Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
1.
Cell ; 161(6): 1293-1305, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-26046437

RESUMO

Dendritic cells (DCs) play a critical role in the immune response to viral infection through the facilitation of cell-intrinsic antiviral activity and the activation of adaptive immunity. HIV-1 infection of DCs triggers an IRF3-dependent innate immune response, which requires the activity of cyclic GAMP synthase (cGAS). We report the results of a targeted RNAi screen utilizing primary human monocyte-derived DCs (MDDCs) to identify immune regulators that directly interface with HIV-1-encoded features to initiate this innate response. Polyglutamine binding protein 1 (PQBP1) emerged as a strong candidate through this analysis. We found that PQBP1 directly binds to reverse-transcribed HIV-1 DNA and interacts with cGAS to initiate an IRF3-dependent innate response. MDDCs derived from Renpenning syndrome patients, who harbor mutations in the PQBP1 locus, possess a severely attenuated innate immune response to HIV-1 challenge, underscoring the role of PQBP1 as a proximal innate sensor of a HIV-1 infection.


Assuntos
Proteínas de Transporte/metabolismo , HIV-1/imunologia , Imunidade Inata , Proteínas Nucleares/metabolismo , Nucleotidiltransferases/metabolismo , Sequência de Bases , Linhagem Celular , Paralisia Cerebral/imunologia , DNA Viral/genética , Proteínas de Ligação a DNA , HIV-1/fisiologia , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/imunologia , Dados de Sequência Molecular
2.
Mol Cell ; 82(15): 2871-2884.e6, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35809572

RESUMO

We have previously described polyglutamine-binding protein 1 (PQBP1) as an adapter required for the cyclic GMP-AMP synthase (cGAS)-mediated innate response to the human immunodeficiency virus 1 (HIV-1) and other lentiviruses. Cytoplasmic HIV-1 DNA is a transient and low-abundance pathogen-associated molecular pattern (PAMP), and the mechanism for its detection and verification is not fully understood. Here, we show a two-factor authentication strategy by the innate surveillance machinery to selectively respond to the low concentration of HIV-1 DNA, while distinguishing these species from extranuclear DNA molecules. We find that, upon HIV-1 infection, PQBP1 decorates the intact viral capsid, and this serves as a primary verification step for the viral nucleic acid cargo. As reverse transcription and capsid disassembly initiate, cGAS is recruited to the capsid in a PQBP1-dependent manner. This positions cGAS at the site of PAMP generation and sanctions its response to a low-abundance DNA PAMP.


Assuntos
HIV-1 , Capsídeo/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , HIV-1/genética , Humanos , Imunidade Inata , Nucleotidiltransferases/metabolismo , Moléculas com Motivos Associados a Patógenos/metabolismo
3.
Curr Opin Cell Biol ; 20(3): 334-40, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18513937

RESUMO

P-TEFb (CycT1:Cdk9), the metazoan RNA polymerase II Ser2 C-terminal domain (CTD) kinase, regulates transcription elongation at many genes and integrates mRNA synthesis with histone modification, pre-mRNA processing, and mRNA export. Recruitment of P-TEFb to target genes requires deubiquitination of H2Bub, phosphorylation of H3S10, and the bromodomain protein, Brd4. Brd4 activates growth-related genes in the G1 phase of the cell cycle and can also tether P-TEFb to mitotic chromosomes, possibly to mark sites of active transcription throughout cell division. P-TEFb co-operates with c-Myc during transactivation and cell transformation, and also requires SKIP (c-Ski-interacting protein), an mRNA elongation and splicing factor. Some functions of the P-TEFb/Ser2P CTD are executed by the Spt6 transcription elongation factor, which binds directly to the phosphorylated CTD and recruits the Iws1 ('interacts with Spt6') protein. Iws1, in turn, interacts with the REF1/Aly nuclear export adaptor and stimulates the kinetics of mRNA export. Given the prominent role of Spt6 in regulating chromatin structure, the CTD-bound Spt6:Iws1 complex may also control histone modifications during elongation. Following transcription, P-TEFb accompanies the mature mRNA to the cytoplasm to promote translation elongation.


Assuntos
Histonas/genética , Fator B de Elongação Transcricional Positiva/genética , Biossíntese de Proteínas/genética , Transcrição Gênica/genética , Animais , Proteínas de Ciclo Celular , Histonas/metabolismo , Humanos , Metilação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
J Mol Biol ; 436(4): 168409, 2024 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-38128824

RESUMO

Human immunodeficiency virus type 1 (HIV-1) stimulates innate immune responses upon infection, including cyclic GMP-AMP synthase (cGAS) signaling that results in type I interferon production. HIV-1-induced activation of cGAS requires the host cell factor polyglutamine binding protein 1 (PQBP1), an intrinsically disordered protein that bridges capsid recognition and cGAS recruitment. However, the molecular details of PQBP1 interactions with the HIV-1 capsid and their functional implications remain poorly understood. Here, we show that PQBP1 binds to HIV-1 capsids through charge complementing contacts between acidic residues in the N-terminal region of PQBP1 and an arginine ring in the central channel of the HIV-1 CA hexamer that makes up the viral capsid. These studies reveal the molecular details of PQBP1's primary interaction with the HIV-1 capsid and suggest that additional elements are likely to contribute to stable capsid binding.


