Figure 7: Identification of adaptive mutations in NEP of other human isolates.
From: Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells
(a) Sequence alignment of NEPs of human and avian H5N1 viruses as well as the NEP of the 2009 pandemic H1N1 virus to the H5N1 consensus sequence. Bold letters indicate amino acids exclusively found in human H5N1 isolates. (b) Reporter activities after reconstitution of the AvianPr polymerase in human HEK293T cells and co-transfection of 10 ng of expression plasmids coding for AvianPr NEP harbouring the indicated amino-acid substitution. Error bars indicate the s.e. of the mean of three independent experiments. Student's t-test was performed to determine the P-value. *P<0.05, **P<0.01, ***P<0.001, NS, not significant. NEP-M16I corresponds to NEP-KAN-1. (c) Western blot analysis of the NEP expression levels after transient transfection of HEK 293 T cells with equal amounts of expression plasmids (500 ng) encoding the indicated NEP variants. (d) Determination of the mRNA, cRNA and vRNA levels by primer extension analysis after reconstitution of the AvianPr polymerase in human HEK293 T cells and co-transfection of either empty plasmid (Vector) or AvianPr NEP harbouring the indicated amino-acid substitution using primers specific for segment 6. Determination of the 5sRNA levels served as an internal loading control.
