Figure 5: Influence of NS segment-encoded proteins on polymerase activity.
From: Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells
(a) Cartoon depicting the amino-acid mutations in NS1 and NEP of KAN-1 caused by a single nucleotide mutation in the KAN-1 NS segment. (b) Reporter activities after reconstitution of the AvianPr polymerase in HEK293 T cells and co-transfection of the indicated amounts of NS1-AvianPr, or NS1-KAN-1 encoding plasmids. Vector, empty plasmid. (c) Reporter activities after reconstitution of the AvianPr polymerase and co-transfection of the indicated amounts of NEP-AvianPr, or NEP-KAN-1 encoding plasmids. Student's t-test was performed to determine the P-value. *P<0.05, **P<0.01. Vector, empty plasmid. (d) Determination of the mRNA, cRNA and vRNA levels by primer extension analysis after reconstitution of the AvianPr polymerase in HEK293T cells and co-transfection of either empty plasmid (Vector), NEP-AvianPr or NEP-KAN-1 using primers specific for segment 6. Determination of the 5sRNA levels served as an internal loading control. (e) Time course of reporter activities of AvianPr, KAN-1 and Vn-1203 polymerases in HEK293T cells after co-transfection of 10 ng of the indicated NEP-expressing plasmids. (f) Interferon induction in infected cells. Mouse 3T3 cells stably transfected with a plasmid coding for firefly luciferase under the control of the interferon β-promoter were infected with AvianPr or AvianPr harbouring the NS-segment of KAN-1 (AvianPr-NS-KAN-1) at the indicated MOI. Mock infection or infection with either SC35M-WT or SC35M-delNS1 at a MOI of 0.5 known to induce β-interferon served as internal controls. Luciferase activity was determined after 16 h post infection. (g) Complex formation of NEP with either PB1, PA or PB2. Human 293T cells were transiently transfected with Strep-tagged KAN-1 NEP and the indicated HA-tagged polymerase subunits and subjected to co-immunoprecipitations (IP). Precipitated polymerase subunits were identified by western blot analysis, using specific antibodies against the HA- and the Strep-tag. (h) Binding of Strep-tagged KAN-1 NEP to the indicated PB2 variants and (i) to PB2 truncation mutants.
