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[This corrects the article DOI: 10.1371/journal.ppat.1006851.].
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Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin ß, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.
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Núcleo Celular/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Células A549 , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Cães , Regulação para Baixo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Proteínas do Nucleocapsídeo , Ligação Proteica , Transporte ProteicoRESUMO
The technology of pattern recognition for vibration events based on the phase-sensitive optical time domain reflectometer (Φ-OTDR) has been significantly improved, thanks to plenty of valuable research in recent years. However, it remains challenging to develop an efficient algorithm for it with computing resources that are simpler to achieve at lower cost. To the best of our knowledge, this paper, for the first time, analyzes the superiority of using graphics processing unit (GPU) parallel computing to improve time-consuming performance in pattern recognition for vibration events based on Φ-OTDR. And the pattern-recognition algorithm, including spectral subtraction and artificial neural networks, is implemented by CPU and GPU, respectively. Then, the time consumption of the CPU-based method and the time consumption of the GPU-based method are, respectively, recorded and compared. As a result of our experiments, we concluded that using GPU parallel computing can develop an efficient algorithm with a computing resource that is simpler to achieve at a lower cost.
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Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice. IMPORTANCE: The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.
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Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Infecções por Orthomyxoviridae/virologia , Proteínas Recombinantes de Fusão/química , Proteínas de Transporte Vesicular/química , Proteínas da Matriz Viral/química , Animais , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/virologia , Cães , Células Epiteliais/virologia , Feminino , Expressão Gênica , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/patogenicidade , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Neuroglia/virologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/mortalidade , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Análise de Sobrevida , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/imunologia , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/imunologia , Liberação de Vírus/genética , Liberação de Vírus/imunologia , Replicação Viral/genética , Replicação Viral/imunologia , Rede trans-Golgi/virologiaRESUMO
During their circulation in nature, H5N1 avian influenza viruses (AIVs) have acquired the ability to kill their natural hosts, wild birds and ducks. The genetic determinants for this increased virulence are largely unknown. In this study, we compared two genetically similar H5N1 AIVs, A/duck/Hubei/49/05 (DK/49) and A/goose/Hubei/65/05 (GS/65), that are lethal for chickens but differ in their virulence levels in ducks. To explore the genetic basis for this difference in virulence, we generated a series of reassortants and mutants of these two viruses. The virulence of the reassortant bearing the PA gene from DK/49 in the GS/65 background increased 10(5)-fold relative to that of the GS/65 virus. Substitution of two amino acids, S224P and N383D, in PA contributed to the highly virulent phenotype. The amino acid 224P in PA increased the replication of the virus in duck embryo fibroblasts, and the amino acid 383D in PA increased the polymerase activity in duck embryo fibroblasts and delayed the accumulation of the PA and PB1 polymerase subunits in the nucleus of virus-infected cells. Our results provide strong evidence that the polymerase PA subunit is a virulence factor for H5N1 AIVs in ducks.
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Virus da Influenza A Subtipo H5N1/enzimologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Influenza Aviária/virologia , Doenças das Aves Domésticas/virologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Animais Domésticos/virologia , Galinhas , Patos , Virus da Influenza A Subtipo H5N1/genética , Dados de Sequência Molecular , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , VirulênciaRESUMO
Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.
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Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Humana , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Humanos , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/genéticaRESUMO
The duck (Anas platyrhynchos) is one of the principal natural hosts of influenza A viruses. We present the duck genome sequence and perform deep transcriptome analyses to investigate immune-related genes. Our data indicate that the duck possesses a contractive immune gene repertoire, as in chicken and zebra finch, and this repertoire has been shaped through lineage-specific duplications. We identify genes that are responsive to influenza A viruses using the lung transcriptomes of control ducks and ones that were infected with either a highly pathogenic (A/duck/Hubei/49/05) or a weakly pathogenic (A/goose/Hubei/65/05) H5N1 virus. Further, we show how the duck's defense mechanisms against influenza infection have been optimized through the diversification of its ß-defensin and butyrophilin-like repertoires. These analyses, in combination with the genomic and transcriptomic data, provide a resource for characterizing the interaction between host and influenza viruses.