Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice

Abstract

The influenza virus PB1-F2 protein is a novel protein previously shown to be involved in induction of cell death. Here we characterize the expression and the function of the protein within the context of influenza viral infection in tissue culture and a mouse model. We show that the C-terminal region of the protein can be expressed from a downstream initiation codon and is capable of interaction with the full-length protein. Using this knowledge, we generated influenza viruses knocked out for the expression of PB1-F2 protein and its downstream truncation products. Knocking out the PB1-F2 protein had no effect on viral replication in tissue culture but diminished virus pathogenicity and mortality in mice. The viruses replicated to similar levels in mouse lungs by day 3 postinfection, suggesting that the knockout did not impair viral replication. However, while the PB1-F2 knockout viruses were cleared after day 5, the wild-type viruses were detectable in mouse lungs until day 7, implying that expression of PB1-F2 resulted in delayed clearance of the viruses by the host immune system. Based on our findings and on the fact that the PB1 genomic segment was always newly introduced into some pandemic influenza viruses of the last century, we speculate that the PB1-F2 protein plays an important role in pathogenesis of influenza virus infection and may be an important contributor to pathogenicity of pandemic influenza viruses.

FIG. 1.

The C-terminal region of PB1-F2 interacts with the full-length protein and can be expressed from a downstream initiation site. (A) Pulse-chase for PB1-F2 in wt PR8 virus- and PR8delF2 virus-infected MDCK cells. MDCK cells were infected with the corresponding virus at an MOI of 1 for 12 h, pulse-labeled for 10 min, and chased for another 4 h. (B) Immunoprecipitation (IP) and Western blotting (WB) with the 26D3 PB1-F2 antibody recognizing the N terminus of the protein do not reveal additional bands of PB1-F2. HC, immunoglobulin heavy chain; LC, immunoglobulin light chain. (C) The PB1-F2 C terminus (C-T) can be expressed separately from a plasmid from a downstream initiation site. Plasmids encoding either N- or C-terminally Flag-tagged PB1-F2 were transfected into 293T cells for 24 h, and lysates were probed by Western blotting with monoclonal anti-Flag antibody. The diagram depicts the expected proteins and their downstream truncation products. N-T, N terminus; *, C-terminal domain of the protein. In panels B and C, numbers to the left of blots are molecular size markers in kilodaltons. (D) Coimmunoprecipitation of PB1-F2 N and C termini with full-length PB1-F2 protein. (Left) IP against HA-tagged N and C termini and WB against Flag-tagged full-length PB1-F2. (Right) IP against PB1-F2 with anti-F2 antibody and WB against HA-tagged C terminus. (E) Expression of PB1-F2 from the WP virus and the corresponding virus knocked out for downstream PB1-F2 initiation codons (WP-F2-1S). Cell lysates from virus-infected cells were immunoprecipitated with either anti-PB1-F2 polyclonal antiserum (F2) or anti-M1 monoclonal antibody (M), which recognizes M1 and M2 proteins.

FIG. 2.

Knocking out PB1-F2 from a highly virulent mouse-adapted WSN virus. (A) PB1-F2 knockout strategy. Arrows indicate the positions of mutations: 1, T120C; 2, C153G; 3, G291A. (B) Expression of PB1-F2 by virus. MDCK cells were infected at an MOI of 5 for 12 h, and lysates were probed by Western blotting with 26D3 antibody. Numbers to the left of blots are molecular size markers in kilodaltons. (C) Growth of WSN and WSNdelF2 viruses in MDCK tissue culture. Cells were inoculated at indicated MOIs, and infection supernatants were collected at 24, 48, and 72 h. (D) Pathogenesis of WSN and WSNdelF2 viruses in mice. Five mice per virus group were inoculated with 5 × 10² PFU of the indicated viruses, and mouse weights were assessed every day after infection. (E) Survival of mice infected with WSN and WSNdelF2 viruses as described for panel D.

FIG. 3.

Knocking out PB1-F2 from WSN virus possessing the PR8 PB1 gene (WP). (A) Growth of WP and WPdelF2 viruses in MDCK tissue culture. Cells were inoculated at the indicated MOIs, and infection supernatants were collected at 24, 48, and 72 h. (B) Pathogenesis of viruses in mice. Five mice per virus group were inoculated with 1 × 10⁶ PFU of the indicated viruses, and mouse weights were assessed every day after infection. (C) Survival of mice infected with WP and WPdelF2 viruses as described for panel B. (D) Lung virus titers from mice infected with WP and WPdelF2 viruses. Ten mice per virus group were infected with 1 × 10⁶ PFU of each virus. Five mice from each group were sacrificed on days 3 and 6, and lung virus titers were assessed by infection of MDCK cell monolayers. Error bars represent standard errors of the means.

FIG. 4.

Contribution of PB1-F2 to pathogenicity of a virus with an intermediate virulent phenotype (WPW). (A) Construction of a chimeric WSN-PR8 PB1 gene (WPW) and a WSN-PR8 PB1 gene knocked out for PB1-F2 (WP-WdelF2). The PB1-F2 protein was knocked out in a manner analogous to that described in the legend for Fig. 2. (B) Replication of WPW and WP-WdelF2 viruses in tissue culture. Cells were inoculated at the indicated MOIs, and infection supernatants were collected at 24, 48, and 72 h. (C) Pathogenesis of viruses in mice. Five mice per virus group were inoculated with 4 × 10⁵ PFU of the indicated viruses, and mouse weights were assessed every day after infection. (D) Survival of mice infected with WPW and WP-WdelF2 viruses as described for panel C. (E) Mouse lung virus titers. Twenty-five mice per virus group were infected with 4 × 10⁵ PFU of each virus. Five mice from each group were sacrificed on days 3, 5, 6, 7, and 9, and lung virus titers were assessed by infection of MDCK cell monolayers. Error bars represent standard errors of the means. (F) Viral pathogenesis in BALB/c mice. Five mice per group were inoculated with 2 × 10⁵ PFU of the indicated viruses, and mouse weights were assessed every day after infection.

FIG. 5.

Contribution of A/HK/156/97 PB1-F2 to viral virulence. (A) Growth of the WH and WHdelF2 viruses in tissue culture. Cells were inoculated at the indicated MOIs, and infection supernatants were collected at 24, 48, and 72 h. (B) Pathogenesis of the WH and the WHdelF2 viruses in mice at 4 × 10⁵ PFU. Eight mice per virus group were inoculated with 4 × 10⁵ PFU of the indicated viruses, and mouse weights were monitored over the next 12 days. (C) Pathogenesis of the WH and the WHdelF2 viruses in mice at 2 × 10³ PFU. The experiment was performed as described for panel B, with eight mice per virus group. (D and E) Survival of mice infected with WH and WHdelF2 viruses, as described for panels B and C.