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The influenza virus genome consists of eight viral ribonucleoproteins (vRNPs), each consisting of a copy of the polymerase, one of the genomic RNA segments and multiple copies of the nucleoprotein arranged in a double helical conformation. vRNPs are macromolecular machines responsible for messenger RNA synthesis and genome replication, that is, the formation of progeny vRNPs. Here, we describe the structural basis of the transcription process. The mechanism, which we call the 'processive helical track', is based on the extreme flexibility of the helical part of the vRNP that permits a sliding movement between both antiparallel nucleoprotein-RNA strands, thereby allowing the polymerase to move over the genome while bound to both RNA ends. Accordingly, we demonstrate that blocking this movement leads to inhibition of vRNP transcriptional activity. This mechanism also reveals a critical role of the nucleoprotein in maintaining the double helical structure throughout the copying process to make the RNA template accessible to the polymerase., This work was supported by the Spanish Ministry of Science, Innovation and Universities (Ministerio de Ciencia, Innovación y Universidades) grant nos. BFU2017-90018-R and BFU2011-25090/BMC (J.M.-B.) and Integrative Biology of Emerging Infectious Diseases LabEx grant no. 10-LABX-0062 (N.N.)

Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and economy. To establish a productive infection, influenza viruses interact with cellular factors to favour their own replication and to suppress antiviral cell responses. Although most virus-host interaction studies have been centred on cell protein factors, most of the human transcriptome comprises non-coding RNAs, as miRNAs and lncRNAs. The latter are key cellular regulators in many cellular processes, including transcriptional and post-transcriptional regulation. Influenza virus infection induces the differential expression of hundreds of potential lncRNAs, some of which are related to the antiviral pathways activated by the cell while others may be deregulated by the infection to allow efficient virus multiplication. Although our knowledge on the role of cellular lncRNAs for influenza virus replication and pathogenesis is still at its infancy, several lncRNAs have been described to influence the cell innate response to the virus by altering the histone modification at specific sites, by interaction with specific transcription factors or directly stimulating in cis the expression of specific IFN-induced genes. In addition, at least one lncRNA appears to be required for virus multiplication in an IFN-independent way.

13 p.-5 fig.3 tab. Martínez, Isidoro et al., Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here., Research activities in the participating laboratories received further funding from the following sources: Centro Nacional de Microbiología, ISCIII, PI15CIII/00024 and MINECO (SAF2015- 67033-R); Centro Nacional de Biotecnología, MINECO (BFU2014-57797-R); Hospital Universitari Germans Trias I Pujol, Spanish Society of Pneumology and Thoracic Surgery (SEPAR 054/2011); Departamento de Bioquímica y Biología Molecular I, MINECO (SAF2015-65307-R); Centro de Investigaciones Biológicas, MINECO (SAF2012-39444-C01/02); Fundación de Investigación Sanitaria de las Islas Baleares, MINECO (SAF2012-39841); Instituto de Agrobiotecnología, MINECO (SAF2015-66520-R); Instituto de Química Física Rocasolano, MINECO (BFU2015-70052-R) and the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA- 2012-317297). Subprograma Estatal de Formación (BES-2013- 065355).

La búsqueda de paradigmas explicativos totales en Ciencias Sociales es una constante de la que tampoco puede desentenderse el caso de las migraciones como proceso social total que es. No por ello debe ignorarse la existencia de paradigmas teórico-conceptuales de referencia y de utilidad en el saber instrumental, en la caracterización y aún en la significación socio-cultural -sin olvidar la económica-, del proceso en sus dimensiones espacio-temporales estructurales actuales. En el artículo se intentan significar algunas consistencias de los procesos migratorios en general y que a la vez suponen ciertas limitaciones en la articulación micro-macro, investigación-teorías, sobre las migraciones. Consistencias relativas a la condición-consideración, construida, política y socio-cultural del migrante, el papel representado por las escalas locales como espacios de referencia en la que ésta finalmente se caracteriza, significa y considera, y el grado de discrecionalidad con el que se resuelven las negociaciones de rol de migrantes y autóctonos.

