Immunopathogenic and antibacterial effects of H3N2 influenza A virus PB1-F2 map to amino acid residues 62, 75, 79, and 82

Abstract

The influenza A virus protein PB1-F2 has been linked to the pathogenesis of both primary viral and secondary bacterial infections. H3N2 viruses have historically expressed full-length PB1-F2 proteins with either proinflammatory (e.g., from influenza A/Hong Kong/1/1968 virus) or noninflammatory (e.g., from influenza A/Wuhan/359/1995 virus) properties. Using synthetic peptides derived from the active C-terminal portion of the PB1-F2 protein from those two viruses, we mapped the proinflammatory domain to amino acid residues L62, R75, R79, and L82 and then determined the role of that domain in H3N2 influenza virus pathogenicity. PB1-F2-derived peptides containing that proinflammatory motif caused significant morbidity, mortality, and pulmonary inflammation in mice, manifesting as increased acute lung injury and the presence of proinflammatory cytokines and inflammatory cells in the lungs compared to peptides lacking this motif, and better supported bacterial infection with Streptococcus pneumoniae. Infections of mice with an otherwise isogenic virus engineered to contain this proinflammatory sequence in PB1-F2 demonstrated increased morbidity resulting from primary viral infections and enhanced development of secondary bacterial pneumonia. The presence of the PB1-F2 noninflammatory (P62, H75, Q79, and S82) sequence in the wild-type virus mediated an antibacterial effect. These data suggest that loss of the inflammatory PB1-F2 phenotype that supports bacterial superinfection during adaptation of H3N2 viruses to humans, coupled with acquisition of antibacterial activity, contributes to the relatively diminished frequency of severe infections seen with seasonal H3N2 influenza viruses in recent decades compared to their first 2 decades of circulation.

Fig. 1.

Amino acid sequences of H3N2 lineage PB1-F2 proteins. Sequences are pictured for selected H3N2 strains that are representative of the evolution of this protein. Black shading indicates identity within this set of strains; gray and white highlight differences, dark blue shading highlights amino acids determined to be critical for the proinflammatory phenotype, and light blue shading highlights amino acids that differ between the pandemic strain HK68 and the seasonal strain Wuh95 but that were not necessary for the phenotype. Udo72, A/Udorn/307/72; Mem74, A/Memphis/101/74; Alb78, A/Albany/14/78; HK87, A/Hong Kong/7/87; Pan99, A/Panama/2007/99; NY10, A/New York/20343/10.

Fig. 2.

Weight loss of mice exposed to H3N2 PB1-F2-derived peptides. C-terminal peptides derived from the PB1-F2 sequences of HK68 (A) and Wuh95 (B) with the indicated alterations to the amino acid sequence were administered to groups of BALB/c mice (n = 10), and mice were monitored individually for weight loss; results are presented as mean percentages of starting weight ± standard deviations (SD). HK68-4 contained the L62P, R75H, R79Q, and L82S changes, whereas Wuh95+4 had the P62L, H75R, Q79R, and S82L changes. An asterisk indicates a significant (P < 0.05) difference in weight data on the indicated day for groups exposed to altered peptides compared to the results determined for the group exposed to the wild-type sequence of the HK68 (A) or Wuh95 (B) peptide.

Fig. 3.

Histopathologic changes in the lungs of mice exposed to H3N2 PB1-F2-derived peptides. BALB/c mice were exposed to peptides derived from HK68 (A) or Wuh95 (B), a version of HK68 altered at L62P, R75H, R79Q, and L82S (HK68-4) (D), a version of Wuh95 altered at P62L, H75R, Q79R, and S82L (Wuh95+4) (E), or PBS (C). Sections of lungs stained with hematoxylin and eosin are pictured at ×20 magnification. (F) A semiquantitative grading system was used to assess the degree of lung injury present in each set of lungs. The scores (means ± SD) determined for 3 mice per group are shown. An asterisk indicates a significant (P < 0.05) difference by ANOVA compared to the results determined for the PBS, Wuh95, and HK68-4 groups.

Fig. 4.