Assuntos
Capsídeo , Proteínas de Ligação a DNA , HIV-1 , Humanos , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas de Ligação a DNA/química , HIV-1/química , Imunidade Inata , Nucleotidiltransferases/química , Ligação Proteica , Conformação Proteica
5.
ACS Infect Dis ; 8(7): 1265-1279, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35766385

RESUMO

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.


Assuntos
Tratamento Farmacológico da COVID-19 , Vírus da Influenza A , Infecção por Zika virus , Zika virus , Antivirais/farmacologia , Antivirais/uso terapêutico , Depsipeptídeos , Humanos , Pandemias , SARS-CoV-2 , Infecção por Zika virus/tratamento farmacológico
6.
Cell Rep ; 34(2): 108628, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33440148

RESUMO

Recent studies have profiled the innate immune signatures in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggest that cellular responses to viral challenge may affect disease severity. Yet the molecular events that underlie cellular recognition and response to SARS-CoV-2 infection remain to be elucidated. Here, we find that SARS-CoV-2 replication induces a delayed interferon (IFN) response in lung epithelial cells. By screening 16 putative sensors involved in sensing of RNA virus infection, we found that MDA5 and LGP2 primarily regulate IFN induction in response to SARS-CoV-2 infection. Further analyses revealed that viral intermediates specifically activate the IFN response through MDA5-mediated sensing. Additionally, we find that IRF3, IRF5, and NF-κB/p65 are the key transcription factors regulating the IFN response during SARS-CoV-2 infection. In summary, these findings provide critical insights into the molecular basis of the innate immune recognition and signaling response to SARS-CoV-2.


Assuntos
Imunidade Inata , Helicase IFIH1 Induzida por Interferon/metabolismo , SARS-CoV-2/fisiologia , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferons/genética , Interferons/metabolismo , RNA Helicases/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Replicação Viral
7.
Cells ; 9(1)2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31968566

RESUMO

Innate immunity represents the human immune system's first line of defense against a pathogenic intruder and is initiated by the recognition of conserved molecular structures known as pathogen-associated molecular patterns (PAMPs) by specialized cellular sensors, called pattern recognition receptors (PRRs). Human immunodeficiency virus type 1 (HIV-1) is a unique human RNA virus that causes acquired immunodeficiency syndrome (AIDS) in infected individuals. During the replication cycle, HIV-1 undergoes reverse transcription of its RNA genome and integrates the resulting DNA into the human genome. Subsequently, transcription of the integrated provirus results in production of new virions and spreading infection of the virus. Throughout the viral replication cycle, numerous nucleic acid derived PAMPs can be recognized by a diverse set of innate immune sensors in infected cells. However, HIV-1 has evolved efficient strategies to evade or counteract this immune surveillance and the downstream responses. Understanding the molecular underpinnings of the concerted actions of the innate immune system, as well as the corresponding viral evasion mechanisms during infection, is critical to understanding HIV-1 transmission and pathogenesis, and may provide important guidance for the design of appropriate adjuvant and vaccine strategies. Here, we summarize current knowledge of the molecular basis for sensing HIV-1 in human cells, including CD4+ T cells, dendritic cells, and macrophages. Furthermore, we discuss the underlying mechanisms by which innate sensing is regulated, and describe the strategies developed by HIV-1 to evade sensing and immune responses.


Assuntos
HIV-1/imunologia , Imunidade Inata , Moléculas com Motivos Associados a Patógenos/metabolismo , Infecções por HIV/imunologia , Humanos , Interferons/metabolismo , Transdução de Sinais
8.
Cell Rep ; 22(9): 2493-2503, 2018 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-29490283

RESUMO

Accessory proteins of lentiviruses, such as HIV-1, target cellular restriction factors to enhance viral replication. Systematic analyses of proteins that are targeted for degradation by HIV-1 accessory proteins may provide a better understanding of viral immune evasion strategies. Here, we describe a high-throughput platform developed to study cellular protein stability in a highly parallelized matrix format. We used this approach to identify cellular targets of the HIV-1 accessory protein Vpu through arrayed coexpression with 433 interferon-stimulated genes, followed by differential fluorescent labeling and automated image analysis. Among the previously unreported Vpu targets identified by this approach, we find that the E2 ligase mediating ISG15 conjugation, UBE2L6, and the transmembrane protein PLP2 are targeted by Vpu during HIV-1 infection to facilitate late-stage replication. This study provides a framework for the systematic and high-throughput evaluation of protein stability and establishes a more comprehensive portrait of cellular Vpu targets.


