Nucleoprotein S69 phosphorylation regulates influenza A virus replication

RationaleHypercapnia, the elevation of CO2 in blood and tissue, commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary bacterial and viral infections, including influenza. We previously reported that hypercapnia downregulates several innate immune genes and increases the mortality of bacterial pneumonia and influenza A virus (IAV) infection in mice. High CO2 also increases viral nucleoprotein (NP) expression in the mouse lung, primarily in alveolar macrophages (MØs). In the current study, we explored the effect of hypercapnia on the PI3K/Akt pathway during IAV infection in MØs. In addition, using pharmacologic PI3K and Akt inhibitors we studied the role of hypercapnia‐induced activation of Akt on the expression of antiviral proteins, including MX dynamin like GTPase 1 (MX1), 2′‐5′‐oligoadenylate synthetase 1 (OAS1) and viperin; viral nonstructural 1 protein 1 (NS1) and NP.MethodsHuman alveolar and PMA‐differentiated THP‐1 MØs as well as murine alveolar and bone marrow derived MØs were cultured in 5% CO2 (PCO2 36 mmHg; normocapnia) or 15% CO2 (PCO2 108 mmHg; hypercapnia) and infected with IAV. Akt activation was assessed by immunoblot and immunofluorescence microscopy (IF). MX1, OAS1 and viperin mRNA and protein levels were determined using qPCR and immunoblot or IF, respectively. IAV replication was assessed using MDCK plaque assay, and NS1 and NP expression were determined by immunoblot or IF. For in vivo studies, mice were pre‐exposed to normoxic hypercapnia (10% CO2/21% O2) for 3 days, or air as control, then infected with IAV. Mice were sacrificed 4 days after infection for determination of antiviral and viral proteins expression in lung tissue by immunoblot and IF.ResultsHypercapnia potentiated IAV‐induced Akt activation in MØs in vivo and in vitro, an effect blocked by the PI3K inhibitor LY294002 and the Akt inhibitor MK2206. In addition, elevated CO2 decreased MX1, OAS1 and viperin mRNA and protein expression and increased viral replication and NS1 and NP proteins in IAV infected human and mouse MØs. Hypercapnia also increased viral protein expression in alveolar MØs, and decreased antiviral proteins and augmented viral titers in the lungs of IAV‐infected mice. We also found that the PI3K and Akt inhibitors blocked the hypercapnia‐induced suppression of antiviral proteins and the increase on viral proteins and IAV replication in vitro.ConclusionHypercapnia decreases antiviral proteins and increases viral NS1 and NP proteins and virus replication in MØs following IAV infection in vivo and in vitro. The effect of elevated CO2 on the macrophage antiviral response and virus production can be blocked by small molecule inhibitors of PI3K and Akt, suggesting a link between hypercapnia‐induced activation of the PI3K/Akt pathway and an impaired antiviral host response. Therefore, PI3K/Akt inhibitors may hold promise as pharmacotherapy to improve antiviral host defense in patients with advanced lung disease and hypercapnia.Support or Funding InformationR01HL107629, R56HL131745, P01AG049665

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function. A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase protein 1 (SAMHD1) is an intracellular protein that regulates a stable deoxynucleoside triphosphates (dNTPs) for normal cellular function. Many studies have documented SAMHD1-mediated restriction of RNA retroviruses and different DNA viruses in non-dividing myeloid cells, where it acts to deplete cellular dNTP pools to inhibit replication of the viral genome. Less is currently known regarding its ability to restrict the replication of other RNA viruses in different cell types. In this study, we investigate the role of SAMHD1 in modulating the replication of RNA viruses associated with respiratory virus infection, with a particular focus on seasonal influenza A virus (IAV). Moreover, our studies have assessed the ability of SAMHD1 to modulate IAV replication in two key cell types associated with the pathogenesis of IAV, namely epithelial and macrophage-like cells. qPCR was used to examine expression of endogenous SAMHD1 in response to IAV infection and/or treatment with recombinant type I interferon (IFN) in epithelial (A549) and macrophage-like (THP-1) cells. CRISPR/Cas9 knockout (KO) of endogenous SAMHD1 was performed to assess the role of SAMHD1 in modulating IAV replication in A549 cells or in differentiated (d)THP-1. IAV infection and replication were also assessed in A549 and dTHP-1 SAMHD1 KO cells with stable doxycycline (DOX)-induced expression of SAMHD1 or of phosphorylation mutants of SAMHD1. CRISPR/Cas9 KO of endogenous SAMHD1 in dTHP-1 or A549 cells did not enhance virus release following infection with seasonal IAV. Moreover, reconstitution of KO cells with DOX-inducible SAMHD1 did not restrict the growth of seasonal IAV in dTHP-1 or A549 cells, despite SAMHD1-mediated restriction of a DNA virus (HSV-1) in both cell types. In contrast to seasonal IAV, we also observed restriction of highly pathogenic avian influenza (HPAI) A(H5N1) IAV replication in dTHP-1 cells expressing DOX-inducible SAMHD1. Together, these studies indicate that neither endogenous nor DOX-inducible SAMHD1 restricts the growth of seasonal IAV in macrophage-like or epithelial cells.

Influenza A virus (IAV) is a major public health problem responsible for the death of half
\na million people every year worldwide. Zoonotic transmissions of the virus from swine and avian
\norigin have occurred and can in the worst-case result in pandemics. Prediction of the next
\npandemic strain is still a major challenge as mechanisms of antigenic shift and the zoonotic
\nnature of influenza viruses are still poorly understood. On the other hand, the current vaccination
\nstrategy treating seasonal influenza viruses, given its own problems in efficacy, is not well suited
\nto mitigate an impending pandemic. While there are few clinically approved anti-IAV drugs for
\ntherapeutic and prophylactic use, these drugs have also been challenged by the emergence of
\ndrug resistance, toxicity, adverse effects and low efficacy. Hence, development of a broad range
\nanti-IAV drug that could target all of the IAV strains irrespective of their source is critically
\nimportant.
\nTo discover effective IAV vaccines and alternative anti-IAV therapeutics, it is crucial to
\nunderstand how IAVs adapt to different species’ host ranges and vice versa, how different
\nspecies hosts respond to different IAV subtypes. Additionally, it is crucial to understand IAVhost interactions and identifying critical viral or host factors that could support the replication of
\nthe virus or induce pathologic conditions. Recently, high throughput technologies such as mRNA
\nmicroarray-based gene expression, genome-wide siRNA screening, and proteomic analysis are
\nproviding in-depth insights into host-pathogen interactions of IAVs. Hence, this study aimed to
\ncomputationally investigate IAV-host interactions using three data set (transcriptome, genomewide siRNA screens and interactome) and identify 1) virus and host specific responses, 2) host
\ndeterminants in host adaptations and 3) host targets for IAV therapeutics.
\nThis thesis contains four main projects. In the first project (chapter 3), we investigated the
\nhost gene expression changes of eight IAVs (H1N1/WSN, pH1N1, H5N2/F118, H5N2/F59,
\nH5N2/F189, H5N3, H7N9 and H9N2 viruses) in A549 cells at different time points of infections.
\nThen we integrated the differentially expressed genes (DEGs) in at least 3 viruses, with 1713 and
\n1780 host factors comprehensively curated from 11 siRNA and 13 interactome studies
\nrespectively. The integration of the three data set highlighted plausible influenza A virus required
\nhost factors (IHFs) that could be targeted against IAVs. The up-regulated IHFs (e.g. TRIM21,
\nTRIM26, IRF2, and SAMHD1) might support the replication of IAV through suppression of the innate and adaptive antiviral immune responses. The other up-regulated IHFs could also enhance
\nthe replication of IAV at different stages of the virus lifecycle: endocytosis (BTC), prevention of
\napoptosis (TIMM17A), nuclear import (JAK2), and translation elongation of viral proteins
\n(HEXIM1). Although several of these IHFs have been implicated in other viruses, the detailed
\nmechanisms of how several of these up-regulated IHFs could support IAV replication require
\nfurther investigation.
\nThe second project (chapter 4), explored a comprehensive analysis of host gene
\nexpression changes in different IAV infections in different host species. We used host gene
\nexpression signatures of cell lines from three species (A549 (human), CEF (chicken), and
\nMDCK (canine) in response to six IAVs (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189,
\nH5N3 and H9N2 viruses). To compare the expression changes between the three species, we
\nperformed comprehensive probe set re-annotation and human ortholog mapping. The result
\nshowed that the expression signatures of different IAV isolates in a single species cell type are
\nmore similar to each other compared to the expression signatures of a single isolate in different
\nspecies’ cell types. The functional annotations (pathways) and the highly expressed cell-specific
\nsignatures indicated that IAVs up-regulated host factors could induce virus infectivity (e.g.
\nOSBPL1A and ARHGAP21), reduce apoptosis (e.g. MRPS27) and increase cell proliferation (e.g.
\nCOPS2) in CEF cells. Conversely, increased antiviral, pro-apoptotic and inflammatory
\nsignatures have been identified in A549 cells. Except in H5N3 virus infections, generally IAV
\ninfections down-regulates genes associated with cell cycle and metabolic pathways with the
\nstrongest effect in MDCK cells, followed by A549 cells in a strain dependent manner, but not in
\nCEF cells. Previously our group demonstrated that the replication of the examined viruses was
\nsignificantly higher in CEF cells than the other cell types. Thus, we hypothesise that this could
\npartially explain the mechanism how infectious LPAI viruses shed by chickens lack the
\ninflammatory response and cellular disruption that may lead to disease conditions.
\nIn the third project (chapter 5), we used a systems-based approach to investigate changes
\nto the transcriptome of primary murine lung macrophages (PMФ) in response to infection with
\nthe mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2
\nand H5N3. The results showed that while all viruses induced antiviral responses, the H5N3
\ninfection resulted in higher expression levels of cytokines and chemokines associated with
\ninflammatory responses. Previously, our group showed that the LPAI H5N2 and H5N3 were able to infect murine lung macrophages and together with increased expression of inflammatory
\nmediators particularly in H5N3 virus could impose threats to human health in the future.
\nThe IHFs identified by the IAV genome-wide siRNA screening studies could be used as
\npotential anti-IV targets. However, these studies were not consistent (reproducible) mainly due to
\nfalse negative results. Hence, in the fourth project (chapter 6), we applied computational gene
\nnetwork growing for discovering gene network links that could have been missed by these
\nexperimental investigations. Using the known IHFs we compared the network growing function
\nof two free tools GeneMANIA and STRING and the commercial IPA for their performance of
\nrecovering other known IHFs previously identified from siRNA screens. The result showed that,
\ngiven small (~30 genes) or medium (~150 genes) input sets, all three network growing tools
\ndetected significantly more known host factors than random human genes with STRING overall
\nperforming strongest. Extending the networks with all the three tools significantly improved the
\ndetection of GO biological processes of known host factors compared to not growing networks.
\nNotably, the rate of identification of true host factors using computational network growing is
\nequal or better to doing another experimental siRNA screening study which could also be true
\nand applied to other biological pathways/processes.
\nFinally, using the known IHFs and the "new IHF candidates" (genes connected to the
\nIHFs from the network growing analysis), we predicted computational drug-target interactions
\nusing MetaCore. We identified 343 US Food and Drug Administration (FDA) approved drugs
\nthat had an inhibitory effect on either the known or new candidate IHFs, of which 258 were new
\npredictions. Furthermore, using different criteria, we computationally ranked the 343 FDA
\napproved drugs for further experimental validation.

Caspases and poly (ADP-ribose) polymerase 1 (PARP1) have been shown to promote influenza A virus (IAV) replication. However, the relative importance and molecular mechanisms of specific caspases and their downstream substrate PARP1 in regulating viral replication in airway epithelial cells (AECs) remains incompletely elucidated. Here, we targeted caspase 2, 3, 6, and PARP1 using specific inhibitors to compare their role in promoting IAV replication. Inhibition of each of these proteins caused significant decline in viral titer, although PARP1 inhibitor led to the most robust reduction of viral replication. We previously showed that the pro-apoptotic protein Bcl-2 interacting killer (Bik) promotes IAV replication in the AECs by activating caspase 3. In this study, we found that as compared with AECs from wild-type mice, bik-deficiency alone resulted in ~ 3 logs reduction in virus titer in the absence of treatment with the pan-caspase inhibitor (Q-VD-Oph). Inhibiting overall caspase activity using Q-VD-Oph caused additional decline in viral titer by ~ 1 log in bik-/- AECs. Similarly, mice treated with Q-VD-Oph were protected from IAV-induced lung inflammation and lethality. Inhibiting caspase activity diminished nucleo-cytoplasmic transport of viral nucleoprotein (NP) and cleavage of viral hemagglutinin and NP in human AECs. These findings suggest that caspases and PARP1 play major roles to independently promote IAV replication and that additional mechanism(s) independent of caspases and PARP1 may be involved in Bik-mediated IAV replication. Further, peptides or inhibitors that target and block multiple caspases or PARP1 may be effective treatment targets for influenza infection.

Objective To investigate the antiviral effects of antisense phosphorothioate oligodeoxynucleotide (ASODN)in 9HTEO infected with influenza A virus (IFAV) in vitro. Methods The ASODN which complemented to genomic PB1, M2, NS, PB2, HA mRNA of IFAV was used to investigate antiviral effection in vitro. The cytopathic effect (CPE) was observed, and the cell survival rates were measured by MTF assay, plaque assay, RT-PCR, Western blot and Immunofluorescence were performed to test anti-viral efficiency of PB1, M2, NS in cells mRNA and protein level. Results 9HTEO cells infected with IFAV almost all died when the multipicity of infection (MOI) is aboved after 5 days cell culture. The ASODN could increase the cell survival rates. The IFAV PB1, M2, NS significantly reduced CPE of IFAV infected 9HTEO cells, reduced the viral replication of IFAV in the cells (P <0.05). Conclusion The ASODN which targeted the mRNA of IFAV gene showed a significant and specific anti-IFAV effect both in mRNA and protein level in cells culture system. The study indicates that the PB1, M2 and NS mRNA may play an important role in regulating IFAV replication, and ASODN may have inhibitory activity on IFAV replication. The results established the basis for further study on new drugs against IFAV infection include the highly pathogenic H5N1 influenza virus. Key words: Influenza A virus; Antisense oligodeoxynucleotide; 9HTEO cell; Virus replication

ABSTRACTThe M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research.IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Influenza A viruses (IAVs) rely on viral ribonucleoprotein (vRNP) complexes to control transcription and replication. Each vRNP consists of one viral genomic RNA segment associated with multiple nucleoproteins (NP) and a trimeric IAV RNA polymerase complex. Previous studies showed that post-translational modifications of vRNP components, such as NP, by host factors would in turn affect the IAV life cycle or modulate host anti-viral response. In this study, we found host E3 ubiquitin ligase Pirh2 interacted with NP and mediated short-chain ubiquitination of NP at lysine 351, which suppressed NP-PB2 interaction and vRNP formation. In addition, we showed that knockdown of Pirh2 promoted IAV replication, whereas overexpression of Pirh2 inhibited IAV replication. However, Pirh2-ΔRING lacking E3 ligase activity failed to inhibit IAV infection. Moreover, we showed that Pirh2 had no effect on the replication of a rescued virus, WSN-K351R, carrying lysine-to-arginine substitution at residue 351. Interestingly, by analyzing human and avian IAVs from 2011 to 2020 in influenza research databases, we found that 99.18% of 26 977 human IAVs encode lysine, but 95.3% of 9956 avian IAVs encode arginine at residue 351 of NP protein. Consistently, knockdown of Pirh2 failed to promote propagation of two avian-like influenza viruses, H9N2-W1 and H9N2-C1, which naturally encode arginine at residue 351 of NP. Taken together, we demonstrated that Pirh2 is a host factor restricting IAV infection by modulating short-chain ubiquitination of NP. Meanwhile, it is noteworthy that residue 351 of NP targeted by Pirh2 may associate with the evasion of human anti-viral response against avian-like influenza viruses.

Infection of influenza A virus (IAV) can trigger exaggerated pulmonary inflammation and induce acute lung injury (ALI). Limiting IAV replication and alleviation of pulmonary inflammation are two important therapeutic strategies for influenza virus infection. Recent studies have shown that hypoxia inducible factor-1α (HIF-1α) is an essential factor for the development and repair of ALI; however, the role and the underlying mechanisms of HIF-1α in IAV-induced ALI remain elusive. Here, we demonstrated that lung epithelial cell-specific Hif1α knockout mice infected with IAV developed more lung IAV replication and severe lung inflammation, which led to increased mortality compared to IAV-infected control mice. Moreover, knockdown of HIF1A in A549 cells (human alveolar type II epithelial cell line) promoted IAV replication in vitro. Mechanistically, knockdown of HIF1A reduced glycolysis by regulating transcription of glycolysis-related enzymes, which subsequently activated the AMPKα-ULK1 signalling pathway. Interestingly, AMPKα-ULK1 signalling promoted autophagy and augmented IAV replication. Taken together, deficiency of HIF-1α in lung epithelial cells reduces glycolysis and enhances AMPKα-ULK1-mediated autophagy, which finally facilitates IAV replication. These findings have deepened our understanding of the role of HIF-1α in regulating IAV replication and provided us novel therapeutic targets for combating influenza infection.

Long noncoding RNA #61 exerts a broad anti-influenza a virus effect by its long arm rings

Influenza A viruses (IAVs) significantly impact animal and human health due to their zoonotic potential. A growing body of evidence indicates that the host’s long noncoding RNAs (lncRNAs) play crucial roles in regulating host–virus interactions during IAV infection. However, numerous lncRNAs associated with IAV infection have not been well characterised. Here, in this study, we identify the LINC01197 as an antiviral host factor. LINC01197 was significantly upregulated after IAV infection, which is controlled by the NF-κB pathway. Functional analysis revealed that overexpression of LINC01197 inhibited IAV replication and virus production, while knockdown of LINC01197 facilitated IAV replication. Mechanistically, LINC01197 directly interacts with poly(A) binding protein cytoplasmic 1 (PABPC1), which in turn sequesters and restricts its functions. This work shows that LINC01197 acts as a protein decoy, suppressing IAV replication and playing a key role in controlling IAV replication.

The Influenza A virus belongs to the Orthomyxoviridae family. Influenza virus infection occurs yearly in all countries of the world. It usually kills between 250,000 and 500,000 people and causes severe illness in millions more. Over the last century alone we have seen 3 global influenza pandemics. The great human and financial cost of this disease has made it the second most studied virus today, behind HIV. Recently, several genome-wide RNA interference studies have focused on identifying host molecules that participate in Influenza infection. We used nine of these studies for this meta-analysis. Even though the overlap among genes identified in multiple screens was small, network analysis indicates that similar protein complexes and biological functions of the host were present. As a result, several host gene complexes important for the Influenza virus life cycle were identified. The biological function and the relevance of each identified protein complex in the Influenza virus life cycle is further detailed in this paper.

Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication.

Influenza virus induces apoptosis in infected cells to promote viral replication by manipulating the host cell death signaling pathway. Although some Bcl-2 family proteins play a role in the replication of influenza A virus (IAV), the role of cell death pathways in the viral replication cycle is unclear. We investigated whether deficiency of the proapoptotic Bcl-2 family protein, Bik, plays a role in IAV replication. IAV replication was attenuated in mouse airway epithelial cells (MAECs) from bik(-/-) compared with bik(+/+) mice, as indicated by reduced viral titers. Bik(-/-) MAECs showed more stable transepithelial resistance after infection than did bik(+/+) MAECs, were less sensitive to infection-induced cell death, and released fewer copies of viral RNA. Similar results were obtained when Bik expression was suppressed in human airway epithelial cells (HAECs). Bik(+/+) mice lost weight drastically and died within 8 days of infection, whereas 75% of bik(-/-) mice survived infection for 14 days and were 10-fold less likely to die from infection compared with bik(+/+) mice. IAV infection activated caspase 3 in bik(+/+) but not in bik(-/-) MAECs. Cleavage of viral nucleoprotein and M2 proteins were inhibited in bik(-/-) MAECs and when caspase activation was inhibited in HAECs. Furthermore, Bik deficiency impaired cytoplasmic export of viral ribonucleoprotein. These studies suggest a link between Bik-mediated caspase activation and cleavage of viral proteins. Thus, inhibition of proapoptotic host factors such as Bik and downstream mediators of cell death may represent a novel approach to influenza treatment.