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1.
PLoS Pathog ; 16(4): e1008407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32240278

RESUMO

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/farmacologia , Núcleo Celular/virologia , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Vírus da Influenza A/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos
2.
Proc Natl Acad Sci U S A ; 115(52): E12218-E12227, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30538201

RESUMO

The influenza virulence factor NS1 protein interacts with the cellular NS1-BP protein to promote splicing and nuclear export of the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix protein and is alternatively spliced into the M2 mRNA, which is translated into the M2 ion channel. These proteins have key functions in viral trafficking and budding. To uncover the NS1-BP structural and functional activities in splicing and nuclear export, we performed proteomics analysis of nuclear NS1-BP binding partners and showed its interaction with constituents of the splicing and mRNA export machineries. NS1-BP BTB domains form dimers in the crystal. Full-length NS1-BP is a dimer in solution and forms at least a dimer in cells. Mutations suggest that dimerization is important for splicing. The central BACK domain of NS1-BP interacts directly with splicing factors such as hnRNP K and PTBP1 and with the viral NS1 protein. The BACK domain is also the site for interactions with mRNA export factor Aly/REF and is required for viral M mRNA nuclear export. The crystal structure of the C-terminal Kelch domain shows that it forms a ß-propeller fold, which is required for the splicing function of NS1-BP. This domain interacts with the polymerase II C-terminal domain and SART1, which are involved in recruitment of splicing factors and spliceosome assembly, respectively. NS1-BP functions are not only critical for processing a subset of viral mRNAs but also impact levels and nuclear export of a subset of cellular mRNAs encoding factors involved in metastasis and immunity.


Assuntos
Vírus da Influenza A/metabolismo , Influenza Humana/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cristalografia por Raios X , Dimerização , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , Proteínas Nucleares/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , Domínios Proteicos , Splicing de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
3.
J Virol ; 92(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30258002

RESUMO

The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCE A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.


Assuntos
Proteína DEAD-box 58/genética , Vírus da Influenza A/metabolismo , Precursores de RNA/metabolismo , Proteínas não Estruturais Virais/fisiologia , Células A549 , Sítios de Ligação , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteína DEAD-box 58/metabolismo , Células HEK293 , Humanos , Vírus da Influenza A/química , Íntrons , Ligação Proteica , Processamento Pós-Transcricional do RNA , Receptores Imunológicos , Análise de Sequência de RNA
4.
PLoS Pathog ; 13(9): e1006635, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28953980

RESUMO

Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress.


Assuntos
Complexos Multiproteicos/metabolismo , Orthomyxoviridae/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Replicação Viral , Proteínas de Transporte/metabolismo , Movimento Celular/fisiologia , Replicação do DNA , Regulação para Baixo/efeitos dos fármacos , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosforilação/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo
5.
PLoS Pathog ; 12(1): e1005370, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26735921

RESUMO

Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in uis2 conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both uis1 and uis2 were highly transcribed in the salivary gland sporozoites but uis2 expression was inhibited by the Pumilio protein Puf2. The repression of uis2 expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Malária/parasitologia , Monoéster Fosfórico Hidrolases/metabolismo , Plasmodium berghei/enzimologia , Plasmodium berghei/crescimento & desenvolvimento , Esporozoítos/enzimologia , Esporozoítos/crescimento & desenvolvimento , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Inativação de Genes , Humanos , Immunoblotting , Imunoprecipitação , Estágios do Ciclo de Vida , Camundongos , Fosforilação
6.
Sci Rep ; 5: 17655, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26631972

RESUMO

The Sec13 protein functions in various intracellular compartments including the nuclear pore complex, COPII-coated vesicles, and inside the nucleus as a transcription regulator. Here we developed a mouse model that expresses low levels of Sec13 (Sec13(H/-)) to assess its functions in vivo, as Sec13 knockout is lethal. These Sec13 mutant mice did not present gross defects in anatomy and physiology. However, the reduced levels of Sec13 in vivo yielded specific immunological defects. In particular, these Sec13 mutant mice showed low levels of MHC I and II expressed by macrophages, low levels of INF-γ and IL-6 expressed by stimulated T cells, and low frequencies of splenic IFN-γ+CD8+ T cells. In contrast, the levels of soluble and membrane-bound TGF-ß as well as serum immunoglobulin production are high in these mice. Furthermore, frequencies of CD19+CD5-CD95+ and CD19+CD5-IL-4+ B cells were diminished in Sec13(H/-) mice. Upon stimulation or immunization, some of the defects observed in the naïve mutant mice were compensated. However, TGF-ß expression remained high suggesting that Sec13 is a negative modulator of TGF-ß expression and of its immunosuppressive functions on certain immune cells. In sum, Sec13 regulates specific expression of immune factors with key functions in inflammation.


Assuntos
Proteínas de Transporte/genética , Fatores Imunológicos/metabolismo , Inflamação/genética , Inflamação/imunologia , Proteínas Nucleares/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/imunologia , Fatores Imunológicos/genética , Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-6/metabolismo , Macrófagos/imunologia , Camundongos Mutantes , Mycobacterium tuberculosis/patogenicidade , Proteínas Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/metabolismo , Tuberculose/genética , Tuberculose/imunologia
7.
Nat Commun ; 5: 4963, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25232931

RESUMO

MicroRNAs (miRNAs) have been shown to regulate viral infection, but the miRNAs that target intracellular sensors and adaptors of innate immunity have not been fully uncovered. Here we conduct an miRNA mimic screen and validation with miRNA inhibitors in cells infected with vesicular stomatitis virus (VSV) to identify miRNAs that regulate viral-host interactions. We identify miR-576-3p as a robust regulator of infection by VSV and other RNA and DNA viruses. While an miR-576-3p mimic sensitizes cells to viral replication, inhibition of endogenous miR-576-3p prevents infection. miR-576-3p is induced by IRF3 concomitantly with interferon and targets STING, MAVS and TRAF3, which are critical factors for interferon expression. Interestingly, miR-576-3p and its binding sites are primate-specific and miR-576-3p levels are reduced in inflammatory diseases. These findings indicate that induction of miR-576-3p by IRF3 triggers a feedback mechanism to reduce interferon expression and set an antiviral response threshold to likely avoid excessive inflammation.


Assuntos
Interações Hospedeiro-Patógeno , Fator Regulador 3 de Interferon/metabolismo , MicroRNAs/metabolismo , Vesiculovirus/imunologia , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Brônquios/virologia , Caenorhabditis elegans , Sobrevivência Celular , Chlorocebus aethiops , Cães , Células Epiteliais/virologia , Células HEK293 , Humanos , Imunidade Inata , Inflamação/metabolismo , Interferons/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/metabolismo , Células Vero
8.
Traffic ; 15(2): 127-40, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24289861

RESUMO

Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Because of its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, whereas viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. As viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. This review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens.


Assuntos
Núcleo Celular/metabolismo , Vírus/patogenicidade , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/virologia , Citoplasma/metabolismo , Citoplasma/virologia , Humanos , Transporte de RNA , Vírus/metabolismo
9.
Proc Natl Acad Sci U S A ; 109(10): 3956-61, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22355110

RESUMO

In response to environmental stresses, the mammalian serine threonine kinases PERK, GCN2, HRI, and PKR phosphorylate the regulatory serine 51 of the eukaryotic translation initiation factor 2α (eIF2α) to inhibit global protein synthesis. Plasmodium, the protozoan that causes malaria, expresses three eIF2α kinases: IK1, IK2, and PK4. Like GCN2, IK1 regulates stress response to amino acid starvation. IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands. Here we show that the phosphorylation by PK4 of the regulatory serine 59 of Plasmodium eIF2α is essential for the completion of the parasite's erythrocytic cycle that causes disease in humans. PK4 activity leads to the arrest of global protein synthesis in schizonts, where ontogeny of daughter merozoites takes place, and in gametocytes that infect Anopheles mosquitoes. The implication of these findings is that drugs that reduce PK4 activity should alleviate disease and inhibit malaria transmission.


Assuntos
Plasmodium falciparum/metabolismo , eIF-2 Quinase/metabolismo , Animais , Anopheles , Códon , DNA/genética , Proteínas Fúngicas/química , Células Hep G2 , Humanos , Malária/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Mutação , Fosforilação , Serina/química
10.
Hum Mol Genet ; 20(21): 4155-66, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21816948

RESUMO

Hedgehog (Hh) is a core signaling pathway implicated in fundamental processes during embryonic kidney development. We previously found that loss-of-function mutations in the transcription factor GLIS2, a putative vertebrate ortholog of Drosophila Ci, cause nephronophthisis type 7 in humans and mice. Kidney tubular cells in Glis2-knockout mice acquire mesenchymal phenotype, but the cellular mechanisms of this transition are unknown. Here, we demonstrate that Glis2 is a functional component of Hh signaling and is necessary to suppress this pathway in the postnatal kidney. In the epithelial compartment, Glis2 opposes Gli1 activity by binding cis-acting regulatory sequences in the 5' flanking regions of Snai1 and Wnt4, thereby inhibiting de-differentiation of tubular cells. We conclude that Glis2 is necessary to inhibit Hh signaling and to maintain the mature tubular epithelial phenotype in the adult kidney. This is the first description of a molecular mechanism that links the Hh signaling pathway to cystic kidney diseases and can open new avenues for the treatment of diverse ciliopathies.


Assuntos
Proteínas Hedgehog/metabolismo , Néfrons/crescimento & desenvolvimento , Néfrons/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Néfrons/patologia , Proteínas do Tecido Nervoso/metabolismo , Fator de Transcrição PAX2/metabolismo , Fenótipo , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Wnt4/genética , Proteína Wnt4/metabolismo , Proteína GLI1 em Dedos de Zinco
11.
J Exp Med ; 207(7): 1465-74, 2010 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-20584882

RESUMO

Sporozoites, the invasive form of malaria parasites transmitted by mosquitoes, are quiescent while in the insect salivary glands. Sporozoites only differentiate inside of the hepatocytes of the mammalian host. We show that sporozoite latency is an active process controlled by a eukaryotic initiation factor-2alpha (eIF2alpha) kinase (IK2) and a phosphatase. IK2 activity is dominant in salivary gland sporozoites, leading to an inhibition of translation and accumulation of stalled mRNAs into granules. When sporozoites are injected into the mammalian host, an eIF2alpha phosphatase removes the PO4 from eIF2alpha-P, and the repression of translation is alleviated to permit their transformation into liver stages. In IK2 knockout sporozoites, eIF2alpha is not phosphorylated and the parasites transform prematurely into liver stages and lose their infectivity. Thus, to complete their life cycle, Plasmodium sporozoites exploit the mechanism that regulates stress responses in eukaryotic cells.


Assuntos
Culicidae/parasitologia , Plasmodium berghei/enzimologia , Glândulas Salivares/parasitologia , Esporozoítos/enzimologia , eIF-2 Quinase/metabolismo , Animais , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica , Marcação de Genes , Estágios do Ciclo de Vida , Fígado/metabolismo , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Plasmodium berghei/citologia , Plasmodium berghei/patogenicidade , Plasmodium berghei/ultraestrutura , Biossíntese de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glândulas Salivares/citologia , Glândulas Salivares/ultraestrutura , Esporozoítos/citologia , Esporozoítos/ultraestrutura
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