Influenza A virus protein PB1-F2 from different strains shows distinct structural signatures.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian influenza isolates. The structures of full-length PB1-F2 of the influenza strains Pandemic flu 2009 H1N1, 1918 Spanish flu H1N1, Bird flu H5N1 and H1N1 PR8, have been characterized by NMR and CD spectroscopy. The study was conducted using chemically synthesized full-length PB1-F2 protein and fragments thereof. The amino acid residues 30-70 of PR8 PB1-F2 were found to be responsible for amyloid formation of the protein, which could be assigned to formation of ß-sheet structures, although α-helices were the only structural features detected under conditions that mimic a membranous environment. At membranous conditions, in which the proteins are found in their most structured state, significant differences become apparent between the PB1-F2 variants investigated. In contrast to Pandemic flu 2009 H1N1 and PR8 PB1-F2, which exhibit a continuous extensive C-terminal α-helix, both Spanish flu H1N1 and Bird flu H5N1 PB1-F2 contain a loop region with residues 66-71 that divides the C-terminus into two shorter helices. The observed structural differences are located to the C-terminal ends of the proteins to which most of the known functions of these proteins have been assigned. A C-terminal helix-loop-helix motif might be a structural signature for PB1-F2 of the highly pathogenic influenza viruses as observed for 1918 Spanish flu H1N1 and Bird flu H5N1 PB1-F2. This signature could indicate the pathological nature of viruses emerging in the future and thus aid in the recognition of these viruses.

The intriguing cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding.

BACKGROUND: Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined. RESULTS: Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding. CONCLUSIONS: Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP35RIW motif of N-terminal Vpr.

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 PKCalpha interacts with PB1-F2 in yeast two-hybrid assays. (32)P 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.

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.

Antiviral activity of the proteasome inhibitor VL-01 against influenza A viruses.

The appearance of highly pathogenic avian influenza A viruses of the H5N1 subtype being able to infect humans and the 2009 H1N1 pandemic reveals the urgent need for new and efficient countermeasures against these viruses. The long-term efficacy of current antivirals is often limited, because of the emergence of drug-resistant virus mutants. A growing understanding of the virus-host interaction raises the possibility to explore alternative targets involved in the viral replication. In the present study we show that the proteasome inhibitor VL-01 leads to reduction of influenza virus replication in human lung adenocarcinoma epithelial cells (A549) as demonstrated with three different influenza virus strains, A/Puerto Rico/8/34 (H1N1) (EC50 value of 1.7 µM), A/Regensburg/D6/09 (H1N1v) (EC50 value of 2.4 µM) and A/Mallard/Bavaria/1/2006 (H5N1) (EC50 value of 0.8 µM). In in vivo experiments we could demonstrate that VL-01-aerosol-treatment of BALB/c mice with 14.1 mg/kg results in no toxic side effects, reduced progeny virus titers in the lung (1.1 ± 0.3 log10 pfu) and enhanced survival of mice after infection with a 5-fold MLD50 of the human influenza A virus strain A/Puerto Rico/8/34 (H1N1) up to 50%. Furthermore, treatment of mice with VL-01 reduced the cytokine release of IL-α/ß, IL-6, MIP-1ß, RANTES and TNF-α induced by LPS or highly pathogen avian H5N1 influenza A virus. The present data demonstrates an antiviral effect of VL-01 in vitro and in vivo and the ability to reduce influenza virus induced cytokines and chemokines.

The proapoptotic influenza A virus protein PB1-F2 forms a nonselective ion channel.

BACKGROUND: PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1) (IAV(PR8)), from A/Brevig Mission/1/1918(H1N1) (IAV(SF2)) or the H5N1 consensus sequence (IAV(BF2)) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. CONCLUSION/SIGNIFICANCE: The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development.

Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein.

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.