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.     

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.     

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.     

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.     

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.     

Molecular interactions between mitochondrial membrane proteins and the C-terminal domain of PB1-F2: an in silico approach

PB1-F2 is a recently described influenza A viral protein that induces apoptosis by binding with two mitochondrial membrane proteins, i.e. VDAC1 (outer membrane) and ANT3 (inner membrane). Knowledge of this binding mechanism could provide insights that would aid in the design of novel inhibitors against this protein. Therefore, to better understand these interactions, we have undertaken this study to model the PB1-F2 protein of the highly pathogenic influenza A virus subtype H5N1. Moreover, a model of human ANT3 was also established. The dynamics of the molecular interactions between the C-terminal region of PB1-F2 protein and VDAC1 and ANT3 were expounded by employing an in silico approach. Our results suggest the involvement of 12 amino acids of PB1-F2 protein, which form hydrophobic contacts with 22 amino acids of VDAC1. Of these, Leu64, Arg75 and Val76 were found to be crucial for mitochondrial targetting. In the case of the PB1-F2-ANT3 complex, 14 amino acids of ANT3 were found to make hydrophobic contacts with 9 amino acids of PB1-F2. Furthermore, two hydrogen bonds were predicted in both complexes PB1-F2/VDAC1 and PB1-F2/ANT3. This study reveals the molecular interactions required for PB1-F2-induced apoptosis and suggests a hypothetical model for future study.     

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.     

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.     

Mitochondrial targeting sequence of the influenza A virus PB1-F2 protein and its function in mitochondria

The influenza A virus PB1-F2 protein predominantly localizes in the mitochondria of virus-infected cells. A series of cDNAs encoding N- and C-terminal deletion mutants and site-directed mutagenesis of the basic residues of PB1-F2 appended to 3xFLAG revealed the domain from residues 46 to 75 to be both necessary and sufficient for mitochondrial targeting. In addition, the subdomain residues 63-75 and both Lys73 and Arg75 are minimally required for mitochondrial localization. Transfection of untagged- and 3xFLAG tagged-PB1-F2 into Vero, HeLa and MDCK cells changed the mitochondrial morphology from a filamentous to a dotted structure and suppressed the inner-membrane potential.     

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.     

Molecular basis for mitochondrial localization of viral particles during beet necrotic yellow vein virus infection

During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.     

Influenza A Virus PB1-F2 Protein Expression Is Regulated in a Strain-Specific Manner by Sequences Located Downstream of the PB1-F2 Initiation Codon

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.     

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.     

A cell death assay for assessing the mitochondrial targeting of proteins

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Von Willebrand factor A domain-containing protein 8 (VWA8) localizes to the matrix side of the inner mitochondrial membrane

VWA8 is a poorly characterized mitochondrial AAA + ATPase protein. The specific submitochondrial localization of VWA8 remains unclear. The purpose of this study was to determine the specific submitochondrial compartment within which VWA8 resides in order to provide more insight into the function of this protein. Bioinformatics analysis showed that VWA8 has a 34 amino acid N-terminal Matrix-Targeting Signal (MTS) that is similar to those in proteins known to localize to the mitochondrial matrix. Experiments in C2C12 mouse myoblasts using confocal microscopy showed that deletion of the VWA8 MTS (vMTS) resulted in cytosolic, rather than mitochondrial, localization of VWA8. Biochemical analysis using differential sub-fractionation of mitochondria isolated from rat liver showed that VWA8 localizes to the matrix side of inner mitochondrial membrane, similar to the inner mitochondrial membrane protein Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETFDH). The results of these experiments show that the vMTS is essential for localization to the mitochondrial matrix and that once there, VWA8 localizes to the matrix side of inner mitochondrial membrane.     

The N-terminal helix of Bcl-xL targets mitochondria

Anti- and pro-apoptotic Bcl-2 family members regulate the mitochondrial phase of apoptotic cell death. The mitochondrial targeting mechanisms of Bcl-2 family proteins are tightly regulated. Known outer mitochondrial membrane targeting sequences include the C-terminal tail and central helical hairpin. Bcl-x_L also localizes to the inner mitochondrial membrane, but these targeting sequences are unknown. Here we investigate the possibility that the N-terminus of Bcl-x_L also contains mitochondrial targeting information. Amino acid residues 1–28 of Bcl-x_L fused to EGFP are sufficient to target mitochondria. Although positive charges and helical propensity are required for targeting, similar to import sequences the N-terminus is not sufficient for efficient mitochondrial import.     

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.     

Non-Avian Animal Reservoirs Present a Source of Influenza A PB1-F2 Proteins with Novel Virulence-Enhancing Markers

PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses'' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.