRs. Our results indicate that imatinib and nilotinib may differ significantly with regard to their influence on antitumor immunity. Thus, for future combinatory approaches and particularly stop studies in CML treatment, choice of the most suitable BCR-ABL inhibitor requires careful consideration.
Assuntos
Células Dendríticas/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Mesilato de Imatinib/farmacologia , Monócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Monócitos/citologia , Monócitos/imunologia , FenótipoRESUMO
BACKGROUND: Elderly HIV-1 infected individuals progress to AIDS more frequently and rapidly than people becoming infected at a young age. To identify possible reasons for these differences in clinical progression, we performed comprehensive phenotypic analyses of CD4+ T cells from uninfected young and elderly individuals, and examined their susceptibility to HIV-1 infection and programmed death. RESULTS: Peripheral blood mononuclear cells (PBMCs) from older people contain an increased percentage of central memory and Th17 CD4+ T cells that are main target cells of HIV-1 and strongly reduced proportions of naïve T cells that are poorly susceptible to HIV-1. Unstimulated T cells from elderly individuals expressed higher levels of activation markers, death receptors, and the viral CXCR4 co-receptor than those from young individuals but responded poorly to stimulation. CD4+ T cells from older individuals were highly susceptible to CXCR4- and CCR5-tropic HIV-1 infection but produced significantly lower quantities of infectious virus than cells from young individuals because they were highly prone to apoptosis and thus presumably had a very short life span. The increased susceptibility of T cells from the elderly to HIV-1 infection correlated directly with CXCR4 and inversely with CD4 expression. The levels of apoptosis correlated with the cell surface expression of FAS but not with the expression of programmed death receptor 1 (PD1) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). CONCLUSIONS: Increased levels of activated and highly susceptible HIV-1 target cells, reduced CD4 and enhanced CXCR4 cell surface expression, together with the high susceptibility to FAS-induced programmed cell death may contribute to the rapid CD4+ T cell depletion and accelerated clinical course of infection in elderly HIV-1-infected individuals.
Assuntos
Envelhecimento , Antígenos CD4/genética , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , Receptores CXCR4/genética , Receptor fas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Feminino , Infecções por HIV/imunologia , HIV-1/fisiologia , Humanos , Memória Imunológica , Leucócitos Mononucleares/imunologia , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/genética , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND: Dendritic cells (DC) are the most potent antigen-presenting cells (APC) with the unique ability to activate naïve T cells and to initiate and maintain primary immune responses. Immunosuppressive and anti-inflammatory stimuli on DC such as the cytokine IL-10 suppress the activity of the transcription factor NF-κB what results in downregulation of costimulatory molecules, MHC and cytokine production. Glycoprotein NMB (GPNMB) is a transmembrane protein, which acts as a coinhibitory molecule strongly inhibiting T cell responses if present on APC. Interestingly, its expression on human monocyte-derived dendritic cells (moDC) is dramatically upregulated upon treatment with IL-10 but also by the BCR-ABL tyrosine kinase inhibitors (TKI) imatinib, nilotinib or dasatinib used for the treatment of chronic myeloid leukemia (CML). However, the molecular mechanisms responsible for GPNMB overexpression are yet unknown. RESULTS: The immunosuppressive cytokine IL-10 and the BCR-ABL TKI imatinib or nilotinib, that were examined here, concordantly inhibit the PI3K/Akt signaling pathway, thereby activating the downstream serine/threonine protein kinase GSK3ß, and subsequently the microphthalmia-associated transcription factor (MITF) that is phosphorylated and translocated into the nucleus. Treatment of moDC with a small molecule inhibitor of MITF activity reduced the expression of GPNMB at the level of mRNA and protein, indicating that GPNMB expression is in fact facilitated by MITF activation. In line with these findings, PI3K/Akt inhibition was found to result in GPNMB overexpression accompanied by reduced stimulatory capacity of moDC in mixed lymphocyte reactions (MLR) with allogeneic T cells that could be restored by addition of the GPNMB T cell ligand syndecan-4 (SD-4). CONCLUSIONS: In summary, imatinib, nilotinib or IL-10 congruently inhibit the PI3K/Akt signaling pathway thereby activating MITF in moDC, resulting in a tolerogenic phenotype. These findings extend current knowledge on the molecular mechanisms balancing activating and inhibitory signals in human DC and may facilitate the targeted manipulation of T cell responses in the context of DC-based immunotherapeutic interventions.
Assuntos
Células Dendríticas/metabolismo , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/biossíntese , Fator de Transcrição Associado à Microftalmia/metabolismo , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Células Cultivadas , Células Dendríticas/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Interleucina-10/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologiaRESUMO
Most simian immunodeficiency viruses use their Nef protein to antagonize the host restriction factor tetherin. A deletion in human tetherin confers Nef resistance, representing a hurdle to successful zoonotic transmission. HIV-1 group M evolved to utilize the viral protein U (Vpu) to counteract tetherin. Although HIV-1 group O has spread epidemically in humans, it has not evolved a Vpu-based tetherin antagonism. Here we show that HIV-1 group O Nef targets a region adjacent to this deletion to inhibit transport of human tetherin to the cell surface, enhances virion release, and increases viral resistance to inhibition by interferon-α. The Nef protein of the inferred common ancestor of group O viruses is also active against human tetherin. Thus, Nef-mediated antagonism of human tetherin evolved prior to the spread of HIV-1 group O and likely facilitated secondary virus transmission. Our results may explain the epidemic spread of HIV-1 group O.