Análisis estructural y funcional de la polimerasa del virus de la gripe

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular y Celular. Fecha de lectura: 17-06-201

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

Novel Methodology for the Detection of Enveloped Viruses

Presented at Viruses 2020—Novel Concepts in Virology, Barcelona, Spain, 5–7 February 2020 (abstract)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

Understanding the neurological implications of acute and long COVID using brain organoids

As early as in the acute phase of the coronavirus disease 2019 about the long-term implications of infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), like many other viruses, can trigger chronic disorders that last months or even years. Long COVID, the chronic and persistent disorder lasting more than 12 weeks after the primary infection with SARS-CoV-2, involves a variable number of neurological manifestations, ranging from mild to severe and even fatal. In vitro and in vivo modeling suggest that SARS-CoV-2 infection drives changes within neurons, glia and the brain vasculature. In this Review, we summarize the current understanding of the neuropathology of acute and long COVID, with particular emphasis on the knowledge derived from brain organoid models. We highlight the advantages and main limitations of brain organoids, leveraging their humanoerived origin, their similarity in cellular and tissue architecture to human tissues, and their potential to decipher the pathophysiology of long COVID

Identification of PatL1, a human homolog to yeast P body component Pat1

AbstractIn 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

Structural and Functional Characterization of an Influenza Virus RNA Polymerase-Genomic RNA Complex ▿

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 negative-polarity 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

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein

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

Alternative interaction sites in the influenza A virus nucleoprotein mediate viral escape from the importin-alpha 7 mediated nuclear import pathway

Influenza A viruses are able to adapt to restrictive conditions due to their high mutation rates. Importin-alpha 7 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-alpha 7. To address this question, we assessed viral evolution in mice lacking the importin-alpha 7 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-alpha 7 requirement likely due to the utilization of alternative interaction sites in NP beyond the classical nuclear localization signal. However, viral escape from importin-alpha 7 by mutations in NP is, at least in part, associated with reduced viral replication highlighting the crucial contribution of importin-alpha 7 to replicative fitness in human cells