Assuntos
HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteólise , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antivirais/metabolismo , Regulação para Baixo , Células HEK293 , Células HeLa , Humanos , Interferons/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Reprodutibilidade dos Testes , Vírion/metabolismo
9.
Nat Microbiol ; 2: 17022, 2017 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-28248290

RESUMO

Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.


Assuntos
Proteína DEAD-box 58/antagonistas & inibidores , RNA Viral/imunologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/imunologia , Ligação Proteica , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Receptores Imunológicos
10.
Cell Host Microbe ; 19(4): 515-528, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-27078069

RESUMO

Understanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1(-/-) mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses.


Assuntos
Infecções por Vírus de DNA/virologia , Vírus de DNA/fisiologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Interferon beta/imunologia , Proteínas de Membrana/imunologia , Proteínas Mitocondriais/metabolismo , Replicação Viral , Animais , Infecções por Vírus de DNA/imunologia , Regulação para Baixo , Feminino , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Humanos , Imunidade Inata , Interferon beta/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas Mitocondriais/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
12.
Genes Dev ; 22(24): 3422-34, 2008 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19141475

RESUMO

Many steps in gene expression and mRNA biosynthesis are coupled to transcription elongation and organized through the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAPII). We showed recently that Spt6, a transcription elongation factor and histone H3 chaperone, binds to the Ser2P CTD and recruits Iws1 and the REF1/Aly mRNA export adaptor to facilitate mRNA export. Here we show that Iws1 also recruits the HYPB/Setd2 histone methyltransferase to the RNAPII elongation complex and is required for H3K36 trimethylation (H3K36me3) across the transcribed region of the c-Myc, HIV-1, and PABPC1 genes in vivo. Interestingly, knockdown of either Iws1 or HYPB/Setd2 also enhanced H3K27me3 at the 5' end of the PABPC1 gene, and depletion of Iws1, but not HYPB/Setd2, increased histone acetylation across the coding regions at the HIV-1 and PABPC1 genes in vivo. Knockdown of HYPB/Setd2, like Iws1, induced bulk HeLa poly(A)+ mRNAs to accumulate in the nucleus. In vitro, recombinant Spt6 binds selectively to a stretch of uninterrupted consensus repeats located in the N-terminal half of the CTD and recruits Iws1. Thus Iws1 connects two distinct CTD-binding proteins, Spt6 and HYPB/Setd2, in a megacomplex that affects mRNA export as well as the histone modification state of active genes.


Assuntos
Regulação da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/biossíntese , Acetilação , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , HIV-1/genética , Humanos , Células Jurkat , Fatores de Transcrição Kruppel-Like/genética , Metilação , Camundongos , Proteína I de Ligação a Poli(A)/genética , Ligação Proteica , Proteínas/genética , RNA Polimerase II/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição
13.
Genes Dev ; 21(2): 160-74, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17234882

RESUMO

Spt6 promotes transcription elongation at many genes and functions as a histone H3 chaperone to alter chromatin structure during transcription. We show here that mammalian Spt6 binds Ser2-phosphorylated (Ser2P) RNA polymerase II (RNAPII) through a primitive SH2 domain, which recognizes phosphoserine rather than phosphotyrosine residues. Surprisingly, a point mutation in the Spt6 SH2 domain (R1358K) blocked binding to RNAPIIo without affecting transcription elongation rates in vitro. However, HIV-1 and c-myc RNAs formed in cells expressing the mutant Spt6 protein were longer than normal and contained splicing defects. Ectopic expression of the wild-type, but not mutant, Spt6 SH2 domain, caused bulk poly(A)+ RNAs to be retained in the nucleus, further suggesting a widespread role for Spt6 in mRNA processing or assembly of export-competent mRNP particles. We cloned the human Spt6-interacting protein, hIws1 (interacts with Spt6), and found that it associates with the nuclear RNA export factor, REF1/Aly. Depletion of endogenous hIws1 resulted in mRNA processing defects, lower levels of REF1/Aly at the c-myc gene, and nuclear retention of bulk HeLa poly(A)+ RNAs in vivo. Thus binding of Spt6 to Ser2-P RNAPII provides a cotranscriptional mechanism to recruit Iws1, REF1/Aly, and associated mRNA processing, surveillance, and export factors to responsive genes.


Assuntos
Proteínas/metabolismo , RNA Polimerase II/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Domínios de Homologia de src , Substituição de Aminoácidos , Animais , Far-Western Blotting , Núcleo Celular/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , HIV-1/genética , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fosfosserina , Mutação Puntual , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA , Proteínas Recombinantes/metabolismo , Fatores de Transcrição , Ativação Transcricional
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA