Role of the PB2 627 Domain in Influenza A Virus Polymerase Function

FIG 1. Design of PB2 deletion mutants and their expression. (A) Crystal structure of the (apo) influenza C/Johannesburg/1/66 virus polymerase (PDB code 5D98) and the (vRNA promoter bound) influenza A/little yellow-shouldered bat/Guatemala/060/10 (H17N10) virus polymerase (PDB code 4WSB) with the PB2 subunit domains shown in different colors. (B) Schematic of full-length, K627E, and truncated PB2 subunits with domains colored as in panel A. (C) Complex formation of K627E and PB2 truncation mutants with PB1 and PA. Polymerase subunits PB1 and PA were coexpressed with K627E or PB2 truncation mutants in HEK-293T cells and purified by IgG Sepharose chromatography. Purified recombinant polymerase was analyzed by SDS-PAGE. PA, PB1, and full-length PB2 were detected by silver staining. Copurification of the K627E mutant and truncated PB2 subunits with PB1 and PA was detected by Western blotting with a primary antibody targeting the N-terminal (positions 1 to 180) region of PB2 (36). Molecular masses, indicated on the left, are in kilodaltons. (D) Polymerase subunits PA, PB1, and PB2 lacking the 627 domain were coexpressed in HEK-293T cells, and PB2 was detected by immunofluorescence using a…

FIG 2. Polymerase lacking the PB2 627 domain is active in vitro. Purified recombinant polymerases were incubated in reaction mixtures containing radiolabeled capped RNA and vRNA template in the absence (left) or presence (right) of nucleotides (A) or ATP, UTP, CTP, [α-32P]GTP, ApG primer, and either vRNA or cRNA as the template (B). Reaction products were analyzed by 20% PAGE and autoradiography. The graphs show the mean intensity signal relative to that of wild-type polymerase with K627 PB2 from three independent biological replicates (n = 3), with error bars representing the standard errors of the means and the asterisks indicating a significant difference from 627K (two-tailed one-sample t test) as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

FIG 3. The PB2 627 domain is required for polymerase activity in the cell. Human HEK-293T (A) or chicken DF-1 (B) cells were cotransfected with plasmids expressing PA, PB1, wild-type or mutant PB2, NP, and segment 6 vRNA. Human HEK-293T cells (C) were cotransfected with plasmids expressing PA, PB1, wild-type or mutant PB2, and a 47-nt-long segment 6-derived vRNA. Accumulation of mRNA, cRNA, and vRNA was assessed by primer extension. The graphs show the mean intensity signal (with the mean intensity signal with no PB2 expressed subtracted) relative to that of wild-type PB2 627K polymerase from three independent biological replicates (n = 3), with error bars representing the standard errors of the means and the asterisks indicating a significant difference from the wild type (two-tailed one-sample t test) as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (D) Polymerase subunits PA-TAP, PB1 with an active-site mutation (PB1a), wild-type and mutant PB2, and segment 6-derived 47-nt-long vRNA or cRNA were coexpressed in HEK-293T cells and purified by IgG Sepharose chromatography. Levels of vRNA or cRNA that copurified with polymerase were assessed by primer extension. The graphs show…

FIG 4. The PB2 627 domain is required for cRNA accumulation in infected cells. PA, PB1, and wild-type or mutant PB2 were coexpressed in HEK-293T cells in the presence (left) or absence (right) of NP. Twenty-four hours posttransfection, cells were infected with influenza A/WSN/33 virus at an MOI of 10 in the presence of actinomycin D. Six hours postinfection, total RNA was extracted and analyzed by primer extension. The graphs show the mean intensity signal relative to that of wild-type 627K polymerase from three independent biological replicates (n = 3), with error bars representing the standard errors of the means and the asterisks indicating a significant difference from 627K (two-tailed one-sample t test) as follows: *, P < 0.05, and **, P < 0.01.