PB1-F2 is a small influenza A virus (IAV) protein encoded by an alternative (+1) reading frame of the PB1 gene. While dispensable for IAV replication in cultured cells, PB1-F2 has been implicated in IAV pathogenicity. To better understand PB1-F2 expression in vivo and its immunogenicity, we analyzed anti-PB1-F2 antibodies (Abs) in sera of mice infected intranasally (i.n.) with A/PR/8/34 (H1N1) virus and human acute and convalescent sera collected from the influenza H3N2 winter 2003-2004 epidemic. We explored a number of methods for detecting anti-PB1-F2 Abs, finding that PB1-F2-specific Abs could clearly be detected via immunoprecipitation or immunofluorescence assays using both immune mouse and human convalescent sera. Importantly, paired human sera exhibited similar increases in HI titers and PB1-F2-specific Abs. This study indicates that PB1-F2 is expressed in sufficient quantities in mice and humans infected with IAV to elicit an Ab response, supporting the biological relevance of this intriguing accessory protein.

Summary. -Influenza A virus (IAV) PB1-F2 protein is encoded by an alternative reading frame (+1) within the PB1 gene. PB1-F2 has been shown to contribute to the pathogenesis of influenza virus infection as well as to the secondary bacterial infection. More recently has been shown that PB1-F2 protein may regulate a viral RNA (vRNA) polymerase activity by the interaction with PB1 protein. We proved that PB1-F2 protein increased the level of expression of PB1 protein and vRNA in the infected cells. Moreover, we demonstrated that a higher level of vRNA expression resulted in the increase of expression of multiple viral proteins, including NP, M1, and NS1. Finally, we used plasmids expressing N-terminal (1-50 aa) or C-terminal (51-87 aa) region of the PB1-F2 molecule for transfection of MDCK cells co-infected with influenza A/Puerto Rico/8/34 (H1N1) virus deficient in the PB1-F2 protein expression (PR8ΔPB1-F2). These experiments clearly showed that N-terminal region of PB1-F2 protein was responsible for the increase in PB1 protein expression. C-terminal region of PB1-F2 protein had no effect. Thus, we have identified the important function for N-terminal region of PB1-F2 protein.

PB1-F2 is a small influenza A virus (IAV) protein encoded by an alternative reading frame of the PB1 gene. During IAV infection, antibodies to PB1-F2 proteins are induced. To determine their function and contribution to virus infection, three distinct approaches were employed: passive transfer of anti-PB1-F2 MAbs and polyclonal antibodies, active immunization with PB1-F2 peptides and DNA vaccination with plasmids expressing various parts of PB1-F2. Mostly N-terminal specific antibodies were detected in polyclonal sera raised to complete PB1-F2. Passive and active immunization revealed that antibodies recognizing the N-terminal part of the PB1-F2 molecule have no remarkable effect on the course of IAV infection. Interestingly antibodies against the C-terminal region of PB1-F2, obtained by immunization with KLH-PB1-F2 C-terminal peptide or DNA immunization with pC-ter.PB1-F2 plasmid, partially protected mice against virus infection. To our knowledge, this is the first report demonstrating the biological relevance of humoral immunity against PB1-F2 protein in vivo.