Unstable polymerase-nucleoprotein interaction is not responsible for avian influenza virus polymerase restriction in human cells

Abstract

Avian-origin influenza virus polymerase activity can be dramatically increased in human cells with the PB2 E627K mutation. Previously, others have proposed that this mutation increases the stability of the viral ribonucleoprotein complex (vRNP) measured by the interaction between PB2 and NP. However, we demonstrate here that a variety of PB2 adaptive mutations, including E627K, do not enhance the stability of the vRNP but rather increase the amount of replicated RNA that results in more PB2-NP coprecipitation.

Fig 1

Polymerase activity supported by PB2 mutants. (A to C) 293T (A), NPTr (B), and DF-1 (C) cells were transfected with pCAGGS 5092 PB1, PA, NP, and WT or mutated PB2 as well as pCAGGS Renilla and a virus-like firefly luciferase minigenome-expressing plasmid. Luciferase production was measured 12 h posttransfection. Values were normalized to Renilla expression and to the activity of the WT polymerase. (D to F) The levels of viral mRNA, cRNA, and vRNA synthesized by different RNA polymerase PB2 mutants normalized to that of the WT polymerase in 293T (D), NPTr (E), and DF-1 (F) cells. Expression of 50-92 NP mRNA was used as an internal control. Results are from one experiment, undertaken in triplicate, representative of three independent experiments. The statistical significance of differences in polymerase activity or RNA synthesis from that of WT was assessed by a two-tailed, unpaired Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 2

Activity of 50-92 with untagged or FLAG-tagged PB2. 293T cells were transfected with pCAGGS 50-92 NP, PB1, PA, and WT (A) or E627K (B) PB2 (untagged or FLAG tagged), as well as a virus-like firefly luciferase reporter plasmid and pCAGGS Renilla. Firefly luciferase production was measured 12 h posttransfection. Values were normalized to Renilla expression and to the activity of the untagged polymerase. Results shown are the means ± standard deviations from three independent experiments.

Fig 3

Effect of PB2 mutations on polymerase-NP interaction in 293T cells. (A) 293T cells were transfected with pCAGGS 50-92 NP and WT or mutated PB2-FLAG. At 12 h posttransfection, cell lysates were prepared and subjected to NP immunoprecipitation prior to Western blot analysis. Results are from one experiment representative of three independent experiments. (B) The levels of coimmunoprecipitated PB2 in panel A were normalized to precipitated NP using ImageJ. (C) 293T cells were transfected with pCAGGS 50-92 NP, PB1, PA, and WT or mutated PB2-FLAG and a virus-like firefly luciferase reporter plasmid. At 12 h posttransfection, cell lysates were prepared and subjected to NP immunoprecipitation prior to Western blot analysis. Results are from one experiment representative of three independent experiments. (D) The levels of coimmunoprecipitated PB2 in panel C were normalized to precipitated NP using ImageJ. (E) Polymerase activity supported by mutated PB2-FLAG compared to WT PB2-FLAG. Values were normalized to Renilla expression and to the activity of the WT polymerase. Results shown are the means ± standard deviations from three independent experiments. The statistical significance of differences in polymerase activity compared to that of WT was assessed by a two-tailed, unpaired Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (F) Level of vRNA isolated from cell immunoprecipitates analyzed in panel C for PB2 E158G-FLAG and PB2 E627K-FLAG normalized to PB2 WT.

Fig 4

In the absence of transcription/replication, PB2 mutations do not affect the amount of PB2 precipitated by NP. (A) PB1 mutation D446Y inhibits 50-92 polymerase activity. 293T cells were transfected with pCAGGS 50-92 NP, FLAG-tagged PB2 WT or 627K, PA, and PB1 WT or PB1 D446Y as well as a virus-like firefly luciferase reporter plasmid and pCAGGS Renilla. Firefly luciferase production was measured 12 h posttransfection. Values were normalized to Renilla expression. Results shown are the means ± standard deviations from three independent experiments. RLU, relative light units. (B) 293T cells were transfected with pCAGGS 50-92 NP, PB1 D446Y, PA, and WT or mutated PB2-FLAG and a virus-like firefly luciferase reporter plasmid. At 12 h posttransfection, cell lysates were prepared and subjected to NP immunoprecipitation prior to Western blot analysis. Results are from one experiment representative of three independent experiments. (C) The levels of coimmunoprecipitated PB2 in panel A were normalized to precipitated NP using ImageJ.

Fig 5

Mutated 3-5-8 virus-like firefly luciferase reporter enhances luciferase activity and the amount of PB2 precipitated by NP. (A) 293T cells were transfected with pCAGGS 50-92 NP, PB1, PA, and PB2 (WT) as well as a WT or mutated 3-5-8 firefly luciferase reporter plasmid. At 12 h posttransfection, cell lysates were prepared and subjected to NP immunoprecipitation prior to Western blot analysis. Results are from one experiment representative of three independent experiments. (B) The levels of coimmunoprecipitated PB2 in panel A were normalized to precipitated NP using ImageJ. (C) Luciferase activity supported by 50-92 polymerase with 3-5-8 luciferase reporter. Values were normalized to Renilla expression and to the activity of the WT polymerase. Results shown are the means ± standard deviations from three independent experiments. The statistical significance of differences in polymerase activity compared to that of WT was assessed by a two-tailed, unpaired Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).