Host restriction of influenza virus polymerase activity by PB2 627E is diminished on short viral templates in a nucleoprotein-independent manner

Abstract

Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. Nevertheless, this impediment can be overcome through an E→K adaptive mutation at residue 627 of the PB2 subunit of the polymerase. Accordingly, viral ribonucleoprotein (RNP) reconstitution assays show that a viral polymerase containing PB2 627E has impaired activity in mammalian cells compared to a viral polymerase that contains PB2 627K, characteristic of mammalian-adapted influenza viruses. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that require no RNP assembly. We sought to reconcile these conflicting observations by using an NP-independent cell-based transcription/replication assay to assess viral polymerase activity. We found that PB2 627E polymerase restriction in mammalian cells is independent of NP expression but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase was overcome by mutations specific to the viral RNA template promoter sequence. Consequently, we propose that PB2 627E affects recruitment of the viral RNA promoter by the viral polymerase in mammalian cells.

FIG 1

Effects of PB2 K627E mutation on polymerase assembly and activity in cultured cells and in vitro. (A) HEK-293T (human) or DF1 (chicken) cells were cotransfected with plasmids expressing PA, PB1, PB2 627K or 627E, NP, and a segment 6 vRNA. Accumulation of mRNA and vRNA was assessed by primer extension 48 h posttransfection (22). The RNA levels detected for the E627K polymerase were set to 100. The error bars indicate standard errors of the mean (SEM), and the asterisks indicate a significant difference from 100 (one-sample t test; ***, P < 0.001). Expression of PB2 was analyzed by Western blotting, with RanBP5 serving as a loading control. (B) HEK-293T cells were cotransfected with plasmids expressing PA, PB1-TAP, and PB2 627K or 627E, and polymerase was purified using IgG Sepharose chromatography 48 h posttransfection (26). The purified polymerase was analyzed by SDS-PAGE, and PB2 levels were assessed by using AIDA software (Raytest). PB2 627K levels were set to 100. The error bar indicates SEM of three experiments. (C) The activity of the purified polymerase from panel B was assessed in an in vitro transcription assay using ApG primer or β-globin mRNA as a cap donor (26, 27). The RNA levels detected for the E627K polymerase were set to 100. The error bars indicate SEM of three experiments.

FIG 2

Transcription and replication of short vRNA templates in the presence or absence of NP by influenza virus RNA polymerase containing PB2 627K or 627E in a cell-based replication assay. HEK-293T cells were cotransfected with plasmids expressing short segment 5-based vRNA-like templates (22) of the indicated lengths, PA, PB1, PB2 627K or 627E, and a plasmid expressing NP (A) or empty vector (B) (22). Accumulation of vRNA and mRNA was assessed by primer extension 48 h posttransfection (22). The level of vRNA template produced by cellular transcription was determined by the omission of the PB2 subunit of the viral polymerase and set to zero. The RNA levels detected for the 627K polymerase were set to 100 for each template length. The error bars indicate SEM of three experiments, and the asterisks indicate a significant difference from 100 (one-sample t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Pearson's correlation coefficient was calculated for the PB2 627E polymerase activities, producing the r values shown.

FIG 3

Transcription and replication of short NA segment vRNA templates by PB2 627K or 627E in human and avian cells. HEK-293T (A) or DF1 (B) cells were cotransfected with plasmids expressing short segment 6-based vRNA-like templates of the indicated lengths, as well as PA, PB1, and PB2 627K or 627E. Accumulation of vRNA and mRNA was assessed by primer extension 48 h posttransfection. The level of vRNA template produced by cellular transcription was determined by the omission of the PB2 subunit of the viral polymerase and set to zero. The RNA levels detected for the 627K polymerase were set to 100 for each template length. The error bars indicate SEM of three experiments, and the asterisks indicate a significant difference from 100 (one-sample t test; **, P < 0.01; ***, P < 0.001).

FIG 4

Effects of vRNA promoter mutations on transcription and replication by viral polymerase with PB2 627K or 627E. Shown is a schematic representation of wild-type (W) and mutant (i to iv) influenza virus vRNA promoter structures according to the panhandle model. Mutated residues are indicated by circles. HEK-293T cells were cotransfected with plasmids expressing PA, PB1, PB2 627K or 627E, NP, and segment 5 vRNA with the indicated promoter mutations and mutations to prevent expression of NP (22). Accumulation of mRNA and vRNA was assessed by primer extension 48 h posttransfection (22). The level of vRNA template produced by cellular transcription was determined by the omission of the PB2 subunit of the viral polymerase and set to zero. The RNA levels detected for the 627K polymerase were set to 100. The error bars indicate SEM of three experiments, and the asterisks indicate a significant difference from 100 (one-sample t test; ***, P < 0.001). a, no mRNA signal was detected above background levels for either PB2 627K or PB2 627E polymerases when template iii was used.

FIG 5

Effects of vRNA promoter mutations on PB2 627K or 627E polymerase activity in human and avian cells. HEK-293T (A) or DF1 (B) cells were cotransfected with plasmids expressing full-length segment 6 containing the indicated mutations, as well as PA, PB1, and PB2 627K or 627E. Accumulation of vRNA and mRNA was assessed by primer extension 48 h posttransfection. The level of vRNA template produced by cellular transcription was determined by the omission of the PB2 subunit of the viral polymerase and set to zero. The RNA levels detected for the 627K polymerase were set to 100 for each template length. The error bars indicate SEM of three experiments, and the asterisks indicate a significant difference from 100 (one-sample t test; ***, P < 0.001).