Biochemical impact of the host adaptation-associated PB2 E627K mutation on the temperature-dependent RNA synthesis kinetics of influenza A virus polymerase complex

Abstract

Most 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), whereas the human viruses, which optimally replicate at the low temperatures observed in the human respiratory tract, have a lysine (Lys-627). The mechanism of action for this mutation is still not understood, although interaction with host factors has been proposed to play a major role. In this study, we explored an alternative, yet related, hypothesis that this PB2 mutation may alter the temperature-dependent enzymatic polymerase activity of the viral polymerase. First, the avian polymerase protein, which was purified from baculovirus expression system, indeed remained significantly active at higher temperatures (i.e. 37 and 42 °C), whereas the human E627K mutant drastically lost activity at these high temperatures. Second, our steady-state kinetics data revealed that the human E627K mutant polymerase is catalytically more active than the avian Glu-627 polymerase at 34 °C. Importantly, 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 the interaction with the incoming NTP. Third, this temperature-dependent kinetic impact of the human E627K mutation was also observed with different RNA templates, with different primers and also in the presence of nucleoprotein. In conclusion, our study suggests that the amino acid sequence variations at residue 627 of PB2 subunit can directly alter the enzyme kinetics of influenza polymerase.

FIGURE 1.

Temperature-dependent ApG primer-initiated RNA synthesis profiles of avian Glu-627 and human E627K PB2 influenza A virus RNA polymerase complex proteins purified from baculovirus expression system. A, ApG primer was extended by Glu-627 and E627K Pol proteins purified from insect cells at five different physiologically relevant temperatures (30, 32, 34, 37, and 42 °C) as described under “Experimental Procedures.” The 30-nt reaction products are radioactively labeled by the incorporation of [α-³²P]NTPs during RNA synthesis and analyzed by urea-denaturing gels. An equal amount of 5′ end ³²P-labeled 11-nt primer was added in each reaction and used as a loading control (LC) to avoid loading errors. The asterisks are used for comparison only. Lane C, no polymerase control. B and C, the 30-nt product band intensity at each temperature with avian Glu-627 and human E627K Pol complexes was normalized by the band intensity at 42 °C (asterisk), and the normalized relative enzyme activities were plotted. At least three independent reactions were conducted for the analysis. The data represent mean values; the error bars denote standard deviations.

FIGURE 2.

Steady-state kinetic analysis of the avian Glu-627 PB2 influenza A virus RNA polymerase complex protein at varying temperatures. A, ApG-initiated RNA synthesis was conducted with the avian Pol complex protein at 34, 37, and 42 °C with varying concentrations of NTP substrates (10, 25, 100, 250, and 500 μm). The reactions were conducted as described in the legend to Fig. 1, and the products were analyzed by 20% denaturing gels. Lane C, no polymerase control. LC, loading control. B–D, the reaction rates at each of the temperature and NTP concentration settings were normalized with the maximum rate (apparent K_cat = 1/mol min) at 42 °C at the highest NTP concentration of 500 μm, and the calculated relative reaction rates were plotted for the determination of K_m values by the Michaelis-Menten equation. The PB2 concentration in the avian Glu-627 Pol complex was used for determining the apparent K_cat values. At least three independent reactions were conducted for the analysis. The data represent the mean values; the error bars denote standard deviations.

FIGURE 3.

Steady-state kinetic analysis of ApG-initiated RNA synthesis by human E627K PB2 influenza A virus RNA polymerase complex at varying temperatures. A, ApG-initiated RNA synthesis was conducted with the human Pol complex at 34, 37, and 42 °C with varying concentrations of NTP substrates (10, 25, 100, 250, and 500 μm). The reactions were conducted as described in the legend to Fig. 2, and the products were analyzed by 20% denaturing gels. LC, loading control. B–D, the reaction rates at each of the temperature and NTP concentration settings were normalized with the maximum rate (apparent K_cat = 1/mol min) at 42 °C at the highest NTP concentration of 500 μm, and the calculated relative reaction rates were plotted for the determination of K_m values by the Michaelis-Menten equation. The PB2 concentration of the human E627K Pol complex was used for determining the apparent K_cat values. At least three independent reactions were conducted for the analysis. The data represent mean values; the error bars denote standard deviations.

FIGURE 4.

Comparison of steady-state kinetic values of avian Glu-627 and human E627K Pol complex proteins summarized in Table 1. The fold differences between the K_m values (top left panel), apparent K_cat values (top right panel), and polymerase efficiency (apparent K_cat/K_m) (bottom panel) of avian and human Pol complex proteins at the three temperatures (Table 1) were calculated and compared.

FIGURE 5.

Temperature-dependent capped primer-initiated RNA synthesis profiles of the avian Glu-627 and human E627K PB2 influenza A virus RNA polymerase complexes. A, globin mRNA was used as a primer and extended by purified Glu-627 and E627K Pol proteins at four different physiologically relevant temperatures (30, 34, 37, and 42 °C) as described under “Experimental Procedures.” The reaction products (Cap+30nt) are radioactively labeled by the incorporation of [α-³²P]NTPs during RNA synthesis and analyzed by urea-denaturing gels. An equal amount of 5′ end ³²P-labeled 11-nt primer was added in each reaction and used as a loading control (LC) for avoiding loading errors. The asterisks are used for comparison only. Lane C, no polymerase control. B and C, the product band intensity (Cap+30nt) at each temperature with avian Glu-627 and human E627K Pol complexes was normalized by the band intensity at 42 °C (asterisks), and the normalized relative enzyme activities were plotted. At least three independent reactions were conducted for the analysis. The data represent mean values; the error bars denote standard deviations.