RESUMO
The nuclear pore complex is the primary conduit for nuclear import and export of molecules. In this issue, Gu et al. uncover a novel mechanism in which immune signaling and programmed cell death require nuclear pore rearrangement and release of sequestered cyclin-dependent kinase inhibitors to elicit immunity and death.
Assuntos
Transporte Ativo do Núcleo Celular , Poro Nuclear/metabolismo , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transdução de SinaisRESUMO
The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
Assuntos
Transporte Ativo do Núcleo Celular , COVID-19 , Proteínas de Transporte Nucleocitoplasmático , RNA Mensageiro , Proteínas de Ligação a RNA , SARS-CoV-2 , Proteínas não Estruturais Virais , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Animais , COVID-19/virologia , COVID-19/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Replicação Viral , Núcleo Celular/metabolismo , Células Vero , Virulência , Chlorocebus aethiops , Células HEK293RESUMO
The ability of proteins and nucleic acids to undergo liquid-liquid phase separation has recently emerged as an important molecular principle of how cells rapidly and reversibly compartmentalize their components into membrane-less organelles such as the nucleolus, processing bodies or stress granules1,2. How the assembly and turnover of these organelles are controlled, and how these biological condensates selectively recruit or release components are poorly understood. Here we show that members of the large and highly abundant family of RNA-dependent DEAD-box ATPases (DDXs)3 are regulators of RNA-containing phase-separated organelles in prokaryotes and eukaryotes. Using in vitro reconstitution and in vivo experiments, we demonstrate that DDXs promote phase separation in their ATP-bound form, whereas ATP hydrolysis induces compartment turnover and release of RNA. This mechanism of membrane-less organelle regulation reveals a principle of cellular organization that is conserved from bacteria to humans. Furthermore, we show that DDXs control RNA flux into and out of phase-separated organelles, and thus propose that a cellular network of dynamic, DDX-controlled compartments establishes biochemical reaction centres that provide cells with spatial and temporal control of various RNA-processing steps, which could regulate the composition and fate of ribonucleoprotein particles.
Assuntos
Adenosina Trifosfatases/metabolismo , Compartimento Celular , RNA Helicases DEAD-box/metabolismo , Células Eucarióticas/enzimologia , Organelas/enzimologia , Organelas/metabolismo , Células Procarióticas/enzimologia , Biocatálise , Linhagem Celular , Sequência Conservada , Grânulos Citoplasmáticos/metabolismo , Células Eucarióticas/citologia , Evolução Molecular , Humanos , Células Procarióticas/citologia , RNA/metabolismo , Transporte de RNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.
Assuntos
Núcleo Celular , Salpicos Nucleares , Proteínas Quinases , Splicing de RNA , Transporte Ativo do Núcleo Celular , Núcleo Celular/enzimologia , Células HeLa , Humanos , Proteínas Quinases/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.
Assuntos
COVID-19/metabolismo , Interferons/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Transdução de Sinais , Células VeroRESUMO
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 fármacosRESUMO
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/metabolismoRESUMO
In this issue, Singer et al. (2012) reveal that the nucleoporin Nup98 supports adaptation to genotoxic stress by protecting specific p53-induced mRNAs from exosome-dependent degradation, suggesting that wild-type Nup98 may possess tumor suppressor function.
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 RNARESUMO
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/metabolismoRESUMO
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 , FosforilaçãoRESUMO
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/metabolismoRESUMO
Influenza A virus is a major human pathogen with a genome comprised of eight single-strand, negative-sense, RNA segments. Two viral RNA segments, NS1 and M, undergo alternative splicing and yield several proteins including NS1, NS2, M1 and M2 proteins. However, the mechanisms or players involved in splicing of these viral RNA segments have not been fully studied. Here, by investigating the interacting partners and function of the cellular protein NS1-binding protein (NS1-BP), we revealed novel players in the splicing of the M1 segment. Using a proteomics approach, we identified a complex of RNA binding proteins containing NS1-BP and heterogeneous nuclear ribonucleoproteins (hnRNPs), among which are hnRNPs involved in host pre-mRNA splicing. We found that low levels of NS1-BP specifically impaired proper alternative splicing of the viral M1 mRNA segment to yield the M2 mRNA without affecting splicing of mRNA3, M4, or the NS mRNA segments. Further biochemical analysis by formaldehyde and UV cross-linking demonstrated that NS1-BP did not interact directly with viral M1 mRNA but its interacting partners, hnRNPs A1, K, L, and M, directly bound M1 mRNA. Among these hnRNPs, we identified hnRNP K as a major mediator of M1 mRNA splicing. The M1 mRNA segment generates the matrix protein M1 and the M2 ion channel, which are essential proteins involved in viral trafficking, release into the cytoplasm, and budding. Thus, reduction of NS1-BP and/or hnRNP K levels altered M2/M1 mRNA and protein ratios, decreasing M2 levels and inhibiting virus replication. Thus, NS1-BP-hnRNPK complex is a key mediator of influenza A virus gene expression.
Assuntos
Regulação Viral da Expressão Gênica/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Vírus da Influenza A/fisiologia , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA/fisiologia , RNA Viral/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cães , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Células Madin Darby de Rim Canino , Proteínas Nucleares/genética , Proteômica , Precursores de RNA/genética , RNA Viral/genética , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Replicação Viral/fisiologiaRESUMO
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 , Proteínas Serina-Treonina Quinases/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 , Proteínas Serina-Treonina Quinases/genética , Serina/químicaRESUMO
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.
Assuntos
Transporte Ativo do Núcleo Celular , COVID-19 , Poro Nuclear , Proteínas de Transporte Nucleocitoplasmático , SARS-CoV-2 , Humanos , COVID-19/metabolismo , Interferons/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismoRESUMO
A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.
Assuntos
Antivirais/farmacologia , Naftalimidas/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Vesiculovirus/efeitos dos fármacos , Animais , Antivirais/química , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Naftalimidas/química , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/deficiência , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.
Assuntos
Transporte Ativo do Núcleo Celular , Influenza Humana , Orthomyxoviridae , Transporte de RNA , Humanos , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Influenza Humana/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Orthomyxoviridae/genética , Transporte de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
mRNA in eukaryotic cells is packaged into highly compacted ribonucleoprotein particles (mRNPs) in the nucleus and exported to the cytoplasm for translation. mRNP packaging and export require the evolutionarily conserved transcription-export (TREX) complex. TREX facilitates loading of various RNA-binding proteins on mRNA through the action of its DDX39B subunit. SARNP (Tho1 [transcriptional defect of Hpr1 by overexpression 1] in yeast) is shown to interact with DDX39B and affect mRNA export. The molecular mechanism of how SARNP recognizes DDX39B and functions in mRNP assembly is unclear. Here, we determine the crystal structure of a Tho1/DDX39B/RNA complex, revealing a multivalent interaction mediated by tandem DDX39B interacting motifs in SARNP/Tho1. The high-order complex of SARNP and DDX39B is evolutionarily conserved, and human SARNP can engage with five DDX39B molecules. RNA sequencing (RNA-seq) from SARNP knockdown cells shows the most affected RNAs in export are GC rich. Our work suggests the role of the high-order SARNP/DDX39B/RNA complex in mRNP assembly and export.
Assuntos
Proteínas Nucleares , Ribonucleoproteínas , Humanos , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Fatores de Transcrição/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.
Assuntos
COVID-19 , SARS-CoV-2 , Proteínas Virais , Humanos , COVID-19/virologia , Imunidade Inata , Interferons/genética , Interferons/metabolismo , SARS-CoV-2/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Kapbeta2 (also called transportin) recognizes PY nuclear localization signal (NLS), a new class of NLS with a R/H/Kx((2-5))PY motif. Here we show that Kapbeta2 complexes containing hydrophobic and basic PY-NLSs, as classified by the composition of an additional N-terminal motif, converge in structure only at consensus motifs, which explains ligand diversity. On the basis of these data and complementary biochemical analyses, we designed a Kapbeta2-specific nuclear import inhibitor, M9M.