Figure 3: Activity of H5N1 polymerases in cell cultures.
From: Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells
(a) Cartoon depicting the amino-acid differences between the polymerase subunits PB2, PB1 and PA of the H5N1 isolates KAN-1 and Vn-1203. (b) To determine the polymerase activity of the indicated viruses in a time-dependent manner post-transfection (p.t.), HEK293T cells were transiently transfected with expression plasmids coding for the corresponding PB2, PB1, PA and NP proteins, a human polymerase I-driven vRNA-luciferase reporter plasmid and a renilla-expressing plasmid. Omission of PB2 (−PB2) was used as a negative control. Renilla activity was used to normalize variation in transfection efficiency. Error bars indicate the standard error of the mean of three independent experiments. (c) Relative polymerase activity in avian LMH cells. (d–f) Relative polymerase activity in HEK293 T cells after exchange of the indicated polymerase subunits (d) or mutation of the indicated amino-acid positions in PB2 (e) or PA (f). (g–i) Relative polymerase activity in avian LMH cells after exchange of the indicated polymerase subunits (g) or mutation of the indicated amino-acid positions in PB2 (h) or PA (i). (j, k) RNP formation of reconstituted polymerases in HEK293T. Reconstitution of the AvianPr polymerase complex was carried out, as described in (b), using expression plasmids coding for PB2-HA with the annotated mutation. A cell extract was prepared 24 h post-transfection determining the reporter activity (j) and subjected for co-immunoprecipitation (IP) experiments using anti-HA antibodies. Polymerase activity was normalized to renilla activity, and the levels obtained with the AvianPr-HA-PB2 were set to 100%. Error bars indicate the standard error of the mean of three independent experiments. The viral NP and PB2 were detected by western blot. (l) Determination of the mRNA, cRNA and vRNA levels by primer extension analysis after reconstitution of the AvianPr, KAN-1 and AvianPr-PB2-E627K polymerases 24 h post-transfection, using segment 6. Determination of the 5sRNA levels served as an internal loading control.