Regulated splicing of some influenza virus RNAs is necessary for the synthesis of various essential proteins. Processing of these transcripts is now found to occur in nuclear speckles, previously considered storage sites for cellular splicing factors.

Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK) as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection., IMPORTANCE Influenza A viruses are responsible for annual epidemics and occasional pandemics with important consequences for human health and the economy. The unfolded protein response is a defense mechanism fired by cells when the demand of protein synthesis and folding is excessive, for instance, during an acute virus infection. In this report, we show that influenza virus downregulates the unfolded protein response mediated by the PERK sensor, while Montelukast, a drug used to treat asthma in humans, specifically stimulated this response and downregulated viral protein synthesis and multiplication. Accordingly, we show that PERK phosphorylation was reduced in virus-infected cells and increased in cells treated with Montelukast. Hence, our studies suggest that modulation of the PERK-mediated unfolded protein response is a target for influenza virus inhibition.

Rapid discrimination of avian vs. human phenotypes of emerging influenza A virus isolates with pandemic potential is an important issue in pathogenesis and epidemiology studies of the infection. In this work, functional architectures are tailored on the surface of a gold electrode, introducing receptor molecules as a sensing entity that mimics those found in the membrane of target cells of the influenza A virus and with the aim of developing an impedimetric-based detector for influenza A virus phenotyping. In a bottom-up approach, the artificial receptors are built by sequential assembly of a 1-octanethiol/octyl- galactoside hybrid bilayer, followed by an enzyme-mediated functionalization of the terminal galactoside groups with sialic acid molecules. The detection mechanism relies hence on the specific affinity between the sialic acid-galactose receptor moieties anchored on the modified electrode surface and the hemagglutinin (HA) viral surface protein. By using the appropriate type of sialyltransferase enzyme, sialylation of galactose residues is made through α-2,3 or α-2,6 linkages. This permits the envisaged impedimetric detector to discriminate rapidly between avian vs. human strains of influenza A virus with the absence of elaborate sample preparation steps. In contrast to immunosensors based on antibodies as bioreceptor, the sialylated modified gold electrode is also able to distinguish among influenza phenotypes, which could make the here presented detector a reagentless, label-free diagnostic device for influenza phenotyping. Biomimetic architectures for the impedimetric discrimination of influenza virus phenotypes are tailored by a self-assembly process on gold substrates. Incorporated sialic acid molecules as sensing entities mimic receptors found on target cells of the influenza A virus. The resulting detector rapidly discriminates between avian and human strains of influenza A virus, acting as a reagentless, label-free diagnostic device for influenza phenotyping, which is important in surveillance and prediction of pandemics. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim., This work was supported by the CICYT (Spain, projects MAT2009-09960 and AGL2007-60274) and the CSIC (Spain, projects PIF08-018 and 201060I009). B.W. acknowledges a Personal Investigador contract of the Comunidad de Madrid. M.A.M.B. and M.Y. acknowledge a PIF08-018 contract.

Influenza Revealed Influenza virus, a single-stranded RNA virus, is responsible for substantial morbidity and mortality worldwide. The influenza ribonucleoprotein (RNP) complex, which carries out viral replication and transcription, is central to the virus life-cycle and to viral host adaptation (see the Perspective by Tao and Zheng ). Structural characterization of the viral RNP has been challenging, but Moeller et al. (p. 1631 , published online 22 November) and Arranz et al. (p. 1634 , published online 22 November) now report the structure and assembly of this complex, using cryo-electron microscopy and negative-stain electron microscopy. The structures reveal how the viral polymerase, RNA genome, and nucleoprotein interact in the RNP providing insight into mechanisms for influenza genome replication and transcription.

5 pages.-- PMID: 18198383 [PubMed]., The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG–Sepharose and calmodulin–agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.

