PB2 residue 271 plays a key role in enhanced polymerase activity of influenza A viruses in mammalian host cells

Abstract

The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts.

FIG. 1.

Polymerase activity of Nan, Aichi, and WSN complexes. DF-1 (A) and 293T (B) cells were transfected with pCAGGS-NP, pCAGGS-PA, pCAGGS-PB1, and pCAGGS-PB2 from the indicated strain, along with the appropriate reporter gene plasmid. Transfected cells were cultured at 34, 37, or 39°C for 24 h, and luciferase production was determined. Results shown are the averages with standard deviations from three independent experiments. Mock, untransfected cells; no PB2, PB2 cDNA was omitted from the transfected DNA complex.

FIG. 2.

Polymerase activity of complexes containing single- and double-gene replacements. 293T cells were transfected with pCAGGS-NP, pCAGGS-PA, pCAGGS-PB1, and pCAGGS-PB2 as indicated, along with the reporter gene plasmid pPolI-NP-Luc. Cells were cultured at 34°C (A), 37°C (B), or 39°C (C) for 24 h, and luciferase production was determined. Results shown are the averages with standard deviations from three independent experiments. N, Nan; A, Aichi; mock, untransfected cells.

FIG. 3.

Polymerase activity of Nan complexes containing PB2 mutants. 293T (A, B, and C) or DF-1 (D and E) cells were transfected with Nan pCAGGS-NP, pCAGGS-PA, pCAGGS-PB1, and WT or mutant PB2, along with the appropriate reporter gene plasmid. Cells were cultured for 24 h at 34°C (A and D), 37°C (B), or 39°C (C and E), and luciferase production was determined. Results shown are the averages with standard deviations from three independent experiments. Polymerase activity shown is relative to that of the Nan PB2 WT.

FIG. 4.

Polymerase activity of Nan complexes containing PB2 double mutants. 293T (A, B, and C) or DF-1 (D and E) cells were transfected with Nan pCAGGS-NP, pCAGGS-PA, pCAGGS-PB1, and WT or mutant PB2, along with the appropriate reporter gene plasmid. Transfected cells were cultured for 24 h at 34°C (A and D), 37°C (B), or 39°C (C and E), and luciferase production was determined. Results shown are the averages with standard deviations from three independent experiments. Polymerase activity shown is relative to that of the Nan PB2 WT.

FIG. 5.

Multistep growth of rescued viruses in cultured cells. A549 (A and C) or MDCK (B and D) cells were infected with the indicated viruses at an MOI of 0.01 and cultured at 34°C (A and B) or 37°C (C and D) in the presence of trypsin at 1 μg/ml. Culture supernatants were harvested at the indicated times, and virus titers were determined.

FIG. 6.

Body weight change and lung virus titers of infected mice. (A) BALB/c mice were infected with the indicated viruses at 4 × 10⁵ TCID₅₀, and body weight was measured daily (n ≥ 3). (B) Infected mice were sacrificed on days 2, 4, and 6 postinfection, and virus titers in lung homogenates were measured in MDCK cells (n ≥ 6).

FIG. 7.

Histopathologic changes in lungs of infected mice. The images shown are hematoxylin- and eosin-stained sections of lungs from BALB/c mice infected with 4 × 10⁵ TCID₅₀ of WT (A), 271A (B), 627K (C), or 271A/627K (D) virus. Original magnification, ×20.

FIG. 8.

Polymerase activity of Cal complex containing WT or mutant PB2. 293T cells were transfected with pCAGGS-NP, pCAGGS-PA, pCAGGS-PB1, and pCAGGS-PB2 from WSN, Aichi, Nan, Cal, or Cal with mutant PB2, together with the reporter gene plasmid pPolI-NP-Luc. Transfected cells were cultured for 24 h at 34°C (A), 37°C (B), or 39°C (C), and luciferase production was determined. Results shown are the averages with standard deviations from three independent experiments.