Origin of the 1918 pandemic H1N1 influenza A virus as studied by codon usage patterns and phylogenetic analysis

The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.     

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

The 11^th 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.     

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

The 11^th 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 IAV_PR8 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 IAV_PR8 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.     

Type I interferon antagonistic properties of influenza B virus polymerase proteins

The innate immune system, in particular the type I interferon (IFN) response, is a powerful defence against virus infections. In turn, many if not all viruses have evolved various means to circumvent, resist, or counteract this host response to ensure efficient replication and propagation. Influenza viruses are no exception to this rule, and several viral proteins have been described to possess IFN‐antagonistic functions. Although the viral nonstructural protein 1 appears to be a major antagonist in influenza A and B viruses (IAV and IBV), we have previously shown that a specific motif in the IAV polymerase proteins exerts an IFN‐suppressive function very early in infection. The question remained whether a similar function would also exist in IBV polymerases. Here, we show that indeed a specific amino acid position (A523) of the PB1 protein in the IBV polymerase complex confers IFN‐antagonistic properties. Mutation of this position leads to enhanced activation of the IFN‐mediated signalling pathway after infection and subsequent reduction of virus titres. This indicates that inhibition of innate immune responses is a conserved activity shared by polymerase proteins of IAV and IBV.     

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.     

Avian influenza viruses inhibit the major cellular signalling integrator c‐Abl

The non‐structural protein 1 (NS1) of influenza A viruses (IAV) encodes several src homology (SH) binding motifs (bm) (one SH2bm, up to two SH3bm), which mediate interactions with host cell proteins. In contrast to NS1 of human IAV, NS1 of avian strains possess the second SH3bm (SH3(II)bm) consensus sequence. Since our former studies demonstrated an NS1–CRK interaction, mediated by this motif, here, we addressed the regulatory properties of this SH3bm for cellular signalling. Initially, we observed a reduced basal CRK phosphorylation upon infection with avian IAV harbouring an NS1 with an SH3(II)bm in contrast to human IAV. Reduced activity of the tyrosine kinase c‐Abl was identified to be responsible for reduced CRK phosphorylation. Further, binding of NS1 to c‐Abl was determined, and mutational manipulation of the SH3(II)bm illustrated the necessity of this motif for c‐Abl inhibition. Interestingly, Abl kinase inhibition resulted in impaired avian IAV propagation and pathogenicity and mutational analysis linked the pronounced inhibition of c‐Abl to cytopathogenic cell alterations upon avian IAV infections. Taken together, NS1 proteins of avian IAV interfere with the kinase activity of c‐Abl, a major cellular signalling integrator that controls multiple signalling processes and cell fate regulations apparently including IAV infections.