Ludmilla Wixler

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 regulatory subunit of protein kinase CK2 is a specific A‐Raf activator

Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two‐hybrid screen of a mouse‐embryo cDNA library we detected an interaction between A‐Raf and CK2β subunit. This binding was specific, as no interaction between CK2β and B‐Raf or c‐Raf‐1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A‐Raf kinase domain. A‐Raf kinase activity was enhanced 10‐fold upon coexpression with CK2β in Sf9 cells. The α subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf‐isoform‐specific activation and a regulatory role for CK2β independent of the CK2α subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.© 1997 Federation of European Biochemical Societies.

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.

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.

A new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication

Guanylate‐binding proteins (GBPs) belong to the family of large GTPases that are induced in response to interferons. GBPs contain an N‐terminal globular GTPase domain and a C‐terminal α‐helical regulatory domain that are connected by a short middle domain. Antiviral activity against vesicular stomatitis virus and encephalomyocarditis virus has been shown for hGBP‐1; however, no anti‐influenza virus properties for GBPs have been described to date. Here we show that hGBP‐1 and hGBP‐3 possess anti‐influenza viral activity. Furthermore, we have identified a novel splice variant of hGBP‐3, named hGBP‐3ΔC, with a largely modified C‐terminal α‐helical domain. While all three GBP isoforms were up‐regulated on influenza virus infection, hGBP‐3ΔC showed the most prominent antiviral activity in epithelial cells. Mutational analysis of hGBPs revealed that the globular domain is the principal antiviral effector domain, and GTP‐binding, but not hydrolysis, is necessary for antiviral action. Furthermore, we showed that hGBP‐3ΔC strongly represses the activity of the viral polymerase complex, which results in decreased synthesis of viral vRNA, cRNA, mRNA, and viral proteins, as well.—Nordmann, A., Wixler, L., Boergeling, Y., Wixler, V., Ludwig, S. A new splice variant of the human guanylate‐binding protein 3 mediates anti‐influenza activity through inhibition of viral transcription and replication. FASEB J. 26, 1290‐1300 (2012). www.fasebj.org

Monoclonal Antibodies Against the PB1-F2 Protein of H1N1 Influenza A Virus

In this report we describe the generation of a mouse monoclonal antibody (MAb) against the influenza A virus PB1-F2 protein that is derived from a +1 reading frame of the polymerase basic protein (PB1) gene segment. We further present data that the hybridoma subclone F2-6G10 produces antibodies that specifically recognize the PB1-F2 protein of H1N1 influenza virus types only. The antibody can be used for immunodetection of the PB1-F2 protein in ELISA, Western blot, immunoprecipitation, and immunofluorescence assays.