The E627K mutation in the PB2 subunit of influenza A polymerase affects promoter binding at higher temperatures

Shilpa Aggarwal and Baek Kim

: J Antivir Antiretrovir

Abstract :

M ost avian influenza A viruses, which preferentially replicate at the high temperatures found in the digestive tract of birds, have a glutamic acid at residue 627 of the viral RNA polymerase PB2 subunit (Glu-627). In contrast, human viruses optimally replicate at the low temperatures observed in the human respiratory tract and have a lysine (Lys-627) at this position. The mechanism of action for this mutation is still not understood, although interaction with host factors has been proposed to play a major role. Previously, we have shown that this PB2 mutation may alter the temperature-dependent enzymatic activity of the viral polymerase. Moreover, our steady-state kinetics data revealed that the E627K mutation elevates apparent K(cat) at low temperatures with little effect on K(m), suggesting that the E627K mutation alters the biochemical steps involved in enzyme catalysis rather than interaction with the incoming NTP. To further explore this, we tested the structural integrity of the heterotrimeric polymerase complex at higher temperatures. We also looked at binding of the polymerase with 5? and 3? vRNA conserved promoter sequences at different temperatures. Our data suggests that the polymerase with Lys-PB2 shows increased binding to the 3? vRNA at lower temperatures with no difference in binding to the 5? vRNA. Also, we did not see a difference in the structural stability of the polymerase at the different temperatures tested