Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1

The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC) inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.     

The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function

The 11th influenza A virus gene product is an 87-amino-acid protein provisionally named PB1-F2 (because it is encoded by an open reading frame overlapping the PB1 open reading frame). A significant fraction of PB1-F2 localizes to the inner mitochondrial membrane in influenza A virus-infected cells. PB1-F2 appears to enhance virus-induced cell death in a cell type-dependent manner. For the present communication we have identified and characterized a region near the COOH terminus of PB1-F2 that is necessary and sufficient for its inner mitochondrial membrane localization, as determined by transient expression of chimeric proteins consisting of elements of PB1-F2 genetically fused to enhanced green fluorescent protein (EGFP) in HeLa cells. Targeting of EGFP to mitochondria by this sequence resulted in the loss of the inner mitochondrial membrane potential, leading to cell death. The mitochondrial targeting sequence (MTS) is predicted to form a positively charged amphipathic alpha-helix and, as such, is similar to the MTS of the p13(II) protein of human T-cell leukemia virus type 1. We formally demonstrate the functional interchangeability of the two sequences for mitochondrial localization of PB1-F2. Mutation analysis of the putative amphipathic helix in the PB1-F2 reveals that replacement of five basic amino acids with Ala abolishes mitochondrial targeting, whereas mutation of two highly conserved Leu to Ala does not. These findings demonstrate that PB1-F2 possesses an MTS similar to other viral proteins and that this MTS, when fused to EGFP, is capable of independently compromising mitochondrial function and cellular viability.     

Amyloid Assemblies of Influenza A Virus PB1-F2 Protein Damage Membrane and Induce Cytotoxicity

PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria-mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90 amino acids), its N-terminal domain (52 amino acids), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38 amino acids). Both full-length and N-terminal domain of PB1-F2 are soluble at pH values ≤6, whereas the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥ 7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers, whereas full-length and the N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and the C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nanomolar concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, whereas PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligomers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein.     

Structure-based analysis of VDAC1 protein: defining oligomer contact sites

The outer mitochondrial membrane protein, the voltage-dependent anion channel (VDAC), is increasingly implicated in the control of apoptosis. Oligomeric assembly of VDAC1 was shown to be coupled to apoptosis induction, with oligomerization increasing substantially upon apoptosis induction and inhibited by apoptosis blockers. In this study, structure- and computation-based selection of the predicated VDAC1 dimerization site, in combination with site-directed mutagenesis, cysteine replacement, and chemical cross-linking, were employed to identify contact sites between VDAC1 molecules in dimers and higher oligomers. The predicted weakly stable β-strands were experimentally found to represent the interfaces between VDAC1 monomers composing the oligomer. Replacing hydrophobic amino acids with charged residues in β-strands 1, 2, and 19 interfered with VDAC1 oligomerization. The proximity of β-strands 1, 2, and 19 within the VDAC1 dimer and the existence of other association sites involving β-strand 16 were confirmed when a cysteine was introduced at defined positions in cysteineless VDAC1 mutants, together with the use of cysteine-specific cross-linker bis(maleimido)ethane. Moreover, the results suggest that VDAC1 also exists as a dimer that upon apoptosis induction undergoes conformational changes and that its oligomerization proceeds through a series of interactions involving two distinct interfaces. Dissection of VDAC1 dimerization/oligomerization as presented here provides structural insight into the oligomeric status of cellular VDAC1 under physiological and apoptotic conditions.     

A novel influenza A virus mitochondrial protein that induces cell death.

While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.     

A Novel Cytotoxic Sequence Contributes to Influenza A Viral Protein PB1-F2 Pathogenicity and Predisposition to Secondary Bacterial Infection

Enhancement of cell death is a distinguishing feature of H1N1 influenza virus A/Puerto Rico/8/34 protein PB1-F2. Comparing the sequences (amino acids [aa] 61 to 87 using PB1-F2 amino acid numbering) of the PB1-F2-derived C-terminal peptides from influenza A viruses inducing high or low levels of cell death, we identified a unique I68, L69, and V70 motif in A/Puerto Rico/8/34 PB1-F2 responsible for promotion of the peptide''s cytotoxicity and permeabilization of the mitochondrial membrane. When administered to mice, a 27-mer PB1-F2-derived C-terminal peptide with this amino acid motif caused significantly greater weight loss and pulmonary inflammation than the peptide without it (due to I68T, L69Q, and V70G mutations). Similar to the wild-type peptide, A/Puerto Rico/8/34 elicited significantly higher levels of macrophages, neutrophils, and cytokines in the bronchoalveolar lavage fluid of mice than its mutant counterpart 7 days after infection. Additionally, infection of mice with A/Puerto Rico/8/34 significantly enhanced the levels of morphologically transformed epithelial and immune mononuclear cells recruited in the airways compared with the mutant virus. In the mouse bacterial superinfection model, both peptide and virus with the I68, L69, and V70 sequence accelerated development of pneumococcal pneumonia, as reflected by increased levels of viral and bacterial lung titers and by greater mortality. Here we provide evidence suggesting that the newly identified cytotoxic sequence I68, L69, and V70 of A/Puerto Rico/8/34 PB1-F2 contributes to the pathogenesis of both primary viral and secondary bacterial infections.     

A型流感病毒PB1-F2蛋白与MOAP-1的相互作用

【目的】探讨A型流感病毒PB1-F2蛋白和人类凋亡调节因子1(MOAP-1)之间的相互作用。【方法】构建pACT2-MOAP-1重组质粒,与pGBKT7-PB1-F2质粒共转化酵母AH109,检测转化菌在四缺培养基的生长情况及β半乳糖苷酶报告基因的活性;利用GST pull-down和免疫共沉淀(Co-IP)技术进一步验证PB1-F2与宿主细胞蛋白MOAP-1的相互作用;通过过表达PB1-F2和MOAP-1,检测PB1-F2对MOAP-1蛋白表达水平的影响。【结果】酵母双杂交结果表明,PB1-F2和MOAP-1可以在酵母细胞内特异性结合。GST pull-down和Co-IP实验也进一步证实了这两种蛋白的相互作用,而且PB1-F2可上调外源MOAP-1的蛋白水平。【结论】流感病毒PB1-F2与MOAP-1存在相互作用,PB1-F2可能通过与MOAP-1的相互作用参与调控细胞生长及凋亡过程。     

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.     

Ethanol influences on bax associations with mitochondrial membrane proteins in neonatal rat cerebellum

These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure and focused on interactions between proapoptotic Bax and proteins of the permeability transition pore (PTP), voltage‐dependent anion channel (VDAC) and adenine nucleotide translocator (ANT) of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that, following ethanol exposure, Bax proapoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least 2 h postexposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment and found such interactions were altered by ethanol treatment, but only at 2 h postexposure and only in the P4, ethanol‐sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax but not by a Bax channel blocker. Therefore, we conclude that, at this age, within the constraints of our experimental model, a primary mode of Bax‐induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex and not channel formation independent of PTP constituents. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes

A frameshifted region of the influenza A virus PB1 gene encodes a novel protein, termed PB1-F2, a mitochondrial protein that can induce cell death. Many proapoptotic proteins are believed to act at the mitochondrial outer membrane to form an apoptotic pore with lipids. We studied the interaction of isolated, synthetic PB1-F2 (sPB1-F2) peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing solution induced a transmembrane conductance that increased in a potential-dependent manner. Positive potential on the side of protein addition resulted in a severalfold increase in the rate of change of membrane conductance. sPB1-F2-treated membranes became permeable to monovalent cations, chloride, and to a lesser extent, divalent ions. Despite various experimental conditions, we did not detect the distinctive conductance levels typical of large, stable pores, protein channels, or even pores that are partially proteinaceous. Rather, membrane conductance induced by sPB1-F2 fluctuated and visited almost all conductance values. sPB1-F2 also dramatically decreased bilayer stability in an electric field, consistent with a decrease in the line tension of a lipidic pore. Since similar membrane-destabilizing profiles are seen with proapoptotic proteins (e.g., Bax) and the cytoplasmic helix of human immunodeficiency virus gp41, we suggest that the basis for sPB1-F2-induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis.     

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.     

The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein

PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-β reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-β reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses.     

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.     

New EMBO members' review: viral and bacterial proteins regulating apoptosis at the mitochondrial level

Mitochondrial membrane permeabilization (MMP) is a critical step of several apoptotic pathways. Some infectious intracellular pathogens can regulate (induce or inhibit) apoptosis of their host cells at the mitochondrial level, by targeting proteins to mitochondrial membranes that either induce or inhibit MMP. Pathogen-encoded mitochondrion-targeted proteins may or may not show amino acid sequence homology to Bcl-2-like proteins. Among the Bcl-2-unrelated, mitochondrion-targeted proteins, several interact with the voltage-dependent anion channel (VDAC) or with the adenine nucleotide translocator (ANT). While VDAC-targeted proteins show homology to VDAC/porin, ANT-targeted proteins possess relatively short cationic binding domains, which may facilitate insertion into the negatively charged inner mitochondrial membrane. It may be speculated that such proteins employ pre-existing host-intrinsic mechanisms of MMP control.     

Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential

PB1-F2 is a small, 87- to 90-amino-acid-long protein encoded by the +1 alternate open reading frame of the PB1 gene of most influenza A virus strains. It has been shown to contribute to viral pathogenicity in a host- and strain-dependent manner, and we have previously discovered that a serine at position 66 (66S) in the PB1-F2 protein increases virulence of the 1918 and H5N1 pandemic viruses. Recently, we have shown that PB1-F2 inhibits the induction of type I interferon (IFN) at the level of the MAVS adaptor protein. However, the molecular mechanism for the IFN antagonist function of PB1-F2 has remained unclear. In the present study, we demonstrated that the C-terminal portion of the PB1-F2 protein binds to MAVS in a region that contains the transmembrane domain. Strikingly, PB1-F2 66S was observed to bind to MAVS more efficiently than PB1-F2 66N. We also tested the effect of PB1-F2 on the IFN antagonist functions of the polymerase proteins PB1, PB2, and PA and observed enhanced IFN inhibition by the PB1 and PB2 proteins in combination with PB1-F2 but not by the PA protein. Using a flow cytometry-based assay, we demonstrate that the PB1-F2 protein inhibits MAVS-mediated IFN synthesis by decreasing the mitochondrial membrane potential (MMP). Interestingly, PB1-F2 66S affected the MMP more efficiently than wild-type PB1-F2. In summary, the results of our study identify the molecular mechanism by which the influenza virus PB1-F2 N66S protein increases virulence.     

Differential Localization and Function of PB1-F2 Derived from Different Strains of Influenza A Virus

PB1-F2 is a viral protein that is encoded by the PB1 gene of influenza A virus by alternative translation. It varies in length and sequence context among different strains. The present study examines the functions of PB1-F2 proteins derived from various human and avian viruses. While H1N1 PB1-F2 was found to target mitochondria and enhance apoptosis, H5N1 PB1-F2, surprisingly, did not localize specifically to mitochondria and displayed no ability to enhance apoptosis. Introducing Leu into positions 69 (Q69L) and 75 (H75L) in the C terminus of H5N1 PB1-F2 drove 40.7% of the protein to localize to mitochondria compared with the level of mitochondrial localization of wild-type H5N1 PB1-F2, suggesting that a Leu-rich sequence in the C terminus is important for targeting of mitochondria. However, H5N1 PB1-F2 contributes to viral RNP activity, which is responsible for viral RNA replication. Lastly, although the swine-origin influenza virus (S-OIV) contained a truncated form of PB1-F2 (12 amino acids [aa]), potential mutation in the future may enable it to contain a full-length product. Therefore, the functions of this putative S-OIV PB1-F2 (87 aa) were also investigated. Although this PB1-F2 from the mutated S-OIV shares only 54% amino acid sequence identity with that of seasonal H1N1 virus, it also increased viral RNP activity. The plaque size and growth curve of the viruses with and without S-OIV PB1-F2 differed greatly. The PB1-F2 protein has various lengths, amino acid sequences, cellular localizations, and functions in different strains, which result in strain-specific pathogenicity. Such genetic and functional diversities make it flexible and adaptable in maintaining the optimal replication efficiency and virulence for various strains of influenza A virus.Influenza A viruses contain eight negative-stranded RNA segments that encode 11 known viral proteins. The 11th viral protein was originally found in a search for unknown peptides during influenza A virus infection recognized by CD8⁺ T cells. It was termed PB1-F2 and is the second protein that is alternatively translated by the same PB1 gene (8). PB1-F2 can be encoded in a large number of influenza A viruses that are isolated from various hosts, including human and avian hosts. The size of PB1-F2 ranges from 57 to 101 amino acids (aa) (41). While strain PR8 (H1N1) contains a PB1-F2 with a length of 87 aa, PB1-F2 is terminated at amino acid position 57 in most human H1N1 viruses and is thus a truncated form compared with the length in PR8. Human H3N2 and most avian influenza A viruses encode a full-length PB1-F2 protein, which is at least 87 aa (7). Many cellular functions of the PB1-F2 protein, and especially the protein of the PR8 strain, have been reported (11, 25). For example, PR8 PB1-F2 localizes to mitochondria in infected and transfected cells (8, 15, 38, 39), suggesting that PB1-F2 enhances influenza A virus-mediated apoptosis in human monocytes (8). The phosphorylation of the PR8 PB1-F2 protein has been suggested to be one of the crucial causes of the promotion of apoptosis (30).The rates of synonymous and nonsynonymous substitutions in the PB1-F2 gene are higher than those in the PB1 gene (7, 20, 21, 37, 42). Recent work has shown that both PR8 PB1-F2 and H5N1 PB1-F2 are important regulators of influenza A virus virulence (1). Additionally, the expression of the 1918 influenza A virus (H1N1) PB1-F2 increases the incidence of secondary bacterial pneumonia (10, 28). However, PB1-F2 is not essential for viral replication because the knockout of PB1-F2 in strain PR8 has no effect on the viral titer (40), suggesting that PB1-F2 may have cellular functions other than those that were originally thought (29).PB1-F2 was translated from the same RNA segment as the PB1 protein, whose function is strongly related to virus RNP activity, which is responsible for RNA chain elongation and which exhibits RNA-dependent RNA polymerase activity (2, 5) and endonuclease activity (9, 16, 26). Previous research has already proved that the knockout of PR8 PB1-F2 reduced virus RNP activity, revealing that PR8 PB1-F2 contributes to virus RNP activity (27), even though PB1-F2 has no effect on the virus growth rate (40). In the present study, not only PR8 PB1-F2 but also H5N1 PB1-F2 and putative full-length swine-origin influenza A virus (S-OIV) PB1-F2 contributed to virus RNP activity. However, PR8 PB1-F2 and H5N1 PB1-F2 exhibit different biological behaviors, including different levels of expression, cellular localizations, and apoptosis enhancements. The molecular determinants of the different localizations were also addressed. The function of the putative PB1-F2 derived from S-OIV was also studied. The investigation described here reveals that PB1-F2 proteins derived from various viral strains exhibited distinct functions, possibly contributing to the variation in the virulence of influenza A viruses.     

Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs

In plants, the voltage-dependent anion-selective channel (VDAC) is a major component of a pathway involved in transfer RNA (tRNA) translocation through the mitochondrial outer membrane. However, the way in which VDAC proteins interact with tRNAs is still unknown. Potato mitochondria contain two major mitochondrial VDAC proteins, VDAC34 and VDAC36. These two proteins, composed of a N-terminal α-helix and of 19 β-strands forming a β-barrel structure, share 75% sequence identity. Here, using both northwestern and gel shift experiments, we report that these two proteins interact differentially with nucleic acids. VDAC34 binds more efficiently with tRNAs or other nucleic acids than VDAC36. To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern. This allowed us to show that the β-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding. Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.     

广州地区新型H1N1流感病毒PB1-F2基因进化和变异分析

比较和分析2009～2011年广州地区分离到的甲型H1N1流感病毒PB1-F2基因和世界各地甲型H1N1流感病毒PB1-F2基因的变异情况,为该蛋白的功能和作用机制奠定基础。对分离自中国广州地区2009～2011年人类感染的17株新型H1N1和1株季节性H1N1流感病毒进行了PB1-F2基因克隆和序列测定,通过与GenBank数据库中68株人类新型H1N1和季节性H1N1流感病毒参考株的PB1-F2基因进行比对。结果表明,甲型流感病毒的PB1-F2基因进化树形成了2个不同的进化分支。全部2009～2011年新型H1N1流感病毒为一分支。广州地区PB1-F2基因与其它地区分离到的新型H1N1流感病毒具有高度的同源性,均为截短型变异。本实验室分离的1株季节性H1N1流感病毒也发生了第12位氨基酸截短突变。广州地区新型H1N1流感病毒PB1-F2截短蛋白与其它地区病毒相比未发生氨基酸变异,季节性H1N1流感病毒发现类似新型H1N1流感病毒PB1-F2的截短变异,提示新型H1N1流感病毒和季节性H1N1流感病毒PB1-F2可能发生早期重组。     

Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice

The influenza virus PB1-F2 protein is a novel protein previously shown to be involved in induction of cell death. Here we characterize the expression and the function of the protein within the context of influenza viral infection in tissue culture and a mouse model. We show that the C-terminal region of the protein can be expressed from a downstream initiation codon and is capable of interaction with the full-length protein. Using this knowledge, we generated influenza viruses knocked out for the expression of PB1-F2 protein and its downstream truncation products. Knocking out the PB1-F2 protein had no effect on viral replication in tissue culture but diminished virus pathogenicity and mortality in mice. The viruses replicated to similar levels in mouse lungs by day 3 postinfection, suggesting that the knockout did not impair viral replication. However, while the PB1-F2 knockout viruses were cleared after day 5, the wild-type viruses were detectable in mouse lungs until day 7, implying that expression of PB1-F2 resulted in delayed clearance of the viruses by the host immune system. Based on our findings and on the fact that the PB1 genomic segment was always newly introduced into some pandemic influenza viruses of the last century, we speculate that the PB1-F2 protein plays an important role in pathogenesis of influenza virus infection and may be an important contributor to pathogenicity of pandemic influenza viruses.