PA Mutations Inherited during Viral Evolution Act Cooperatively To Increase Replication of Contemporary H5N1 Influenza Virus with an Expanded Host Range

Figures

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  FIG 1. Polymerase activity of ancestral and contemporary clade 2.2.1 viruses. Viral polymerase activity was measured by minigenome assays in human 293T cells at 33°C and 37°C and in avian DF-1 cells at 37°C. The data are expressed relative to the results for the ancestral D1 virus. Each data point is the mean ± standard deviation (SD) from three independent experiments. Asterisks indicate a P value of <0.01.

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  FIG 2. Polymerase activity of reassortant polymerases formed by subunits from ancestral and contemporary clade 2.2.1 viruses. The viral polymerase activity of reassortant polymerases formed by subunits from ancestral D1 and contemporary EG13 viruses was measured by minigenome assays in human 293T cells at 33°C and 37°C and in avian DF-1 cells at 37°C. (A to C) Polymerase activity of reassortant polymerase complexes composed of one subunit from the D1 virus and the other subunits from the EG13 virus. The data are expressed relative to the results for wild-type EG13. (D to F) Polymerase activity of reassortant polymerase complexes composed of one subunit from the EG13 virus and the other subunits from the D1 virus. The data are expressed relative to the results for wild-type D1. Each data point is the mean ± SD from three independent experiments. (G and H) Western blot analysis of reassortant polymerase complexes without (Input) and with (IP: PB2-flag) immunoprecipitation with PB2-Flag. (G) Reassortant polymerase complexes composed of one subunit from the D1 virus and the other subunits from the EG13 virus. (H) Reassortant polymerase complexes composed of one subunit from the EG13 virus…

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  FIG 3. Phylogeny of the PA gene in clade 2.2.1 viruses isolated in Egypt. The phylogenetic tree of the PA genes of ancestral clade 2.2.1 viruses isolated in Egypt, including the D1 virus, and of contemporary clade 2.2.1.2 viruses isolated in Egypt, including the EG13 virus, was reconstructed from the nucleotide sequences of the PA genes of the Egyptian reference strains in the GISAID database. This reconstruction used the neighbor-joining method with 1,000 bootstrap replicates and was rooted to the prototype A/quail/Hong Kong/G1/1997 (H5N1) strain. The two clade 2.2.1 strains in this study are representative ancestral (D1) and contemporary (EG13) strains and are underlined and marked with black circles. CK, DK, GS, and TK in the virus strain names denote chicken, duck, goose, and turkey hosts, respectively. The PA mutations acquired during the evolution of clade 2.2.1 viruses are shown beside each branch, with the mutations grouped into two categories, i.e., 8 phylogeny-associated mutations and 2 EG13-unique mutations. The three mutations that were shown to act cooperatively in this study to increase clade 2.2.1.2 replication are underlined.

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  FIG 4. Quantification of PA expression. 293T cells were transfected with PA expression plasmids carrying the indicated mutations, and cell lysates were analyzed by Western blotting using anti-PA antibody. (A and C) After quantification of the band intensity, the amount of PA produced by each mutant was expressed relative to that for wild-type PA. (A) Expression of contemporary EG13-PA, each carrying a reverse PA mutation. (C) Expression of ancestral D1-PA, each carrying a forward PA mutation. Each data point is the mean ± SD from five independent experiments. NS indicates no statistically significant difference. (B and D) Representative images of the Western blots of each EG13-PA with a reverse mutation (B) and each D1-PA with a forward mutation (D).

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  FIG 5. Effect of PA mutations on the polymerase activity of clade 2.2.1 viruses in human and avian cells. (A to C) Polymerase activity of contemporary EG13 viruses, each carrying PA with the indicated reverse mutation, measured by minigenome assays in human 293T cells at 33°C (A) and 37°C (B) and in avian DF-1 cells at 37°C (C). The data are expressed relative to the results for wild-type EG13. (D to F) Polymerase activity of ancestral D1 viruses, each carrying PA with the indicated forward mutation, measured by minigenome assays in human 293T cells at 33°C (D) and 37°C (E) and in avian DF-1 cells at 37°C (F). The data are expressed relative to the results for wild-type D1. Each data point is the mean ± SD from five independent experiments. Asterisks indicate a P value of <0.01.

