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 fármacosRESUMO
Coronavirus disease 2019 (COVID-19) in humans has a wide range of presentations, ranging from asymptomatic or mild symptoms to severe illness. Suitable animal models mimicking varying degrees of clinical disease manifestations could expedite development of therapeutics and vaccines for COVID-19. Here we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulted in subclinical disease in rhesus macaques with mild pneumonia and clinical disease in Syrian hamsters with severe pneumonia. SARS-CoV-2 infection was confirmed by formalin-fixed, paraffin-embedded (FFPE) polymerase chain reaction (PCR), immunohistochemistry, or in situ hybridization. Replicating virus in the lungs was identified using in situ hybridization or virus plaque forming assays. Viral encephalitis, reported in some COVID-19 patients, was identified in one macaque and was confirmed with immunohistochemistry. There was no evidence of encephalitis in hamsters. Severity and distribution of lung inflammation were substantially more in hamsters compared with macaques and exhibited vascular changes and virus-induced cytopathic changes as seen in COVID-19 patients. Neither the hamster nor macaque models demonstrated evidence for multisystemic inflammatory syndrome (MIS). Data presented here demonstrate that macaques may be appropriate for mechanistic studies of mild asymptomatic COVID-19 pneumonia and COVID-19-associated encephalitis, whereas Syrian hamsters may be more suited to study severe COVID-19 pneumonia.
Assuntos
COVID-19 , Encefalite , Animais , Vacinas contra COVID-19 , Cricetinae , Modelos Animais de Doenças , Encefalite/patologia , Humanos , Pulmão/patologia , Macaca mulatta , Mesocricetus , SARS-CoV-2RESUMO
The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-ß) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans.IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.
Assuntos
Células Epiteliais/imunologia , Interações Hospedeiro-Patógeno/genética , Imunidade Inata , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Proteínas não Estruturais Virais/genética , Animais , Aves , Células Dendríticas/imunologia , Células Dendríticas/virologia , Cães , Células Epiteliais/virologia , Regulação da Expressão Gênica , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/classificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H7N2/classificação , Vírus da Influenza A Subtipo H7N2/genética , Vírus da Influenza A Subtipo H7N2/imunologia , Subtipo H7N9 do Vírus da Influenza A/classificação , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células Madin Darby de Rim Canino , Filogenia , Cultura Primária de Células , Vírus Reordenados/genética , Vírus Reordenados/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteínas não Estruturais Virais/classificação , Proteínas não Estruturais Virais/imunologiaRESUMO
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
UNLABELLED: African swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection. IMPORTANCE: Since its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Colesterol/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Internalização do Vírus , Animais , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Análise do Fluxo Metabólico , Células VeroRESUMO
The integrity of the cholesterol biosynthesis pathway is required for efficient African swine fever virus (ASFV) infection. Incorporation of prenyl groups into Rho GTPases plays a key role in several stages of ASFV infection, since both geranylgeranyl and farnesyl pyrophosphates are required at different infection steps. We found that Rho GTPase inhibition impaired virus morphogenesis and resulted in an abnormal viral factory size with the accumulation of envelope precursors and immature virions. Furthermore, abundant defective virions reached the plasma membrane, and filopodia formation in exocytosis was abrogated. Rac1 was activated at early ASFV infection stages, coincident with microtubule acetylation, a process that stabilizes microtubules for virus transport. Rac1 inhibition did not affect the viral entry step itself but impaired subsequent virus production. We found that specific Rac1 inhibition impaired viral induced microtubule acetylation and viral intracellular transport. These findings highlight that viral infection is the result of a carefully orchestrated modulation of Rho family GTPase activity within the host cell; this modulation results critical for virus morphogenesis and in turn, triggers cytoskeleton remodeling, such as microtubule stabilization for viral transport during early infection.
Assuntos
Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/metabolismo , Colesterol/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Chlorocebus aethiops , Suínos , Células VeroRESUMO
Influenza A viruses (IAVs) are contagious pathogens and one of the leading causes of respiratory tract infections in both humans and animals worldwide. Upon infection, the innate immune system provides the first line of defense to neutralize or limit the replication of invading pathogens, creating a fast and broad response that brings the cells into an alerted state through the secretion of cytokines and the induction of the interferon (IFN) pathway. At the same time, IAVs have developed a plethora of immune evasion mechanisms in order to avoid or circumvent the host antiviral response, promoting viral replication. Herein, we will review and summarize already known and recently described innate immune mechanisms that host cells use to fight IAV viral infections as well as the main strategies developed by IAVs to overcome such powerful defenses during this fascinating virus-host interplay.
Assuntos
Evasão da Resposta Imune/imunologia , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana , Interferon Tipo I/uso terapêutico , HumanosRESUMO
African Swine Fever virus (ASFV) causes one of the most relevant emerging diseases affecting swine, now extended through three continents. The virus has a large coding capacity to deploy an arsenal of molecules antagonizing the host functions. In the present work, we have studied the only known E2 viral-conjugating enzyme, UBCv1 that is encoded by the I215L gene of ASFV. UBCv1 was expressed as an early expression protein that accumulates throughout the course of infection. This versatile protein, bound several types of polyubiquitin chains and its catalytic domain was required for enzymatic activity. High throughput mass spectrometry analysis in combination with a screening of an alveolar macrophage library was used to identify and characterize novel UBCv1-host interactors. The analysis revealed interaction with the 40S ribosomal protein RPS23, the cap-dependent translation machinery initiation factor eIF4E, and the E3 ubiquitin ligase Cullin 4B. Our data show that during ASFV infection, UBCv1 was able to bind to eIF4E, independent from the cap-dependent complex. Our results provide novel insights into the function of the viral UBCv1 in hijacking cellular components that impact the mTORC signaling pathway, the regulation of the host translation machinery, and the cellular protein expression during the ASFV lifecycle.
RESUMO
Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteína DEAD-box 58/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores Imunológicos/metabolismo , Transdução de Sinais/imunologia , Proteínas não Estruturais Virais/metabolismo , Células A549 , Animais , Citosol/metabolismo , Enzimas Desubiquitinantes/metabolismo , Cães , Deleção de Genes , Células HEK293 , Humanos , Influenza Humana , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/metabolismo , Células Madin Darby de Rim Canino , Masculino , Membranas Mitocondriais/metabolismo , NF-kappa B/metabolismo , Multimerização ProteicaRESUMO
Previous studies demonstrate an association between activation of the maternal immune system during pregnancy and increased risk of neurodevelopmental psychiatric conditions, such as schizophrenia and autism, in the offspring. Relatively recent findings also suggest that the gut microbiota plays an important role in shaping brain development and behavior. Here we show that maternal immune activation (MIA) accomplished by infection with a mouse-adapted influenza virus during pregnancy induced up-regulation of frontal cortex serotonin 5-HT2A receptor (5-HT2AR) density in the adult offspring, a phenotype previously observed in postmortem frontal cortex of schizophrenic subjects. 5-HT2AR agonist-induced head-twitch behavior was also augmented in this preclinical mouse model. Using the novel object recognition (NOR) test to evaluate cognitive performance, we demonstrate that MIA induced NOR deficits in adult offspring. Oral antibiotic treatment of prepubertal mice prevented this cognitive impairment, but not increased frontal cortex 5-HT2AR density or psychedelic-induced head-twitch behavior in adult MIA offspring. Additionally, gut microbiota transplantation from MIA mice produced behavioral deficits in antibiotic-treated mock mice. Adult MIA offspring displayed altered gut microbiota, and relative abundance of specific components of the gut microbiota, including Ruminococcaceae, correlated with frontal cortex 5-HT2AR density. Together, these findings provide a better understanding of basic mechanisms by which prenatal insults impact offspring brain function, and suggest gut-brain axis manipulation as a potential therapeutic approach for neurodevelopmental psychiatric conditions.
Assuntos
Comportamento Animal , Suscetibilidade a Doenças , Microbioma Gastrointestinal , Transtornos do Neurodesenvolvimento/etiologia , Transtornos do Neurodesenvolvimento/psicologia , Comportamento Problema , Maturidade Sexual , Fatores Etários , Animais , Antibacterianos/administração & dosagem , Modelos Animais de Doenças , Memória , Camundongos , Fenótipo , Reconhecimento Psicológico , Esquizofrenia/etiologiaRESUMO
Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1-3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1-nuclear transport factor 2-related export protein 1 (NXF1-NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1-NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1-NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1-NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.
Assuntos
Núcleo Celular/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , RNA Mensageiro/metabolismo , Proteínas não Estruturais Virais/metabolismo , Células A549 , Transporte Ativo do Núcleo Celular , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Influenza A viruses (IAVs) have a remarkable tropism in their ability to circulate in both mammalian and avian species. The IAV NS1 protein is a multifunctional virulence factor that inhibits the type I interferon host response through a myriad of mechanisms. How NS1 has evolved to enable this remarkable property across species and its specific impact in the overall replication, pathogenicity, and host preference remain unknown. Here we analyze the NS1 evolutionary landscape and host tropism using a barcoded library of recombinant IAVs. Results show a surprisingly great variety of NS1 phenotypes according to their ability to replicate in different hosts. The IAV NS1 genes appear to have taken diverse and random evolutionary pathways within their multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAVs to adapt to multiple hosts and aids in our understanding of its global prevalence.
Assuntos
Especificidade de Hospedeiro/genética , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/patogenicidade , Mutação , Infecções por Orthomyxoviridae/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Cães , Feminino , Imunidade Inata , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Camundongos , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/patologia , Filogenia , Proteínas não Estruturais Virais/genéticaRESUMO
Three of the eight RNA segments encoded by the influenza A virus (IAV) undergo alternative splicing to generate distinct proteins. Previously, we found that host proteins hnRNP K and NS1-BP regulate IAV M segment splicing, but the mechanistic details were unknown. Here we show NS1-BP and hnRNP K bind M mRNA downstream of the M2 5' splice site (5'ss). NS1-BP binds most proximal to the 5'ss, partially overlapping the U1 snRNP binding site, while hnRNP K binds further downstream and promotes U1 snRNP recruitment. Mutation of either or both the hnRNP K and NS1-BP-binding sites results in M segment mis-splicing and attenuated IAV replication. Additionally, we show that hnRNP K and NS1-BP regulate host splicing events and that viral infection causes mis-splicing of some of these transcripts. Therefore, our proposed mechanism of hnRNP K/NS1-BP mediated IAV M splicing provides potential targets of antiviral intervention and reveals novel host functions for these proteins.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Influenza Humana/genética , Proteínas Nucleares/genética , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Fatores de Transcrição/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Vírus da Influenza A/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Mutação , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Fatores de Transcrição/metabolismo , Replicação Viral/genéticaRESUMO
African swine fever virus (ASFV) infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns) synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Fosfatidilinositóis/metabolismo , Células VeroRESUMO
Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.
Assuntos
Núcleo Celular/virologia , Vírus da Influenza A/genética , Corpos de Inclusão Intranuclear/metabolismo , Splicing de RNA , RNA Viral/metabolismo , Proteínas da Matriz Viral/genética , Processamento Alternativo , Linhagem Celular , Regulação Viral da Expressão Gênica , Genoma Viral , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Vírus da Influenza A/fisiologia , Corpos de Inclusão Intranuclear/virologia , RNA Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Liberação de Vírus/genética , Liberação de Vírus/fisiologiaRESUMO
The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV.
Assuntos
Vírus da Febre Suína Africana/patogenicidade , Antígenos de Diferenciação/metabolismo , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Internalização do Vírus , Animais , Membrana Celular/metabolismo , Chlorocebus aethiops , Colesterol/metabolismo , Endocitose , Endossomos/metabolismo , Células HEK293 , Humanos , Microscopia de Fluorescência , Vírus de RNA/metabolismo , RNA de Cadeia Dupla , Células VeroRESUMO
African swine fever virus (ASFV) is a double-stranded DNA virus causing a hemorrhagic fever disease with high mortality rates and severe economic losses in pigs worldwide. ASFV replicates in perinuclear sites called viral factories (VFs) that are morphologically similar to cellular aggresomes. This fact raises the possibility that both VFs and aggresomes may be the same structure. However, little is known about the process involved in the formation of these viral replication platforms. In order to expand our knowledge on the assembly of ASFV replication sites, we have analyzed the involvement of both canonical aggresome pathways in the formation of ASFV VFs: HDAC6 and BAG3. HDAC6 interacts with a component of the dynein motor complex (dynactin/p150Glued) and ubiquitinated proteins, transporting them to the microtubule-organizing center (MTOC) and leading to aggresome formation, while BAG3 is mediating the recruitment of non-ubiquitinated proteins through a similar mechanism. Tubacin-mediated HDAC6 inhibition and silencing of BAG3 pathways, individually or simultaneously, did not prevent ASFV VF formation. These findings show that HDAC6 and Bag3 are not required for VFs formation suggesting that aggresomes and VFs are not the same structures. However, alternative unexplored pathways may be involved in the formation of aggresomes.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Histona Desacetilases/metabolismo , SuínosRESUMO
Viruses are strict intracellular pathogens that require the cellular environment to complete a successful infection. Among them, African swine fever virus (ASFV) is an evolutionary ancient DNA virus, endemic in Africa, which is nowadays causing an emergent disease in Europe with a potential high economic impact in the pig industry. It is well known that host-cell components are critical crossroads mapping the virus path for a productive infection, some of them at the endocytic pathway. Considering that ASFV infectious cycle strongly relies in several factors from the host cell, the study of virus-host interactions remains crucial as they will reveal the obstacles, routes and tracks, hints and the target waypoint in the virus journey to destination.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Animais , SuínosRESUMO
The main cellular target for African swine fever virus (ASFV) is the porcine macrophage. However, existing data about the early phases of infection were previously characterized in non-leukocyte cells such as Vero cells. Here, we report that ASFV enters the natural host cell using dynamin-dependent and clathrin-mediated endocytosis. This pathway is strongly pH-dependent during the first steps of infection in porcine macrophages. We investigated the effect of drugs inhibiting several endocytic pathways in macrophages and compared ASFV with vaccinia virus (VV), which apparently involves different entry pathways. The presence of cholesterol in cellular membranes was found to be essential for a productive ASFV infection while actin-dependent endocytosis and the participation of phosphoinositide-3-kinase (PI3K) activity were other cellular factors required in the process of viral entry. These findings improved our understanding of the ASFV interactions with macrophages that allow for successful viral replication.