The Proapoptotic Influenza A Virus Protein PB1-F2 Forms a Nonselective Ion Channel

Abstract

BackgroundPB1-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 FindingsHere, 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) (IAVPR8), from A/Brevig Mission/1/1918(H1N1) (IAVSF2) or the H5N1 consensus sequence (IAVBF2) 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/SignificanceThe 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.

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

“…The findings from numerous studies indicate that, depending on the IAV strain, PB1-F2 may elicit diverse effects such as death in infected cells (10)(11)(12)(13)(14), upregulation of viral polymerase activity (15)(16)(17)(18), increased inflammation (19)(20)(21)(22), and, as recently reported, direct antibacterial activity (8).…”

“…Mitochondrial localization of PB1-F2 is achieved by the mitochondrial targeting sequence, a short ␣-helical arginine-rich motif at the C terminus of the protein, spanning aa 65 to 87 (10,14,23). PB1-F2 initiates the intrinsic pathway of apoptosis through permeabilization of the mitochondrial membranes (6,(11)(12)(13)(14), resulting in the loss of respiratory function, release of intermembrane proteins (such as cytochrome c) into the cytosol, activation of caspase-9 and caspase-3, and cell death. The exact mechanism of PB1-F2-mediated mitochondrial outer membrane permeabilization (MOMP) is not clear.…”

Potassium Ion Channels: Could They Have Evolved from Viruses?

“…The immense power of viruses for gain of functional genes is best described in the case of influenza A. A plus-one frame shift in a gene for an RNA polymerase (PB1) (Chen et al, 2001) is sufficient to generate a second overlapping open reading frame (F2) for a gene product, the PB1-F2 protein, with channel function (Henkel et al 2010). …”

Naturally Occurring Swine Influenza A Virus PB1-F2 Phenotypes That Contribute to Superinfection with Gram-Positive Respiratory Pathogens

“…The PB1-F2 protein contributes to viral polymerase activity (13,31,34,58), induces apoptosis by interacting with host cell mitochondria (24,25,40,61), and affects host immune responses directed toward the virus (29,54). Full-length PB1-F2 is frequently maintained at 87 to 90 amino acids (24,33), with specific amino acid residues that are associated with virulence (1), including a critical one at position 66 (14).…”

“…Interestingly, the pandemic influenza virus isolates from 1918,1957, and 1968 expressed full-length, virulent forms of PB1-F2 (32), but changes in this protein have been correlated with the overall reduction in virulence observed as sustained transmission within humans was established (33). In addition to point mutations within PB1-F2, virulence can be reduced when truncated forms of this protein are expressed, including natural variants that are 11,25,34,57, and 78 amino acids in length (13,33).…”