The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/ C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO; BFU2011-26175 and BFU2014-57797-R) and the Ciber de Enfermedades Respiratorias (ISCIII).

The genome of influenza A virus consists of eight single-stranded RNA molecules of negative polarity. Their replication and transcription take place in the nucleus of infected cells using ribonucleoprotein complexes (RNPs) as templates. Two of the viral transcripts, those generated by RNPs 7 and 8, can be spliced and lead to two alternative protein products (M1 and M2, NS1 and NEP/NS2, respectively). Previous studies have shown that when expressed from cDNA, NS1 protein alters the splicing and transport of RNA polymerase II-driven transcripts. Here we used a transient replication/transcription system, in which RNP 8 is replicated and transcribed by recombinant RNA and proteins, to study the splicing and nucleo-cytoplasmic transport of true viral transcripts. Our results show that the encoded NS1 protein inhibits the splicing of the collinear transcript. This regulation is mediated by the N-terminal region of the protein but does not involve its RNA-binding activity. We also show that NS1 protein preferentially blocks the nucleo-cytoplasmic transport of the collinear RNP 8 transcript in an RNA-binding dependent manner. These results rule out previous models to explain the regulation of mRNA processing and transport by NS1 and underlines the relevance of NS1 protein in the control of virus gene expression.

Transcription and replication of influenza A virus are carried out in the nuclei of infected cells in the context of viral ribonucleoproteins (RNPs). The viral polymerase responsible for these processes is a protein complex composed of the PB1, PB2, and PA proteins. We previously identified a set of polymerase-associated cellular proteins by proteomic analysis of polymerase-containing intracellular complexes expressed and purified from human cells. Here we characterize the role of NXP2/MORC3 in the infection cycle. NXP2/MORC3 is a member of the Microrchidia (MORC) family that is associated with the nuclear matrix and has RNA-binding activity. Influenza virus infection led to a slight increase in NXP2/MORC3 expression and its partial relocalization to the cytoplasm. Coimmunoprecipitation and immunofluorescence experiments indicated an association of NXP2/MORC3 with the viral polymerase and RNPs during infection. Downregulation of NXP2/MORC3 by use of two independent short hairpin RNAs (shRNAs) reduced virus titers in low-multiplicity infections. Consistent with these findings, analysis of virus-specific RNA in high-multiplicity infections indicated a reduction of viral RNA (vRNA) and mRNA after NXP2/MORC3 downregulation. Silencing of NXP2/MORC3 in a recombinant minireplicon system in which virus transcription and replication are uncoupled showed reductions in cat mRNA and chloramphenicol acetyltransferase (CAT) protein accumulation but no alterations in cat vRNA levels, suggesting that NXP2/MORC3 is important for influenza virus transcription. IMPORTANCE Influenza virus infections appear as yearly epidemics and occasional pandemics of respiratory disease, with high morbidity and occasional mortality. Influenza viruses are intracellular parasites that replicate and transcribe their genomic ribonucleoproteins in the nuclei of infected cells, in a complex interplay with host cell factors. Here we characterized the role of the human NXP2/MORC3 protein, a member of the Microrchidia family that is associated with the nuclear matrix, during virus infection. NXP2/MORC3 associates with the viral ribonucleoproteins in infected cells. Downregulation of NXP2/MORC3 reduced virus titers and accumulations of viral genomic RNA and mRNAs. Silencing of NXP2/MORC3 in an influenza virus CAT minireplicon system diminished CAT protein and cat mRNA levels but not genomic RNA levels. We propose that NXP2/MORC3 plays a role in influenza virus transcription.

Contrary to their host cells, many viruses contain RNA as genetic material and hence encode an RNA-dependent RNA polymerase to replicate their genomes. This review discusses the present status of our knowledge on the structure of these enzymes and the mechanisms of RNA replication. The simplest viruses encode only the catalytic subunit of the replication complex, but other viruses also contribute a variable number of ancillary factors. These and other factors provided by the host cell play roles in the specificity and affinity of template recognition and the assembly of the replication complex. Usually, these host factors are involved in protein synthesis or RNA modification in the host cell, but they play roles in remodeling RNA-RNA, RNA-protein, and protein-protein interactions during virus RNA replication. Furthermore, viruses take advantage of and modify previous cell structural elements, frequently membrane vesicles, for the formation of RNA replication complexes.

Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo . In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.

PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.

Conflict is part of the human condition. Therefore, it is neither negative nor positive, but rather a natural phenomena. The negative connotations traditionally attributed to conflict are at odds with current theories that explain the use of the term as an engine of change and generator of competitive advantage. The fact that family business is defined by two different systems, the family and the business, can lead to the emergence of many conflicts, but it can also help a business be successful if it can correctly differentiate between the two. To this end, both parties, the family and the business, must keep their own interests at arm's length and focus on common interests and goals.

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283-6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance. This work was supported in part by grants GR09/0023 (A. N.), GR09/0039 (J. A. M.) and GR09/0040 (I. C.) from Instituto de Salud Carlos III under a special research programme on pandemic flu. Additionally, the Biología Viral Unit is supported currently by grant SAF2012-31217 from Plan Nacional I+D+i. Sí

Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space. IMPORTANCE In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

The PA subunit of the influenza virus polymerase complex is a phosphoprotein that induces proteolytic degradation of coexpressed proteins. Point mutants with reduced proteolysis induction reconstitute viral ribonucleoproteins defective in replication but not in transcriptional activity. To look for cellular factors that could associate with PA protein, we have carried out a yeast two-hybrid screen. Using a human kidney cDNA library, we identified two different interacting clones. One of them was identified as the human homologue of a previously described cDNA clone from Gallus gallus called CLE. The human gene encodes a protein of 36 kDa (hCLE) and is expressed ubiquitously in all human organs tested. The interaction of PA and hCLE was also observed with purified proteins in vitro by using pull-down and pep-spot experiments. Mapping of the interaction showed that hCLE interacts with PA subunit at two regions (positions 493 to 512 and 557 to 574) in the PA protein sequence. Immunofluorescence studies showed that the hCLE protein localizes in both the nucleus and the cytosol, although with a predominantly cytosolic distribution. hCLE was found associated with active, highly purified virus ribonucleoproteins reconstituted in vivo from cloned cDNAs, suggesting that PA-hCLE interaction is functionally relevant. Searches in the databases showed that hCLE has 38% sequence homology to the central region of the yeast factor Cdc68, which modulates transcription by interaction with transactivators. Similar homologies were found with the other members of the Cdc68 homologue family of transcriptional activators, including the human FACT protein.

Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix showed that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we showed that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5′ untranslated region (5′ UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

The influenza virus RNA polymerase is a heterotrimer comprising the PB1, PB2 and PA subunits. PB1 is the core of the complex and accounts for the polymerase activity. We have studied the interaction of PB1 with model cRNA template by in vitro binding and Northwestern analyses. The binding to model cRNA was specific and showed an apparent Kd of approximately 7x10(-8) M. In contrast to the interaction with vRNA, PB1 was able to bind equally the 5' and 3' arm of the cRNA panhandle. The N-terminal 139 amino acids of PB1 and sequences between positions 267 and 493 proved positive for binding to cRNA, whereas the interaction with vRNA template previously was mapped to the N- and C-terminal regions. Competition experiments using the 5' and 3' arms of either the vRNA or cRNA panhandle indicated that the N-terminal binding site is shared by both templates. The data indicate that the PB1 RNA-binding sites are constituted by: (i) residues located at the N-terminus (probably common for vRNA and cRNA binding) and, either (ii) residues from the central part of PB1 (for cRNA) or (iii) residues from the C-terminal region of PB1 (for vRNA), and suggest that PB1 undergoes a conformational change upon binding to cRNA versus vRNA templates.

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis.

The genome of Influenza viruses consists of a set of single-stranded, negative-polarity ribonucleoproteins. Each one is replicated and transcribed as an independent unit in the nucleus of the infected cell. This property allows the existence of both nuclear and cytoplasmic regulatory steps in the control of viral gene expression at the posttranscriptional level. The regulation of mRNA splicing and nucleocytoplasmic mRNA transport and the preferential translation of viral mRNAs in the infected cell are discussed, stressing the possible role of NS1 protein in these processes.

Cellular messenger RNAs (mRNAs) are associated to proteins in the form of ribonucleoprotein particles. The double-stranded RNA-binding (DRB) proteins play important roles in mRNA synthesis, modification, activity and decay. Staufen is a DRB protein involved in the localized translation of specific mRNAs during Drosophila early development. The human Staufen1 (hStau1) forms RNA granules that contain translation regulation proteins as well as cytoskeleton and motor proteins to allow the movement of the granule on microtubules, but the mechanisms of hStau1-RNA recognition are still unclear. Here we used a combination of affinity chromatography, RNAse-protection, deep-sequencing and bioinformatic analyses to identify mRNAs differentially associated to hStau1 or a mutant protein unable to bind RNA and, in this way, defined a collection of mRNAs specifically associated to wt hStau1. A common sequence signature consisting of two opposite-polarity Alu motifs was present in the hStau1-associated mRNAs and was shown to be sufficient for binding to hStau1 and hStau1-dependent stimulation of protein expression. Our results unravel how hStau1 identifies a wide spectrum of cellular target mRNAs to control their localization, expression and fate.

Double-stranded RNA-binding proteins are key elements in the intracellular localization of mRNA and its local translation. Staufen is a double-stranded RNA binding protein involved in the localised translation of specific mRNAs during Drosophila early development and neuronal cell fate. The human homologue Staufen1 forms RNA-containing complexes that include proteins involved in translation and motor proteins to allow their movement within the cell, but the mechanism underlying translation repression in these complexes is poorly understood. Here we show that human Staufen1-containing complexes contain essential elements of the gene silencing apparatus, like Ago1-3 proteins, and we describe a set of miRNAs specifically associated to complexes containing human Staufen1. Among these, miR-124 stands out as particularly relevant because it appears enriched in human Staufen1 complexes and is over-expressed upon differentiation of human neuroblastoma cells in vitro. In agreement with these findings, we show that expression of human Staufen1 is essential for proper dendritic arborisation during neuroblastoma cell differentiation, yet it is not necessary for maintenance of the differentiated state, and suggest potential human Staufen1 mRNA targets involved in this process.

We have previously described the fact that the individual expression of influenza virus PA protein induced a generalized proteolysis (J.J. Sanz-Ezquerro, S. de la Luna, Ortin, and A. Nieto, J. Virol. 69:2420-2426, 1995). In this study, we have further characterized this effect by mapping the regions of PA protein required and have found by deletion analysis that the first 247 amino acids are sufficient to bring about this activity. PA mutants that were able to decrease the accumulation levels of coexpressed proteins also presented lower steady-state levels due to a reduction in their half-lives. Furthermore, the PA wild type produced a decrease in the stationary levels of different PA versions, indicating that is itself a target for its induced proteolytic process. All of the PA proteins that induced proteolysis presented nuclear localization, being the sequences responsible for nuclear transport located inside the first 247 amino acids of the molecule. To distinguish between the regions involved in nuclear localization and those involved in induction of proteolysis, we fused the nuclear localization signal of the simian virus 40 T antigen to the carboxy terminus of the cytosolic versions of PA. None of the cytosolic PA versions affected in the first 247-amino-acid part of PA, which were now located in the nucleus, were able to induce proteolysis, suggesting that conservation of a particular conformation in this region of the molecule is required for the effect observed. The fact that all of the PA proteins able to induce proteolysis presented nuclear localization, together with the observation that this activity is shared by influenza virus PA proteins from two different type A viruses, suggests a physiological role for this PA protein activity in viral infection.

The influenza A viruses cause yearly epidemics and occasional pandemics of respiratory disease, which constitute a serious health and economic burden. Their genome consists of eight single-stranded, negative-polarity RNAs that associate to the RNA polymerase and many nucleoprotein monomers to form ribonucleoprotein complexes (RNPs). Here, we focus on the organization of these RNPs, as well as on the structure and interactions of its constitutive elements and we discuss the mechanisms by which the RNPs transcribe and replicate the viral genome.

Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Barcena et al. (1994) J. Virol. 68, 6900–6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three nondashoverlapping antigenic areas were defined by the anti-PA and anti-PB2 MAbs, respectively. Five of the anti-PA MAbs recognized antigenic determinants located within the amino-terminal 157 amino acids of the PA protein, and 6 others reacted strongly with a PA fragment comprising the first 236 amino acids. All 8 anti-PB2 antibodies reacted strongly with a polypeptide fragment containing amino acids 1–113 of the PB2 protein. Analyses of the reactivities of 4 anti-P antibodies with 23 influenza A virus reference strains isolated over a period of 61 years and recovered from humans, pigs, birds and horses, showed that the epitopes were conserved among all viral isolates. The application of these antibodies as research and diagnostic tools is discussed.

In the process of in vivo reconstitution of influenza virus transcriptase-replicase complex, an inhibitory effect was observed when the level of PA protein expression was increased. This inhibition was paralleled by a decrease in the accumulation of the other influenza virus core proteins. The sole expression of PA protein was sufficient to reduce the accumulation level of the proteins encoded by the coexpressed genes. The PA effect was observed upon influenza virus and non-influenza virus proteins and independently of the expression system chosen and the origin of cell line used. The expression of PA protein did not induce variations in the translation of the target proteins but did induce variations on their half-lives, which were clearly reduced. A functional PA subunit seems to be necessary to induce this negative effect, because an inactive point mutant was unable to decrease the steady-state levels or the half-lives of the reporter proteins. The PA effect was observed as early as 5 h after its expression, and continuous synthesis of proteins was not required for performance of its biological activity. The results presented represent the first biological activity of individually expressed PA polymerase subunit.

The atomic structure of the Borna-disease virus nucleocapsid protein represents the first detailed structural information for such essential element in the negative-stranded RNA virus replication machine.

Lipid-based bio-nanohybrids were evaluated as carriers of viral particles in the development of thermally stable and efficacious vaccines against influenza A. Herein, the surfaces of the microfibrous clay sepiolite and the Mg/Al-type layered double hydroxide were modified with a bilayer lipid membrane on which influenza A virions were immobilised. These vaccines were highly immunogenic in mice and stability studies revealed that thermal and lyophilisation resistance were significantly enhanced when compared with a standard aluminium adjuvant. These findings might be attributable to the colloidal behaviour in combination with the biomimetic interface which improves the carrier-virus interactions and preserves the molecular architecture of the viral particles. All these characteristics make the here presented clay-lipid biohybrid materials promising candidates for vaccine formulations. Clay-lipid bio-nanohybrids were evaluated as carriers of viral antigens in the development of thermally stable and efficacious vaccines against influenza A. The biomimetic lipid interfaces on sepiolite and an Mg/Al-type layered double hydroxide afford improved immunogenicity compared with standard aluminium adjuvants and modulate support-antigen interactions to ensure thermal stability of the immobilised virions. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An in vivo system in which expression of a synthetic influenza virus-like chloramphenicol acetyltransferase (CAT) RNA is driven by influenza virus proteins synthesized from cloned cDNAs has been developed. Expression of the four influenza virus core proteins (nucleoprotein, PA, PB1 and PB2) was performed by transfection of four pGEM recombinant plasmids, each containing one of the four viral genes, into cell cultures previously infected with a vaccinia virus recombinant encoding the T7 RNA polymerase (vTF7-3). When a naked negative-sense influenza virus-like CAT RNA was transfected into cells expressing the four influenza virus proteins, CAT activity was detected in the cell extracts, demonstrating that the expressed proteins had RNA-synthesizing activity. In this system, CAT RNA templates containing additional nucleotides at the 3' end were also expressed, resulting in CAT activity. This showed that the influenza virus polymerase can recognize its promoter when located internally on an RNA template. In influenza virus-infected cells however, CAT activity was detected only when the CAT RNA contained the viral promoter at the exact 3' end and was transfected as in vitro assembled ribonucleoprotein. These results are discussed in terms of the different requirements of the two helper systems for expression of an exogenously added RNA.

The mutation rates to a viable mar (monoclonal antibody-resistant mutant) genotype of wild-type influenza (A/Victoria/3/75; H3N2) virus or its mutator variant strains have been previously determined. In order to estimate the mutation rates per nucleotide position, the sequence alterations present in 44 mar mutants isolated from either the wild-type or the mut43 mutator strain have been determined. These mar mutants were selected with either of two non-overlapping, haemagglutinin-specific, monoclonal antibodies (2G10 and p7). Most of the protein changes were identified as substitutions of large, charged amino acids for glycine residues, as the result of G to A transitions. Particularly interesting amino acid changes, not previously reported, were observed in the p7 monoclonal antibody-specific mutants, in which only Gly to Ser and Gly to Asp at position 226 were detected. The identification of the nucleotide substitutions responsible for the mar phenotype has allowed the calculation of approximate values for the total mutation rates at these positions.

The protein regions involved in the nuclear translocation of the influenza virus PA polymerase subunit have been identified by deletion analysis of the protein expressed from a recombinant simian virus 40. Two regions seem to play a role in the process: region I (amino acids 124 to 139) and region II (amino acids 186 to 247). A nucleoplasmin-like nuclear translocation signal (NLS) has been identified in region I and an additional NLS appears to be present in region II, although no consensus targeting sequence can be detected. Alteration in any of the regions identified by short deletions completely prevented nuclear transport, whereas elimination of the regions I or II by large amino- or carboxy-terminal deletions did not prevent nuclear targeting of the truncated protein. In addition, a point mutation at position 154 completely eliminated nuclear transport. A beta-galactosidase fusion protein containing the 280 amino acid terminal region of the PA protein was partially transported to the nucleus and mutant PA proteins with a cytoplasmic phenotype could not be rescued by superinfection with influenza virus. These results suggest that the PA protein contains a functional nuclear targeting region which is required in influenza virus infection, with two independent NLSs, one in region I and the other in region II.

Objective: To describe the lung pathological changes in influenza A (H1N1) viral pneumonia. We studied morphological changes, nitro-oxidative stress and the presence of viral proteins in lung tissue. Methods and patients: Light microscopy was used to examine lung tissue from 6 fatal cases of pandemic influenza A (H1N1) viral pneumonia. Fluorescence for oxidized dihydroethydium, nitrotyrosine, inducible NO synthase (NOS2) and human influenza A nucleoprotein (NP) (for analysis under confocal microscopy) was also studied in lung tissue specimens. Results: Age ranged from 15 to 50 years. Three patients were women, and 5 had preexisting medical conditions. Diffuse alveolar damage (DAD) was present in 5 cases (as evidenced by hyaline membrane formation, alveolo-capillary wall thickening and PMN infiltrates), and interstitial fibrosis in one case. In the fluorescence studies there were signs of oxygen radical generation, increased NOS2 protein and protein nitration in lung tissue samples, regardless of the duration of ICU admission. Viral NP was found in lung tissue samples from three patients. Type I pneumocytes and macrophages harbored viral NP, as evidenced by confocal immunofluorescence microscopy. Conclusions: Lung tissue from patients with pandemic influenza A (H1N1) viral pneumonia shows histological findings consistent with DAD. Prolonged nitro-oxidative stress is present despite antiviral treatment. Viral proteins may remain in lung tissue for prolonged periods of time, lodged in macrophages and type I pneumocytes.

The influenza A viruses are the causative agents of respiratory disease that occurs as yearly epidemics and occasional pandemics. These viruses are endemic in wild avian species and can sometimes break the species barrier to infect and generate new virus lineages in humans. The influenza A virus genome consists of eight single-stranded, negative-polarity RNAs that form ribonucleoprotein complexes by association to the RNA polymerase and the nucleoprotein. In this review we focus on the structure of this RNA-synthesis machines and the included RNA polymerase, and on the mechanisms by which they express their genetic information as mRNAs and generate progeny ribonucleoproteins that will become incorporated into new infectious virions. New structural, biochemical and genetic data are rapidly accumulating in this very active area of research. We discuss these results and attempt to integrate the information into structural and functional models that may help the design of new experiments and further our knowledge on virus RNA replication and gene expression. This interplay between structural and functional data will eventually provide new targets for controlled attenuation or antiviral therapy.

The influenza A virus RNA polymerase is a heterotrimeric complex responsible for viral genome transcription and replication in the nucleus of infected cells. We recently carried out a proteomic analysis of purified polymerase expressed in human cells and identified a number of polymerase-associated cellular proteins. Here we characterise the role of one such host factors, SFPQ/PSF, during virus infection. Down-regulation of SFPQ/PSF by silencing with two independent siRNAs reduced the virus yield by 2–5 log in low-multiplicity infections, while the replication of unrelated viruses as VSV or Adenovirus was almost unaffected. As the SFPQ/PSF protein is frequently associated to NonO/p54, we tested the potential implication of the latter in influenza virus replication. However, down-regulation of NonO/p54 by silencing with two independent siRNAs did not affect virus yields. Down-regulation of SFPQ/PSF by siRNA silencing led to a reduction and delay of influenza virus gene expression. Immunofluorescence analyses showed a good correlation between SFPQ/PSF and NP levels in infected cells. Analysis of virus RNA accumulation in silenced cells showed that production of mRNA, cRNA and vRNA is reduced by more than 5-fold but splicing is not affected. Likewise, the accumulation of viral mRNA in cicloheximide-treated cells was reduced by 3-fold. In contrast, down-regulation of SFPQ/PSF in a recombinant virus replicon system indicated that, while the accumulation of viral mRNA is reduced by 5-fold, vRNA levels are slightly increased. In vitro transcription of recombinant RNPs generated in SFPQ/PSF-silenced cells indicated a 4–5-fold reduction in polyadenylation but no alteration in cap snatching. These results indicate that SFPQ/PSF is a host factor essential for influenza virus transcription that increases the efficiency of viral mRNA polyadenylation and open the possibility to develop new antivirals targeting the accumulation of primary transcripts, a very early step during infection., Authors Summary The influenza A viruses cause annual epidemics and occasional pandemics of respiratory infections that may be life threatening. The viral genome contains 8 RNA molecules forming ribonucleoproteins that replicate and transcribe in the nucleus of infected cells. Influenza viruses are intracellular parasites that need the host cell machinery to replicate. To better understand this virus-cell interplay we purified the viral RNA polymerase expressed in human cells and identified several specifically associated cellular proteins. Here we characterise the role of one of them, the proline-glutamine rich splicing factor (SFPQ/PSF). Down-regulation of SFPQ/PSF indicated that it is essential for virus multiplication. Specifically, the accumulation of messenger and genomic virus-specific RNAs was reduced by SFPQ/PSF silencing in infected cells. Furthermore, transcription of parental ribonucleoproteins was affected by SFPQ/PSF down-regulation. The consequences of silencing SFPQ/PSF on the transcription and replication of a viral recombinant replicon indicated that it is required for virus transcription but not for virus RNA replication. In vitro transcription experiments indicated that SFPQ/PSF increases the efficiency of virus mRNA polyadenylation. This is the first description of a cellular factor essential for influenza virus transcription and opens the possibility to identify inhibitors that target this host-virus interaction and block virus gene expression.