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  FIG 6. Cooperative effects of PA mutations on clade 2.2.1 polymerase activity in human and avian cells. (A to C) Polymerase activity of contemporary EG13 viruses, each carrying the indicated PA reverse mutation, singly or in combination, measured by minigenome assays in human 293T cells at 33°C (A) and 37°C (B) and in avian DF-1 cells at 37°C (C). The data are expressed relative to the results for wild-type EG13. (D to F) Polymerase activity of ancestral D1 viruses, each carrying the indicated PA forward mutation, singly or in combination, measured by minigenome assays in human 293T cells at 33°C (D) and 37°C (E) and in avian DF-1 cells at 37°C (F). The data are expressed relative to the results for wild-type D1. Each data point is the mean ± SD from five independent experiments. Asterisks indicate a P value of <0.01.

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  FIG 7. Replication kinetics of EG13 and D1 viruses carrying PA mutations in human and avian cells. (A to C) Avian DF-1 cells (A) and human Calu-3 cells (B and C) were infected with the EG13-wt virus or an EG-13 virus carrying the indicated PA mutant at MOIs of 0.003 and 0.03, respectively, and incubated at 37°C (A and B) and 33°C (C). (D to F) Avian DF-1 cells (D) and human Calu-3 cells (E and F) were infected with the reassorted D1/PA viruses carrying PA-wt or PA with the indicated mutation, PB2, PB1, and NP from EG13, and the other genes from D1, at MOIs of 0.003 and 0.03, respectively, and incubated at 37°C (D and E) or 33°C (F). At the indicated times postinfection, the progeny virus titers were measured by FFU assays. Each data point is the mean ± SD from three independent experiments.

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  FIG 8. Virulence and replication of D1 and EG13 viruses carrying PA mutations in the respiratory tract of infected mice. (A to D) Four- to five-week-old BALB/c mice (6 mice per group) were inoculated intranasally with 101 to 105 FFU of the indicated PA mutants in the D1/PA genetic background (A and B) or the EG13 genetic background (C and D). (A and C) The body weight of the infected mice was monitored daily for 14 dpi. The mean ± SD of the percentage of the initial body weight for each group of mice is shown. (B and D) Survival of the infected mice. Survival was calculated, including mice that were humanely sacrificed after they had lost more than 30% of their body weight within a few days. (E) Viral titers in the lungs of mice (6 mice per group) infected with 103 FFU of the EG13 and G1/P viruses carrying the indicated PB2 mutations at 6 dpi. Each symbol marks the titer in an individual mouse. Asterisks indicate a P value of <0.01. (F) Representative photomicrographs of hematoxylin and eosin (HE)-stained (top) and immunohistochemically (IHC) stained (bottom) lung sections from mice infected with the indicated viruses at 6 dpi. In the immunohistochemically stained tissues, the viral…

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  FIG 9. Structural model of the clade 2.2.1 polymerase complex. Shown is a structural model of the D1/PA heterotrimeric polymerase complex. (A and C) Transparent surface diagrams of the D1/PA polymerase complex composed of PB2 (blue), PB1 (pink), and PA (green) with the A448E, S388R, and E18G mutations (red). (A) The transcription preinitiation form (PDB accession number 6RR7 as the modeling template), where the polymerase complex binds to the vRNA promoter (yellow). A close-up of the polymerase complex around the vRNA promoter is also shown. (C) The apo form (without the vRNA promoter) (PDB accession number 6QPF as the modeling template). (B and D) Surface views of the PA structure in the transcription preinitiation form (B) and the apo form (D). The left and right PA structures differ by 180° in orientation. (E to G) Polymerase structure without (top) and with (bottom) the indicated PA mutations in the D1/PA heterotrimeric polymerase complex with vRNA. (E) Close-up of residue 388 in the D1/PA polymerase complex. (F) Close-up of residue 18 in the D1/PA polymerase complex. (G) Close-up of residue 448 in the D1/PA polymerase complex. Potential interactions between residues are represented…