Fig 1.
Co-immunoprecipitation analysis of NP and PB2 N-terminal fragments.
(A) Expression of NP, PB2 and PB2 truncation mutants. Full length PB2 and three truncated constructs (aa 1–483, 1–320 and CBD) of myc-tagged PB2 were co-transfected with NP into HEK 293 cells. Total cell lysate was collected after 48-hour incubation to detect NP and myc-tagged PB2 fragments expression by Western blotting analysis. (B) Co-immunoprecipitation of NP and PB2 or PB2 fragments. Co-immunoprecipitation was performed with overnight incubation of anti-myc antibody with cell lysate, protein A agarose was used to precipitate the antibody. Amount of NP precipitated was detected by anti-NP serum, and the amount of myc-tagged PB2 and PB2 fragments were detected by anti-myc antibody. FL: Full-length PB2. CBD: Cap-binding domain.
Fig 2.
Characterization of cap-binding domain-NP interaction.
(A) Co-immunoprecipitation of NP and myc-tagged PB2 cap-binding domain with and without addition of RNase A. The bar represents the mean ratio ± standard errors from two independent experiments. *The p-values are smaller or equal to 0.05 in two-tailed Student’s t-test. (B) Expression of NP, PB2 cap-binding domain and E361A variant. Wild-type cap-binding domain (CBD WT) or E361A mutant was co-transfected with NP into HEK 293 cells. Protein expression was detected by anti-NP serum and anti-myc antibody. (C) Pull-down assay investigating NP-PB2 cap-binding domain binding. NHS-activated sepharose was used to immobilize 50μM PB2 CBD, BSA was immobilized as control (sample 4). Purified NP and 24-nt 2’-O-methylated RNA were added accordingly, the mixtures were washed extensively after 1 hour incubation. 1.5M NaCl was used to elute the protein captured by CBD. Samples of wash and elution fractions were separated by 12% SDS-PAGE and detected by coomassie blue staining. WT: Wild type.
Fig 3.
Effect of PB2 variants on RNP activity and NP-binding.
(A) Polymerase activity measured by luciferase reporter assay. Plasmids encoding wild-type or mutant PB2, PA, PB1, NP (ie. pcDNA-PA, pcDNA-PB1, pcDNA-PB2 and pcDNA-NP) were co-transfected with a plasmid expressing luciferase reporter gene under viral promoter control, pPOLI-Luc-RT and a plasmid expressing GFP, pEGFP were transfected to HEK 293T, pcDNA3 was used to replace pcDNA-PB2 as negative control. The overall polymerase activity is represented by a ratio of luminescence signal to GFP signal. The activity of polymerase with wild-type PB2 was set to 1. The bar represents the mean ratio ± standard errors from three independent experiments. *The p-values are smaller or equal to 0.05 in two-tailed Student’s t-test. (B) Protein expression of PB2 and mutants. Level of PB2 expression of each mutant was detected by anti-PB2 polyclonal antibodies in Western blotting with ß-actin as loading control. (C) Co-immunoprecipitation of NP and cap-binding domain. Myc-tagged PB2 cap-binding domain and variants were co-transfected and expressed with NP in HEK 293 cells. Myc-tagged empty vector was used as negative control. Co-immunoprecipitation was performed after 48-hour incubation, cell lysates were incubated overnight with or without anti-myc antibody. Expression levels of myc-tagged PB2 cap-binding domain and variants were detected by anti-myc antibody, while anti-NP serum was used to detect NP expression level in the cells. Amounts of NP precipitated by cap-binding domain variants in Co-immunoprecipitation were compared to that precipitated from the wild-type. The bar represents the mean ratio ± standard errors from three independent experiments. *The p-values are smaller or equal to 0.05 in two-tailed Student’s t-test.
Fig 4.
Effects of PB2 variants on NP-polymerase interaction.
(A) Co-immunoprecipitaion of NP-polymerase. Plasmids encoding myc-tagged PA, PB1, PB2 or its variants, pPOLI-NA-RT were co-transfected with NP into HEK 293T cells, myc-tagged empty vector was used as negative control. Cells were harvested after 48-hour incubation. Cell lysates were incubated overnight with or without anti-myc antibody. Amount of NP co-immunoprecipitated with PB2 variants were compared to that precipitated from the wild-type. The bar represents the mean ratio ± standard errors from three independent experiments. *The p-values are smaller or equal to 0.05 in two-tailed Student’s t-test. (B) Expression of RNP subunits. Levels of expression of RNP subunits were detected by corresponding anti-bodies.
Fig 5.
Structure of PB2 and PB2 cap-binding domain.
(A) Schematic of multiple domains of PB2. Schematic of NP was added to indicate NP binding site. Studied regions (aa 1–320, CBD and aa 1–483) were highlighted. (B) (Left) Structure of PB2 from crystal structure of influenza polymerase (PDB ID: 4WSB). Structure showed that domains of PB2 are joined by several flexible linkers and suggested a high degree of flexibility. Studied region was indicated by arrow and studied amino acids of the cap-binding domain were indicated in red. (Right) Structure of PB2 cap-binding domain. Basic residues around the exit of cap-binding pocket (K331, R332, K339, K353, N429, H432) were indicated in red. (PDB ID: 4CB4).