N-terminal region of the PB1-F2 protein is responsible for increased expression of influenza A viral protein PB1

Abstract

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 expedition from the whole protein through the domain to aa residue function

“…(Meunier and von Messling, 2012). In agreement with our observations, N terminus is responsible for increased PB1 and other viral protein expression (Kosik et al, 2011) as well as overall replication kinetics (Smith et al, 2011).Such observation of MDCK cell is important since this cell line has been shown to replicate IAV very efficiently (Ueda et al, 2008) and the contribution of PB1-F2 to replication kinetics is more significantly visible in the A549 cell line. Reconstitution of the PB1-F2 from the pdm H1N1 2009 IAV on the background of PR8 elevated virus growth in comparison to 11aa WT PB1-F2 from the same isolate.…”

“…PB1-F2 possesses both of these factors. Its relation to virus RNA dependent RNA polymerase (vRdRp) fits the cri-teria for virus determined pathogenicity contribution (Kosik et al, 2011;Mazur et al, 2008;McAuley et al, 2010b). In the terms of host immune system contribution, modulation of the cytokines response, selective immune cell pro-apoptosis (Coleman, 2007) and more recently identified neutrophil chemoattraction has been described for PB1-F2 functions (Conenello et al, 2011;Le Goffic et al, 2011).…”

“…It has been shown more recently that single mutation N66S contribute to increased virulence of HPAI H5N1 (HK97) and 1918 IAV (Conenello et al, 2007). It is not yet clear what the pathogenicity determinants of the PB1-F2 are but it is probable that interaction with viral polymerase subunit PB1 (Kosik et al, 2011;Mazur et al, 2008) affecting catalytic activity contributes to the pathogenicity of IAV. Despite some discrepancy surrounded in relation to IFN type I response (Le , it is reasonable to think that PB1-F2 plays an important role in IFN type I response modulation (Conenello et al, 2011;Dudek et al, 2011;Le Goffic et al, 2010;Varga et al, 2011).…”

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana

“…Single mutation in PB1-F2 increased virulence of the IAV (Conenello et al, 2007). The mechanism of action is so far unknown, however, interaction of PB1-F2 with viral RNA polymerase subunit PB1 is proposed to be involved in this process (Mazur et al, 2008;Košík et al, 2011). In vivo, PB1-F2 is very unstable and prone to rapid degradation under physiological conditions (Chen et al, 2001;Schmolke et al, 2011).…”

“…The complete PB1-F2-coding sequence (261 bp) was amplified from the previously prepared construct ptriEX4 PB1-F2 (Košík et al, 2011) using EX taq DNA polymerase (taKaRa) and primers including EagI and KpnI linkers, respectively (underlined in the sequences): AACGGCCGGAtGGGACAGGAACAGGAtAC, AAG GtACCCtCGAGtttGCtGAACAACC. The PCR conditions were as follows: initial denaturation at 94°C for 3 min, 40 cycles of 94°C/15 sec, 58°C/30 sec, 72°C/1 min, and final extension at 72°C for 10 min.…”