The Host-Dependent Interaction of α-Importins with Influenza PB2 Polymerase Subunit Is Required for Virus RNA Replication

The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with α-importins is required for virus RNA replication. To ascertain whether PB2-α-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host

Mutation S110L of H1N1 Influenza Virus Hemagglutinin: A Potent Determinant of Attenuation in the Mouse Model

Characterization of a pandemic 2009 H1N1 influenza virus isolated from a fatal case patient (F-IAV), showed the presence of three different mutations; potential determinants of its high pathogenicity that were located in the polymerase subunits (PB2 A221T and PA D529N) and the hemagglutinin (HA S110L). Recombinant viruses containing individually or in combination the polymerase mutations in the backbone of A/California/04/09 (CAL) showed that PA D529N was clearly involved in the increased pathogenicity of the F-IAV virus. Here, we have evaluated the contribution of HA S110L to F-IAV pathogenicity, through introduction of this point mutation in CAL recombinant virus (HA mut). The HA S110L protein has similar pH stability, comparable mobility, and entry properties both in human and mouse cultured cells that wild type HA. The change HA S110L leads to a non-significant trend to reduce the replication capacity of influenza virus in tissue culture, and HA mut is better neutralized than CAL virus by monoclonal and polyclonal antibodies against HA from CAL strain. In addition, recombinant viruses containing HA S110L alone or in combination with polymerase mutations considerably increased the LD50 in infected mice. Characterization of the lungs of HA mut infected animals showed reduced lung damage and inflammation compared with CAL infected mice. Accordingly, lower virus replication, decreased presence in bronchioli and parenchyma and lower leukocytes and epithelial infected cells were found in the lungs of HA mut-infected animals. Our results indicate that, mutation HA S110L constitutes a determinant of attenuation and suggest that its interaction with components of the respiratory tract mucus and lectins, that play an important role on influenza virus outcome, may constitute a physical barrier impeding the infection of the target cells, thus compromising the infection outcome

Epigenetic control of influenza virus: role of H3K79 methylation in interferon-induced antiviral response

Influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. Using an unbiased search, we analyzed the epigenetic changes at DNA methylation and post-translational histone modification levels induced by the infection. DNA methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. A particular increase in H3K79 methylation was observed and the use of an inhibitor of the specific H3K79 methylase, Dot1L enzyme, or its silencing, increased influenza virus replication. The antiviral response was reduced in conditions of Dot1L downregulation, since decreased nuclear translocation of NF-kB complex, and IFN-β, Mx1 and ISG56 expression was detected. The data suggested a control of antiviral signaling by methylation of H3K79 and consequently, influenza virus replication was unaffected in IFN pathway-compromised, Dot1L-inhibited cells. H3K79 methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. Epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens

Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.

Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.JV is a PhD fellow of the La Caixa Foundation International Fellowship Programme (La Caixa/CNB). This work was supported by the European Molecular Biology Organizat ion (STF-7649 to AF), the Spanish Ministry of Science, Innovation and Universities (MCIU), (BFU2011-26175 and BFU2014-57797-R to AN), and the network Ciber de Enfermedades Respiratorias (CIBERES) including the Improvement and Mobilit y Programme. The CNIC is a Severo Ochoa Center of Excellence (SEV-2015-0505). CNIC is supported by MCIU and the Pro CNIC Foundation. This study was supported by grants from Fondo Europeo de Desarrollo Regional (CB16/11/00458), grants SAF2015-65607-R and SAF2016-80324-R from MCIU (A.H. and D.F-R.) and fellowship SVP-2014-068595 to J.A.N-A. This study was supported by Frankel Cardiovascular Centre, Michigan Medicine (Grant 332475). JJ is supported in part by the National Heart, Lung, and Blood Institute (R01 Grant HL122352). S.F.N is supported in part by the National Heart, Lung, and Blood Institute grants R21HL138064 and R01HL129136.S

La renovación de la palabra en el bicentenario de la Argentina : los colores de la mirada lingüística

El libro reúne trabajos en los que se exponen resultados de investigaciones presentadas por investigadores de Argentina, Chile, Brasil, España, Italia y Alemania en el XII Congreso de la Sociedad Argentina de Lingüística (SAL), Bicentenario: la renovación de la palabra, realizado en Mendoza, Argentina, entre el 6 y el 9 de abril de 2010. Las temáticas abordadas en los 167 capítulos muestran las grandes líneas de investigación que se desarrollan fundamentalmente en nuestro país, pero también en los otros países mencionados arriba, y señalan además las áreas que recién se inician, con poca tradición en nuestro país y que deberían fomentarse. Los trabajos aquí publicados se enmarcan dentro de las siguientes disciplinas y/o campos de investigación: Fonología, Sintaxis, Semántica y Pragmática, Lingüística Cognitiva, Análisis del Discurso, Psicolingüística, Adquisición de la Lengua, Sociolingüística y Dialectología, Didáctica de la lengua, Lingüística Aplicada, Lingüística Computacional, Historia de la Lengua y la Lingüística, Lenguas Aborígenes, Filosofía del Lenguaje, Lexicología y Terminología

Analysis of the interaction of influenza virus polymerase complex with human cell factors

12 pages, 4 figures.-- PMID: 18491320 [PubMed].-- Supplementary information (Suppl. figure S1, 2 pages) available at: http://www.wiley-vch.de/contents/jc_2120/2008/pro200700508_s.pdfThe influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.N. J. was a fellow from Ministerio de Educación y Ciencia. E. T. was a fellow from Instituto de Salud Carlos III. P. G. was a fellow from Gobierno Vasco. This work was supported by the Spanish Ministry of Education and Science (Ministerio de Educación y Ciencia) (grant BFU2004-491), the VIRHOST Program financed by Comunidad de Madrid, European Vigilance Network for the Management of Antiviral Drug Resistance (VIRGIL) and the FLUPOL strep project (SP5B-CT-2007-044263).Peer reviewe

Mutation S110L of H1N1 Influenza Virus Hemagglutinin: A Potent Determinant of Attenuation in the Mouse Model

Characterization of a pandemic 2009 H1N1 influenza virus isolated from a fatal case patient (F-IAV), showed the presence of three different mutations; potential determinants of its high pathogenicity that were located in the polymerase subunits (PB2 A221T and PA D529N) and the hemagglutinin (HA S110L). Recombinant viruses containing individually or in combination the polymerase mutations in the backbone of A/California/04/09 (CAL) showed that PA D529N was clearly involved in the increased pathogenicity of the F-IAV virus. Here, we have evaluated the contribution of HA S110L to F-IAV pathogenicity, through introduction of this point mutation in CAL recombinant virus (HA mut). The HA S110L protein has similar pH stability, comparable mobility, and entry properties both in human and mouse cultured cells that wild type HA. The change HA S110L leads to a non-significant trend to reduce the replication capacity of influenza virus in tissue culture, and HA mut is better neutralized than CAL virus by monoclonal and polyclonal antibodies against HA from CAL strain. In addition, recombinant viruses containing HA S110L alone or in combination with polymerase mutations considerably increased the LD50 in infected mice. Characterization of the lungs of HA mut infected animals showed reduced lung damage and inflammation compared with CAL infected mice. Accordingly, lower virus replication, decreased presence in bronchioli and parenchyma and lower leukocytes and epithelial infected cells were found in the lungs of HA mut-infected animals. Our results indicate that, mutation HA S110L constitutes a determinant of attenuation and suggest that its interaction with components of the respiratory tract mucus and lectins, that play an important role on influenza virus outcome, may constitute a physical barrier impeding the infection of the target cells, thus compromising the infection outcome.info:eu-repo/semantics/publishedVersio

Mutations of the segment-specific nucleotides at the 3’ end of influenza virus NS segment control viral replication

The vRNAs of influenza A viruses contain 12 and 13 nucleotide-long sequences at their 3′ and 5′ termini respectively that are highly conserved and constitute the vRNA promoter. These sequences and the next three segment-specific nucleotides show inverted partial complementarity and are followed by several unpaired nucleotides of poorly characterized function at the 3′ end. We have performed systematic point-mutations at the segment-specific nucleotides 15–18 of the 3′-end of a NS-like vRNA segment. All NS-like vRNAs containing mutations at position 15, and some at positions 16–18 showed reduced transcription/replication efficiency in a transfection/infection system. In addition, the replication of recombinant viruses containing mutations at position 15 was impaired both in single and multi-cycle experiments. This reduction was the consequence of a decreased expression of the NS segment. The data indicate that NS1 plays a role in the transcription/replication of its own segment, which elicits a global defect on virus replication.This work was supported by the Ministerio de Economia y Competitividad, Plan Nacional de Investigacion Científica, Desarrollo e Innovacion Tecnologica (BFU2017-83392R, AEI/FEDER, UE) and Ciber de Enfermedades Respiratorias CIBERES

Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores

Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N 7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m 7 GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m 7 GTP in the active site, and the subsequent covalent transfer of m 7 GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals