Darisuren Anhlan

The proapoptotic influenza A virus protein PB1‐F2 regulates viral polymerase activity by interaction with the PB1 protein

The 11th influenza A virus protein PB1‐F2 was previously shown to enhance apoptosis in response to cytotoxic stimuli. The 87 amino acid protein that is encoded by an alternative reading frame of the PB1 polymerase gene was described to localize to mitochondria consistent with its proapoptotic function. However, PB1‐F2 is also found diffusely distributed in the cytoplasm and in the nucleus suggesting additional functions of the protein. Here we show that PB1‐F2 colocalizes and directly interacts with the viral PB1 polymerase protein. Lack of PB1‐F2 during infection resulted in an altered localization of PB1 and decreased viral polymerase activity. Consequently, mutant viruses devoid of a functional PB1‐F2 reading frame exhibited a small plaque phenotype. Thus, we have identified a novel function of PB1‐F2 as an indirect regulator of the influenza virus polymerase activity via its interaction with PB1.

The influenza virus PB1-F2 protein has interferon antagonistic activity.

Abstract PB1-F2 is a nonstructural protein of influenza viruses encoded by the PB1 gene segment from a +1 open reading frame. It has been shown that PB1-F2 contributes to viral pathogenicity, although the underlying mechanisms are still unclear. Induction of type I interferon (IFN) and the innate immune response are the first line of defense against viral infection. Here we show that influenza A viruses (IAVs) lacking the PB1-F2 protein induce an enhanced expression of IFN-β and IFN-stimulated genes in infected epithelial cells. Studying molecular mechanisms underlying the PB1-F2-mediated IFN antagonistic activity showed that PB1-F2 interferes with the RIG-I/MAVS protein complex thereby inhibiting the activation of the downstream transcription factor IFN regulatory factor 3. These findings were also reflected in in vivo studies demonstrating that infection with PR8 wild-type (wt) virus resulted in higher lung titers and a more severe onset of disease compared with infection with its PB1-F2-deficient counterpart. Accordingly, a much more pronounced infiltration of lungs with immune cells was detected in mice infected with the PB1-F2 wt virus. In summary, we demonstrate that the PB1-F2 protein of IAVs exhibits a type I IFN-antagonistic function by interfering with the RIG-I/MAVS complex, which contributes to an enhanced pathogenicity in vivo.

Pathogenicity of different PR8 influenza A virus variants in mice is determined by both viral and host factors.

Experimental mouse models were used to compare virulence and reproduction rate of three mouse-adapted variants of the PR8 influenza A virus strain. We observed large differences in pathogenicity in two mouse strains. The PR8M variant was lethal in DBA/2J mice but not in C57BL/6J mice, whereas PR8F and hvPR8 variants were lethal in both mouse strains. High lethality of PR8M in DBA/2J correlated with high viral load at early time points after infection and spread of the virus into alveolar regions. Also, higher viral loads and mortality in mice infected with PR8F resulted in a higher number of infiltrating leukocytes. 3D-protein structure predictions of the HA indicated amino acid sequence alterations which may render the HA cleavage site in PR8F more accessible to host proteases. Infection of C57BL/6J mice with a re-assorted PR8 virus revealed that the HA gene is the main determinant of virulence of the PR8F variant.

Phosphorylation of the influenza A virus protein PB1-F2 by PKC is crucial for apoptosis promoting functions in monocytes.

The 11th influenza A virus (IAV) protein PB1‐F2 is encoded by an alternative reading frame of the PB1 polymerase gene and found in the nucleus, cytosol and at the mitochondria of infected cells, the latter is consistent with experimental evidence for its pro‐apoptotic function. Here, the function of PB1‐F2 as a phosphoprotein was characterized. PB1‐F2 derived from isolate IAVPR8 and synthetic fragments thereof were phosphorylated in vitro by purified protein kinase C (PKC) and cellular extract. Constitutively active PKCα interacts with PB1‐F2 in yeast two‐hybrid assays. 32P radiolabelling of transfected 293T cells revealed that phosphorylation of PB1‐F2 is sensitive to inhibitors of PKC and could be increased by the PKC activator PMA. ESI‐MS analysis and cellular expression of PB1‐F2 mutants identified the positions Ser‐35 as the major and the Thr‐27 as an alternative PKC phosphorylation site. Infection of MDCK cells with recombinant IAVPR8 lacking these PKC sites abrogated phosphorylation of PB1‐F2 in vivo. Furthermore, infection of primary human monocytes with mutant viruses lacking these PB1‐F2 phosphorylation sites resulted in impaired caspase 3 activation and reduced progeny virus titres, indicating that the integrity of the identified phosphorylation sites is crucial for a cell‐specific function of PB1‐F2 during virus replication.

Phosphatidylinositol‐3‐kinase (PI3K) is activated by influenza virus vRNA via the pathogen pattern receptor Rig‐I to promote efficient type I interferon production

The phosphatidylinositol‐3‐kinase (PI3K) was identified to be activated upon influenza A virus (IAV) infection. An early and transient induction of PI3K signalling is caused by viral attachment to cells and promotes virus entry. In later phases of infection the kinase is activated by the viral NS1 protein to prevent premature apoptosis. Besides these virus supporting functions, it was suggested that PI3K signalling is involved in dsRNA and IAV induced antiviral responses by enhancing the activity of interferon regulatory factor‐3 (IRF‐3). However, molecular mechanisms of activation remained obscure. Here we show that accumulation of vRNA in cells infected with influenza A or B viruses results in PI3K activation. Furthermore, expression of the RNA receptors Rig‐I and MDA5 was increased upon stimulation with virion extracted vRNA or IAV infection. Using siRNA approaches, Rig‐I was identified as pathogen receptor necessary for influenza virus vRNA sensing and subsequent PI3K activation in a TRIM25 and MAVS signalling dependent manner. Rig‐I induced PI3K signalling was further shown to be essential for complete IRF‐3 activation and consequently induction of the type I interferon response. These data identify PI3K as factor that is activated as part of the Rig‐I mediated anti‐pathogen response to enhance expression of type I interferons.

Viral suppressors of the RIG-I-mediated interferon response are pre-packaged in influenza virions

The type I interferon (IFN) response represents the first line of defence to invading pathogens. Internalized viral ribonucleoproteins (vRNPs) of negative-strand RNA viruses induce an early IFN response by interacting with retinoic acid inducible gene I (RIG-I) and its recruitment to mitochondria. Here we employ three-dimensional stochastic optical reconstruction microscopy (STORM) to visualize incoming influenza A virus (IAV) vRNPs as helical-like structures associated with mitochondria. Unexpectedly, an early IFN induction in response to vRNPs is not detected. A distinct amino-acid motif in the viral polymerases, PB1/PA, suppresses early IFN induction. Mutation of this motif leads to reduced pathogenicity in vivo, whereas restoration increases it. Evolutionary dynamics in these sequences suggest that completion of the motif, combined with viral reassortment can contribute to pandemic risks. In summary, inhibition of the immediate anti-viral response is ‘pre-packaged’ in IAV in the sequences of vRNP-associated polymerase proteins.

The NF-κB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance.

Ongoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine‐origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti‐influenza drugs. The recently identified virus‐supportive function of the cellular IKK/NF‐κB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF‐κB inhibitor SC75741 as a broad and efficient blocker of IV replication in non‐toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF‐κB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro‐apoptotic factors, subsequent inhibition of caspase activation and block of caspase‐mediated nuclear export of viralribonucleoproteins. SC75741 reduces viral replication and H5N1‐induced IL‐6 and IP‐10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus‐induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741‐derived drugs may serve as broadly non‐toxic anti‐influenza agents.

A single point mutation (Y89F) within the non-structural protein 1 of influenza A viruses limits epithelial cell tropism and virulence in mice.

The nonstructural protein 1 (A/NS1) of influenza A viruses (IAV) harbors several src homology (SH)-binding motifs (bm) that mediate interactions with cellular proteins. In contrast to the sequence variability of the second SH3bm, tyrosine 89, within the SH2bm is a highly conserved residue among IAV strains. This prompted us to evaluate the necessity of this SH2bm for IAV virulence. In an in vivo mouse model, we observed drastic reductions in weight loss, mortality, and virus titers in lung and bronchoalveolar lavage fluid after infection with the mutant virus PR8 A/NS1-Y89F (PR8 Y89F) when compared with wild-type virus (PR8 wt). Concomitantly, we observed decreased inflammation and less severe pathologic changes, reflecting reduced levels of virus titers. At histologic analysis, lungs infected with PR8 wt virus showed widespread destruction of the bronchiolar epithelium, with extensive distribution of virus antigen within tracheal, bronchial, bronchiolar, and alveolar epithelium. In marked contrast, the bronchiolar epithelium after infection with the mutant PR8 Y89F virus was entirely intact, and the severity and extent of viral infection was reduced and strongly restricted to alveoli. These findings demonstrate that change of a single residue of the highly conserved SH2bm within the A/NS1 results in restricted virus spread in mouse lung and strongly reduced virulence, which illustrates the necessity of the SH2bm for IAV-induced pathogenicity.

New Virulence Determinants Contribute to the Enhanced Immune Response and Reduced Virulence of an Influenza A Virus A/PR8/34 Variant

The identification of amino acid motifs responsible for increased virulence and/or transmission of influenza viruses is of enormous importance to predict pathogenicity of upcoming influenza strains. We phenotypically and genotypically compared 2 variants of influenza virus A/PR/8/34 with different passage histories. The analysis revealed differences in virulence due to an altered type I interferon (IFN) induction, as evidenced by experiments using IFNAR(-/-) mice. Interestingly, these differences were not due to altered functions of the well-known viral IFN antagonists NS1 or PB1-F2. Using reassortant viruses, we showed that differences in the polymerase proteins and nucleoprotein determined the altered virulence. In particular, changes in PB1 and PA contributed to an altered host type I IFN response, indicating IFN antagonistic properties of these proteins. Thus, PB1 and PA appear to harbor previously unknown virulence markers, which may prove helpful in assessing the risk potential of emerging influenza viruses.

Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1

ABSTRACT A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection.