Evolution and Virulence of Influenza A Virus Protein PB1-F2

PB1-F2 is an accessory protein of most human, avian, swine, equine, and canine influenza A viruses (IAVs). Although it is dispensable for virus replication and growth, it plays significant roles in pathogenesis by interfering with the host innate immune response, inducing death in immune and epithelial cells, altering inflammatory responses, and promoting secondary bacterial pneumonia. The effects of PB1-F2 differ between virus strains and host species. This can at least partially be explained by the presence of multiple PB1-F2 sequence variants, including premature stop codons that lead to the expression of truncated PB1-F2 proteins of different lengths and specific virulence-associated residues that enhance susceptibility to bacterial superinfection. Although there has been a tendency for human seasonal IAV to gradually reduce the number of virulence-associated residues, zoonotic IAVs contain a reservoir of PB1-F2 proteins with full length, virulence-associated sequences. Here, we review the molecular mechanisms by which PB1-F2 may affect influenza virulence, and factors associated with the evolution and selection of this protein

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

<div><p>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.</p></div

Genetic markers of virulence in the PB1-F2 proteins from influenza viruses of domestic mammals.

<p>The IAV isolates of swine, horses, and dogs were categorized into the lineage (outer ring), PB1-F2 length (middle ring), and combination of inflammatory (indicated as “I”) and cytotoxic (indicated as “C”) residues (inner ring). Within the outer ring, the percentage of each grouping is shown relative to the total number of isolates. Within the middle and inner rings, the percentage of each grouping is shown relative to the preceding grouping. The numbers of inflammatory (indicated as “I1” through “I4”) and cytotoxic (indicated as “C1” and “C2”) residues were determined for PB1-F2 proteins with length of 62 or more amino acids (e.g. capable of encoding either inflammatory or cytotoxic residues).</p

Phylogeny overview of equine and canine influenza A PB1 genes.

<p>Maximum likelihood phylogenetic trees for 96 equine and 63 canine H3N8, 11 equine H7N7, and 19 canine H3N2 viruses were generated. 1, 2, 3, and 4 inflammatory residues are indicated in blue, green, fuchsia, and red, respectively. The presence of one or two cytotoxic residues is indicated by one or two asterisks, respectively. Cyan indicates PB1-F2 protein truncated before residue 62. More detail, including strain names and bootstrap values, is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111603#pone.0111603.s002" target="_blank">Figure S2</a>.</p

Phylogeny overview of swine influenza A PB1 gene.

<p>Phylogenetic analysis for 789 H1N1, 529 H3N2, and 329 H1N2 SIVs are shown. 1, 2, 3, and 4 inflammatory residues are indicated in blue, green, fuchsia, and red, respectively. The presence of a cytotoxic residue is indicated by an asterisk. Cyan indicates PB1-F2 proteins truncated before residue 62. More detail, including strain names and bootstrap values, is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111603#pone.0111603.s001" target="_blank">Figure S1</a>.</p