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Sialic-acid-binding immunoglobulin-like lectins are cell surface immune receptors known as Siglecs that play a paramount role as modulators of immunity. In recent years, research has underscored how the underlaying biology of this family of receptors influences the outcome of viral infections. While Siglecs are needed to promote effective antiviral immune responses, they can also pave the way to viral dissemination within tissues. Here, we review how recent preclinical findings focusing on the interplay between Siglecs and viruses may translate into promising broad-spectrum therapeutic interventions or key biomarkers to monitor the course of viral infections.

Avian influenza A viruses (AIV) of the H7 subtype continue to evolve posing a pandemic threat. However, molecular markers of H7N7 AIV pathogenicity and transmission in mammals remain poorly understood. In this study, we performed a systematic in vitro and in vivo analysis by comparing an H7N7 highly pathogenic AIV and its ferret adapted variant. Passaging an H7N7 AIV in ferrets led to six mutations in genes encoding the viral polymerase complex and the viral surface proteins. Here, we show that mutations in the H7 hemagglutinin gene cause increased pathogenicity in mice. Contact transmission between guinea pigs required additional mutations in the gene encoding the polymerase subunit PB1. Thus, particular vigilance is required with respect to HA and PB1 mutations as predictive molecular markers to assess the pandemic risk posed by emerging H7 avian influenza viruses.

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Importin-α7 is a component of the nuclear import machinery required for avian-mammalian adaptation and replicative fitness in human cells. Here, we elucidate the mechanisms by which influenza A viruses may escape replicative restriction in the absence of importin-α7. To address this question, we assessed viral evolution in mice lacking the importin-α7 gene. We show that three mutations in particular occur with high frequency in the viral nucleoprotein (NP) protein (G102R, M105K and D375N) in a specific structural area upon in vivo adaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin-α7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal. However, viral escape from importin-α7 by mutations in NP is, at least in part, associated with reduced viral replication highlighting the crucial contribution of importin-α7 to replicative fitness in human cells.

Viral infections in humans cause a huge burden in worldwide healthcare that has increased due to the emergence of new pathogenic viruses, such as in the recent Ebola virus (EBOV) outbreaks. Viral particles in body fluids are often at very low levels, making diagnosis difficult. In order to address this problem, we have developed a new detection platform to isolate and detect different enveloped viruses. We have recently identified that sialic acid-binding Ig‑like lectin 1 (Siglec-1/CD169) is one cellular receptor used by EBOV and HIV-1 to enter myeloid cells, key target cells for infection and pathogenesis. For viral uptake, the V-set domain of this myeloid cell receptor recognizes the gangliosides of viral membranes that were dragged during viral budding from the plasma membrane of infected cells. We took advantage of this specific interaction between Siglec‑1 and viral gangliosides to develop a new detection methodology. We have generated a recombinant protein that contains the V-set domain of Siglec-1 fused to the human IgG Fc domain for anchoring in latex beads. These coated beads allow the isolation of viral particles and their measurement by flow cytometry. We have tested its efficacy to detect HIV-1 and EBOV and its specificity by using anti-Siglec‑1 antibodies that prevent the interaction and serve as a negative control. To test the capacity of our method, we used synthetic liposomes to assess the effect of ganglioside concentration in membranes as well as the size of viral particles. This methodology would facilitate the diagnosis of infections by concentrating viral particles in a fast and direct method. At a time when global human mobility facilitates the dissemination of infectious agents, our approach represents a rapid and effective method to maximize the identification of both known and emerging enveloped viruses as part of public health viral surveillance strategies.

Summary Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections., Graphical Abstract, Highlights • Importin-α3 is the most abundantly expressed isoform in the mammalian lung • Importin-α3 is highly conserved across species • Importin-α3 is one of the major nuclear transporters of NF-κB • Importin-α3 acts as an immune sensor of influenza A virus infections, Thiele et al. show that importin-α3 is one of the major nuclear transporters of NF-κB in the mammalian lung. High-level TNF-α-inducing HPAIVs inhibit importin-α3 mRNA transcription by interfering with its promoter activity. Thus, HPAIVs may evade antiviral immunity in the respiratory tract by generating a bottleneck in importin-α3 availability.

Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation (’priming state’) and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the ‘apo’ state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the ‘apo’ state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive ‘apo’ state.

Influenza virus is a segmented, negative strand RNA virus with each genome segment being packaged in a distinct ribonucleoprotein particle (RNP). The RNP consists of the heterotrimeric viral RNA-dependent RNA polymerase bound to the conserved 5' and 3' ends of the genome segment (the viral promoter) with the rest of the viral RNA (vRNA) being covered by multiple copies of nucleoprotein. This review focusses on the new insights that recent crystal structures have given into the detailed molecular mechanisms by which the polymerase performs both transcription and replication of the vRNA genome. Promoter binding, in particular that of 5' end, is essential to allosterically activate all polymerase functions. Transcription is initiated by the hijacking of nascent, capped host transcripts by the process of 'cap-snatching', for which the viral polymerase makes an essential interaction with the C-terminal domain (CTD) of cellular RNA polymerase II. The structures allow a coherent mechanistic model of the subsequent cap-snatching, cap-dependent priming, elongation and self-polyadenylation steps of viral mRNA synthesis. During replication, the vRNA is copied without modification into complementary RNA (cRNA) which is packaged into cRNPs. A priming loop located in the polymerase active site is required for the unprimed synthesis of cRNA from vRNA, but is not required for cRNA to vRNA replication due to differences in the mode of initiation of RNA synthesis. Overall a picture emerges of influenza polymerase being a highly complex, flexible and dynamic machine. The challenge remains to understand in more detail how it functions within the RNP and how interacting host factors modulate its activity in the cellular context. Finally, these detailed insights have opened up new opportunities for structure-based antiviral drug design targeting multiple aspects of polymerase function.

The acute respiratory distress syndrome (ARDS) is the leading cause of death in influenza A virus (IAV)-infected patients. Hereby, the cellular importin-α7 gene plays a major role. It promotes viral replication in the lung, thereby increasing the risk for the development of pneumonia complicated by ARDS. Herein, we analyzed whether the recently emerged H7N9 avian IAV has already adapted to human importin-α7 use, which is associated with high-level virus replication in the mammalian lung. Using a cell-based viral polymerase activity assay, we could detect a decreased H7N9 IAV polymerase activity when importin-α7 was silenced by siRNA. Moreover, virus replication was diminished in the murine cells lacking the importin-α7 gene. Consistently, importin-α7 knockout mice presented reduced pulmonary virus titers and lung lesions as well as enhanced survival rates compared to wild-type mice. In summary, our results show that H7N9 IAV have acquired distinct features of adaptation to human host factors that enable enhanced virulence in mammals. In particular, adaptation to human importin-α7 mediates elevated virus replication in the mammalian lung, which might have contributed to ARDS observed in H7N9-infected patients.

The heterotrimeric influenza polymerase (FluPol), comprising subunits PA, PB1 and PB2, binds to the conserved 5' and 3' termini (the 'promoter') of each of the eight single-stranded viral RNA (vRNA) genome segments and performs both transcription and replication of vRNA in the infected cell nucleus. To transcribe viral mRNAs, FluPol associates with cellular RNA polymerase II (Pol II), which enables it to take 5'-capped primers from nascent Pol II transcripts. Here we present a co-crystal structure of bat influenza A polymerase bound to a Pol II C-terminal domain (CTD) peptide mimic, which shows two distinct phosphoserine-5 (SeP5)-binding sites in the polymerase PA subunit, accommodating four CTD heptad repeats overall. Mutagenesis of the SeP5-contacting basic residues (PA K289, R454, K635 and R638) weakens CTD repeat binding in vitro without affecting the intrinsic cap-primed (transcription) or unprimed (replication) RNA synthesis activity of recombinant polymerase, whereas in cell-based minigenome assays the same mutations substantially reduce overall polymerase activity. Only recombinant viruses with a single mutation in one of the SeP5-binding sites can be rescued, but these viruses are severely attenuated and genetically unstable. Several previously described mutants that modulate virulence can be rationalized by our results, including a second site mutation (PA(C453R)) that enables the highly attenuated mutant virus (PA(R638A)) to revert to near wild-type infectivity. We conclude that direct binding of FluPol to the SeP5 Pol II CTD is fine-tuned to allow efficient viral transcription and propose that the CTD-binding site on FluPol could be targeted for antiviral drug development.

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Here, we addressed the question by which mechanisms influenza A viruses may escape restriction by the cellular importin-α7 protein, a component of the nuclear import machinery required for avian-mammalian adaptation and replicative fitness in human cells. Therefore, we assessed viral evolution in mice lacking the importin-α7 gene. Here, we show that particularly three mutations occur with high frequency in the viral NP protein (G102R, M105K and D375N) in a specific structural area uponin vivoadaptation. Moreover, our findings suggest that the adaptive NP mutations mediate viral escape from importin-α7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal and importin-α isoforms. However, viral escape from importin-α7 is, at least in part, associated with reduced replicative fitness in human cells.

Viral drug resistance is believed to be less likely to occur if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely, importin-α7, as a cellular drug target to combat pandemic influenza viruses. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-α7 −/− mice that otherwise are resistant. Viral escape from importin-α7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-α7 −/− mouse-adapted strain became even more virulent for wild-type mice than the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways, eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multitarget approach to reduce viral drug resistance. IMPORTANCE Here, we investigated the long-standing hypothesis based on in vitro studies that viral drug resistance occurrence is less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing, in an in vivo animal model, the feasibility of targeting the cellular factor importin-α7, which is crucial for human influenza virus replication and pathogenesis, as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo , and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multitarget approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence.

After viral entry into the cell, the nuclear envelope poses a major cellular barrier that needs to be overcome upon adaptation of highly pathogenic avian influenza viruses (HPAIV) to the new host. To ensure efficient viral transcription and replication in the nucleus of the host cell, the viral polymerase complex of avian influenza viruses needs to switch from recognition of avian to mammalian components of the nuclear import machinery. Recent evidence suggests that influenza viruses have evolved different mechanisms to utilize importin-α isoforms as components of this machinery, bridging pre- and post-nuclear import on both sides of the nuclear envelope.

Pregnant women are at high risk for severe influenzadisease outcomes, yet insights into the underlyingmechanisms are limited. Here, we present models ofH1N1 infection in syngenic and allogenic pregnantmice; infection in the latter mirrors the severe courseof 2009 pandemic influenza in pregnant women. Wefound that the anti-viral immune response in the pregnanthost was significantly restricted as compared tothe non-pregnant host. This included a reduced type Iinterferon response as well as impaired migration ofCD8+ T cells into the lung. The multi-faceted failureto mount an anti-viral response in allogenic pregnantmice resulted in a less stringent selective environmentthat promoted the emergence of 2009 H1N1 virusvariants that specifically counteract type I interferonresponse and mediate increased viral pathogenicity.These insights underscore the importance of influenzavaccination compliance in pregnant womenand may open novel therapeutic avenues. Fil: Engels, Géraldine. University Medical Center Hamburg-Eppendorf; Alemania. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Hierweger, Alexandra Maximiliane. University Medical Center Hamburg-Eppendorf; Alemania Fil: Hoffmann, Julia. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Thieme, René. University Medical Center Hamburg-Eppendorf; Alemania Fil: Thiele, Swantje. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Bertram, Stephanie. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Dreier, Carola. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Resa-Infante, Patricia. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Jacobsen, Henning. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Thiele, Kristin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Alawi, Malik. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Indenbirken, Daniela. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Grundhoff, Adam. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Siebels, Svenja. University Medical Center Hamburg-Eppendorf; Alemania Fil: Fischer, Nicole. University Medical Center Hamburg-Eppendorf; Alemania Fil: Stojanovska, Violeta. University of Groningen; Países Bajos Fil: Muzzio, Damián Oscar. University of Greifswald; Alemania Fil: Jensen, Cristian Federico. University of Greifswald; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Karimi, Khalil. University Medical Center Hamburg-Eppendorf; Alemania Fil: Mittrücker, Hans-Willi. University Medical Center Hamburg-Eppendorf; Alemania Fil: Arck, Petra Clara. University Medical Center Hamburg-Eppendorf; Alemania Fil: Gabriel, Gülsah. Leibniz Institut Für Experimentelle Virologie; Alemania

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2∆ induces the expression of IFNβ and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo., Author Summary Error-prone polymerase function of RNA viruses can result in expression of defective RNAs harboring internal deletions of various sizes. Small subgenomic RNAs are strong inducers of the antiviral response by serving as pathogen-associated patterns that are predominantly detected by cellular sensors. Recently, it has been shown that influenza A virus defective RNAs are not only generated upon passages in cell culture, but also in infected humans, indicating that these subgenomic RNAs may also be relevant in infections in vivo. Here, we characterize a novel defective RNA derived from the PB2 segment of a highly pathogenic H5N1 influenza A virus. This RNA encodes a 10 kDa peptide (PB2Δ) which activates type I interferon (IFN) responses through direct interaction with the adapter protein MAVS, a key component of the RIG-I-dependent IFN induction. This is the first time that such a function was described for a defective RNA-encoded protein, a finding that has several important implications with regard to deciphering viral protein functions and options for immunostimulatory approaches. Furthermore, this is an example of how influenza viruses may acquire novel polypeptides with altered functions from its limited genome.

Influenza A viruses recruit components of the nuclear import pathway to enter the host cell nucleus and promote viral replication. Here, we analyzed the role of the nuclear import factor importin-α7 in H1N1 influenza virus pulmonary tropism by using various ex vivo imaging techniques (magnetic resonance imaging, confocal laser scanning microscopy, and correlative light-electron microscopy). We infected importin-α7 gene-deficient (α7 −/− ) mice with a recombinant H1N1 influenza virus and compared the in vivo viral kinetics with those in wild-type (WT) mice. In WT mice, influenza virus replication in the bronchial and alveolar epithelium already occurred a few days after infection. Accordingly, extensive mononuclear infiltration and alveolar destruction were present in the lungs of infected WT mice, followed by 100% lethality. Conversely, in α7 −/− mice, virus replication was restricted mostly to the bronchial epithelium with marginal alveolar infection, resulting in significantly reduced lung damage and enhanced animal survival. To investigate the host immune response during alveolar virus replication, we studied the role of primary macrophages in virus propagation and clearance. The ability of macrophages to support or clear the virus infection, as well as the host cellular immune responses, did not significantly differ between WT and α7 −/− mice. However, cytokine and chemokine responses were generally elevated in WT mice, likely reflective of increased viral replication in the lung. In summary, these data show that a cellular factor, importin-α7, is required for enhanced virus replication in the alveolar epithelium, resulting in elevated cytokine and chemokine levels, extensive mononuclear infiltration, and thus, severe pneumonia and enhanced virulence in mice. IMPORTANCE Influenza A viruses are respiratory pathogens that may cause pneumonia in humans. Viral infection and replication in the alveoli of the respiratory tract are believed to be crucial for the development of the acute respiratory distress syndrome associated with fatal outcomes in influenza virus-infected patients. Here, we report the requirement of a cellular factor, importin-α7, for efficient virus replication in the alveolar epithelium of mice. Using complementary ex vivo imaging approaches, we show that influenza virus replication is restricted to the bronchial epithelium, followed by enhanced survival in importin-α7-deficient mice. In contrast, the presence of this gene results in enhanced virus replication in the alveoli, elevated cytokine and chemokine responses, mononuclear infiltration, alveolar destruction, and 100% lethality in wild-type mice. Taken together, our results show that importin-α7 is particularly required for virus replication in the alveolar epithelium in association with severe pneumonia and death in mice.

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 replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negativepolarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex. Copyright © 2010, American Society for Microbiology. All Rights Reserved., This work has been supported by grants BFU2007-60046 (J.O.) and SAF2008-00451 (O.L.) from the Spanish Ministry of Science; project RD06/0020/1001 of the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (O.L.); CIBER de Enfermedades Respiratorias (J.O.), financed by the Instituto de Salud Carlos III; and the VIRHOST Program, financed by Comunidad de Madrid (J.O.). M.A.R.-C. was supported by an FPI fellowship from the Spanish Ministry of Science, and P.R.-I. was supported by a predoctoral fellowship of the Spanish Research Council. O.L.’s group is additionally supported by the Human Frontiers Science Program Organization (grant RGP39/2008), and J.O.’s group is additionally supported by the Fundación Marcelino Botin.

Influenza virus mRNAs are synthesized by the trimeric viral polymerase using short capped primers obtained by a 'cap-snatching' mechanism. The polymerase PB2 subunit binds the 5' cap of host pre-mRNAs, which are cleaved after 10-13 nucleotides by the PB1 subunit. Using a library-screening method, we identified an independently folded domain of PB2 that has specific cap binding activity. The X-ray structure of the domain with bound cap analog m(7)GTP at 2.3-A resolution reveals a previously unknown fold and a mode of ligand binding that is similar to, but distinct from, other cap binding proteins. Binding and functional studies with point mutants confirm that the identified site is essential for cap binding in vitro and cap-dependent transcription in vivo by the trimeric polymerase complex. These findings clarify the nature of the cap binding site in PB2 and will allow efficient structure-based design of new anti-influenza compounds inhibiting viral transcription.

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 alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-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.

In yeast, the activators of mRNA decapping, Pat1, Lsm1 and Dhh1, accumulate in processing bodies (P bodies) together with other proteins of the 5′-3′-deadenylation-dependent mRNA decay pathway. The Pat1 protein is of particular interest because it functions in the opposing processes of mRNA translation and mRNA degradation, thus suggesting an important regulatory role. In contrast to other components of this mRNA decay pathway, the human homolog of the yeast Pat1 protein was unknown. Here we describe the identification of two human PAT1 genes and show that one of them, PATL1, codes for an ORF with similar features as the yeast PAT1. As expected for a protein with a fundamental role in translation control, PATL1 mRNA was ubiquitously expressed in all human tissues as were the mRNAs of LSM1 and RCK, the human homologs of yeast LSM1 and DHH1, respectively. Furthermore, fluorescence-tagged PatL1 protein accumulated in distinct foci that correspond to P bodies, as they co-localized with the P body components Lsm1, Rck/p54 and the decapping enzyme Dcp1. In addition, as for its yeast counterpart, PatL1 expression was required for P body formation. Taken together, these data emphasize the conservation of important P body components from yeast to human cells.