Inflammatory responses in mice exposed to H3N2 PB1-F2-derived peptides. The numbers (means ± SD) of total white blood cells (WBCs) (A), neutrophils (B), and macrophages (C) recovered from bronchoalveolar lavage (BAL) fluid are compared for groups of BALB/c mice (n = 4 or 5) exposed to PBS (as a control) or PB1-F2 peptides derived from HK68, Wuh95, a version of HK68 altered at L62P, R75H, R79Q, and L82S (HK68-4), or a version of Wuh95 altered at P62L, H75R, Q79R, and S82L (Wuh95+4). (D to H) The concentrations (means ± SD) of cytokines and chemokines, including G-CSF, IL-6, KC, MCP-1, and MIP-1α, were determined from the same BAL fluids. An asterisk indicates a significant (P < 0.05) difference by ANOVA compared to the results determined for the PBS, Wuh95, and HK68-4 groups.

Fig. 5.

Inflammatory response of J774 macrophages to H3N2 PB1-F2-derived peptides. J774 macrophages were exposed to PBS (as a control) or peptides derived from HK68 (A) or Wuh95 (B) at a concentration of 100, 250, or 500 μM. Four hours later, the concentration of TNF-α was determined from the supernatants; the data are expressed as the means ± SD of the results of 3 independent experiments. HK68-4 is a version of HK68 altered at L62P, R75H, R79Q, and L82S, and Wuh95+4 is a version of Wuh95 altered at P62L, H75R, Q79R, and S82L. An asterisk indicates a significant (P < 0.05) difference by ANOVA compared to the results determined for the other groups at that peptide concentration.

Fig. 6.

Effect of H3N2 PB1-F2-derived peptides on bacterial pneumonia. Weight loss (A and B), survival (C and D), and bacterial lung titers (E and F) of groups of mice (n = 8 to 10) exposed to PB1-F2 peptides derived from HK68 (A, C, and E) or Wuh95 (B, D, and F) are shown in a bacterial pneumonia model. HK68-4 is a version of HK68 altered at L62P, R75H, R79Q, and L82S, and Wuh95+4 is a version of Wuh95 altered at P62L, H75R, Q79R, and S82L. BALB/c mice were exposed to H3N2 PB1-F2 peptides or PBS and then challenged 1 day later with a dose of 2,000 CFU of S. pneumoniae (SPn) per mouse. (E) Lungs from 3 to 5 mice per group were collected at days 1, 3, and 5 after bacterial challenge and titrated for bacterial load. Error bars indicate the SD of the means. An asterisk indicates a significant (P < 0.05) difference compared to the results determined for the PBS + SPn, HK68-4 + PBS, and HK68-4 + SPn groups (A and B), all other groups (C and D), the HK68-4 group (E), and the Wuh95 group (F) by ANOVA (A, B, E, and F) and the Kaplan-Meier method followed by a log-rank test (C and D). A double asterisk indicates a significant difference in weight loss (A) and in bacterial titers (F) compared to the results determined for all other groups.

Fig. 7.

Characterization of PB1-F2 chimeric viruses in the Wuh95 background. (A) Groups of DBA/2 mice (n = 10) were infected with wild-type Wuh95/PR8 or an isogenic mutant of PB1-F2 altered at P62L, H75R, and S82L (Wuh95+3/PR8) at a dose of 10⁴ (4), 10^4.5 (4.5), 10⁵ (5), or 10⁶ (6) PFU per mouse and monitored for weight loss. (B) Growth kinetics of those viruses for a dose of 10^4.5 PFU per mouse were determined in groups of mice (n = 3 to 5 per time point). Error bars indicate the SD. An asterisk indicates a significant (P < 0.05) difference for the mutant virus compared to the results determined for the wild-type strain.

Fig. 8.

Interactions of Wuh95 PB1-F2 with bacteria. (A) Bacterial cultures (n = 4 per dose and time point) were incubated for 6 h with PBS or wild-type Wuh95 peptide, Wuh95+4 peptide, or melittin as a positive control at doses escalating from 1 to 500 μM, and titers were determined by plating serial dilutions. (B) Groups of mice (n = 10) were infected with wild-type Wuh95/PR8 (Wuh95) or an isogenic mutant of PB1-F2 altered at P62L, H75R, and S82L (Wuh95+3) and then challenged 7 days later with either PBS or 100 CFU of S. pneumoniae and monitored for weight loss. Error bars indicate the SD.