Targeting host calpain proteases decreases influenza A virus infection.

Influenza A viruses (IAV) trigger contagious acute respiratory diseases. A better understanding of the molecular mechanisms of IAV pathogenesis and host immune responses is required for the development of more efficient treatments of severe influenza. Calpains are intracellular proteases that participate in diverse cellular responses, including inflammation. Here, we used in vitro and in vivo approaches to investigate the role of calpain signaling in IAV pathogenesis. Calpain expression and activity were found altered in IAV-infected bronchial epithelial cells. With the use of small-interfering RNA (siRNA) gene silencing, specific synthetic inhibitors of calpains, and mice overexpressing calpastatin, we found that calpain inhibition dampens IAV replication and IAV-triggered secretion of proinflammatory mediators and leukocyte infiltration. Remarkably, calpain inhibition has a protective impact in IAV infection, since it significantly reduced mortality of mice challenged not only by seasonal H3N2- but also by hypervirulent H5N1 IAV strains. Hence, our study suggests that calpains are promising therapeutic targets for treating IAV acute pneumonia.

Replication-competent influenza A virus that encodes a split-green fluorescent protein-tagged PB2 polymerase subunit allows live-cell imaging of the virus life cycle.

Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualization during infection in single cells. This is largely due to the difficulty of encoding fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-green fluorescent protein (split-GFP) system (S. Cabantous, T. C. Terwilliger, and G. S. Waldo, Nat. Biotechnol. 23:102-107, 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large transcomplementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation and intermittent rapid (∼1 µm/s), directional movements in the cytoplasm, dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi-wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new antiviral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and nonviral systems.

HMGB1 protein binds to influenza virus nucleoprotein and promotes viral replication.

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.

Interactions moléculaires entre les ribonucléoprotéines des virus influenza de type A et la cellule hôte.

The study of molecular interactions between viral components and cellular components have become a very active field of research during the recent years. At stake is a better understanding of the determinants of viral pathogenicity and zoonotic potential, and the identification of new therapeutic targets. The viral ribonucleoproteins (vRNPs), which are key components of the viral multiplication cycle and relatively conserved, are of particular interest. Various methodological approaches, including high-throughput screens, have been developed in order to identify cellular factors that are physically or functionally associated to vRNPs. The documented vRNP-host cell interactions are mainly related to the nucleocytoplamic trafficking of vRNPs, to the synthesis and maturation of viral RNAs, and not so expectedly, to the regulation of the innate immune response to viral infection. However, we are far from unraveling the complexity and dynamics of these interactions and from understanding which ones are most critical with respect to host-range.

Multiple hosts and Influenza A viruses genetic mixing.

Influenza A viruses have a segmented, negative-stranded RNA genome. These viruses are classified according to the antigenic properties of the two glycoproteins, expressed on the surface of the virus particles, the hemagglutinin (HA or H) and the neuraminidase (NA or N). To date, 17 H and 10 N have been described and 116 HxNy combinations or subtypes reported. Except for the H17N10 subtype recently identified in bats, all identified subtypes have been identified in wild aquatic birds. These birds are considered to be the natural reservoir of influenza A viruses, from which some subtypes can be transmitted to other bird and mammal species, including humans. Interspecies transmissions seem to occur regularly, and can occasionally lead to the adaptation and stable establishment of a new viral lineage in a given species. This review recalls the genetic diversity of avian, swine and human influenza viruses and focuses on lesser-known influenza A viruses, identified in horses, dogs and very recently in bats. It discusses the genetic mixing that may result from interspecies transmission, and the associated risks of epizootics, zoonosis and pandemics.

Influenza A virus progeny vRNP trafficking in live infected cells studied with the virus-encoded fluorescently tagged PB2 protein.

Dynamic studies of influenza virus infection in the live cells are limited because of the lack of appropriate methods for non-invasive detection of the viral components. Using the split-GFP strategy, we have recently developed and characterized an unimpaired recombinant influenza A virus encoding a tagged PB2 subunit of RNA-dependent RNA polymerase, which enabled continuous real-time visualization of the viral ribonucleoproteins (vRNPs) in living cells (Avilov, Moisy, Munier, Schraidt, Naffakh and Cusack [12]). Here, using this virus, we studied vRNP trafficking and interaction with Rab11 in the context of quasi-wild type infection. In agreement with recent reports, we observed that upon nuclear export, progeny vRNPs accumulate in the particles containing Rab11, a multifunctional protein involved in vesicle trafficking which resides at recycling endosomes. Fluorescence resonance energy transfer microscopy indicated a distance <10nm between PB2 and Rab11, suggesting that a direct interaction occurs. Single particle tracking analysis showed that most of the motions of vRNP-positive particles in infected cells are slow, while rapid directional motions intermittently occur. Analysis focused on these intermittent motions indicated that depolymerization of either microtubules or actin filaments moderately reduced their occurrence, while disruption of both cytoskeleton components in combination suppressed the rapid motions entirely. Thus, the split-GFP based virus enabled us to obtain a live-cell based confirmation for the model of vRNP trafficking which assumes accumulation of vRNP in recycling endosomes through a direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.