Influenza Virus PB1-F2 Protein Induces Cell Death through Mitochondrial ANT3 and VDAC1

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Abstract
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. PB1-F2 is a short polypeptide encoded by influenza viruses. While the role of this viral protein is not completely understood, it is known to localize in the mitochodria of the infected cell and to promote cell death. The authors found that PB1-F2 sensitizes cells to death through interactions with two mitochondrial proteins, ANT3 and VDAC1. These interactions promote the permeabilization of the mitochodria and facilitate the release of mitochondrial products that trigger cell death (apoptosis). PB1-F2-mediated cell death through the mitochondria is likely to contribute to the pathogenicity of the influenza virus.