Fundamental Contribution and Host Range Determination of ANP32A and ANP32B in Influenza A Virus Polymerase Activity

Figures

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  FIG 1. huANP32A&B are indispensable for influenza A virus polymerase activity and viral replication. (A) Wild-type 293T cells and single-gene-knockout 293T cell lines, including BUB3, CLTC, CYC1, NIBP, ZC3H15, C14orf173, CTNNB1, ANP32A, ANP32B, SUPT5H, HTATSF1, and DDX17, were transfected with firefly minigenome reporter, Renilla expression control, and the polymerase of H1N1SC09. The luciferase activity was measured 24 h after transfection, and data indicate the firefly luciferase gene activity normalized to the Renilla luciferase gene activity. Statistical differences between samples are indicated, according to a one-way ANOVA, followed by a Dunnett’s test (NS, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001). Error bars represent the SEM within one representative experiment. (B) Wild-type 293T cells were transfected with pMJ920 vector (a plasmid expressing eGFP and Cas9) and gRNAs targeting huANP32A and huANP32B to generate huANP32A&B double-knockout cells (DKO). The endogenous proteins of different cells (293T cells, huANP32A knockout cells [AKO], huANP32B knockout cells [BKO], and DKO cells) were identified by Western blotting with antibodies against β-actin, huANP32A,…

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  FIG 2. Reconstitutions of huANP32A&B rescue polymerase activity and replication of influenza A virus. We cotransfected 20 ng of huANP32A or huANP32B, 10 ng of huANP32A and 10 ng of huANP32B, or 20 ng of empty vector with either H1N1SC09 polymerase (A), H7N9AH13 polymerase (B), or WSN polymerase (C) into DKO cells and then assayed the luciferase activity at 24 h after transfection. (D and E) Increasing doses of huANP32A or huANP32B were cotransfected with H1N1SC09 polymerase into DKO cells. The luciferase activity was measured at 24 h posttransfection. For panels A to E, the data are the firefly luciferase gene activities normalized to that of the Renilla luciferase activity. Statistical differences between cells are indicated, following a one-way ANOVA and subsequent Dunnett’s test (NS, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent the SEM of the replicates within one representative experiment. (F) DKO cells were transfected with 1 μg of huANP32A and/or huANP32B, or empty vector. After 24 h, the cells were infected with WSN virus at an MOI of 0.01. The supernatants were sampled at 0, 12, 24, 36, and 48 h postinfection, and the virus titers in…

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  FIG 3. huANP32A&B determine viral RNA replication efficiency and viral production. (A to C) Wild-type 293T or DKO cells were transfected with the H1N1SC09 minigenome reporting system, together with 20 ng of empty vector, huANP32A, huANP32B, or huANP32A plus huANP32B. The cells were incubated at 37°C for 24 h before reverse transcription, followed by quantitative PCR (qRT-PCR) for vRNA, mRNA, and cRNA of the luciferase gene. Error bars represent the SD from three independent experiments (NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D and E) Replication kinetics of H1N1SC09 in ANP32 knockout cells. 293T, AKO, BKO, and DKO cells in 6-well plates were transfected with 0.5 μg each of the eight plasmids of H1N1SC09 (D), or, in DKO cells, 40 ng of huANP32A, 40 ng of huANP32B, 20 ng each of huANP32A and huANP32B, or 40 ng of empty vector was cotransfected with the eight plasmids of H1N1SC09 (E). The cells were cultured at 37°C, and supernatants were collected at 0, 12, 24, 48, and 60 h posttransfection and subjected to virus production assay by ELISA as described in Materials and Methods. Error bars indicate the SD from three independent experiments (*, P…

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  FIG 4. Support of influenza A viral replication by ANP32A or ANP32B from different species. (A) One microgram of each ANP32 plasmid was transfected into 293T cells using Lipofectamine 2000. At 48 h posttransfection, the cell lysates were analyzed using SDS-PAGE and Western blotting with antibodies against Flag peptide and β-actin. (B to J) Twenty nanograms of either empty vector or ANP32A or ANP32B from one of several different species was cotransfected with a minigenome reporter, Renilla expression control, and human influenza virus polymerase from H1N1SC09 (B), H7N9AH13 (C), canine influenza virus H3N2GD11 (D), equine influenza virus H3N8XJ07 (E) and an avian origin equine influenza virus H3N8JL89 (F), avian influenza virus H9N2ZJ12 (G), H7N9AH13 with PB2 K627E (H), H3N8XJ07 with PB2 E627K (I), or H9N2ZJ12 with PB2 E627K (J) into DKO cells. The luciferase activity was measured 24 h later. Data indicate the firefly luciferase gene activity normalized to that of Renilla luciferase activity. Statistical differences between cells are indicated, following a one-way ANOVA and subsequent Dunnett’s test (NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error…

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  FIG 5. Mapping of crucial sites in ANP32 proteins that influence viral polymerase activity. (A) ANP32B of chicken, human, and pig sequences were aligned using Geneious R6 software. The notches are marked with dashes. The similarity of amino acid identity is highlighted in different colors. (B) Schematic diagram of chimeric clones between chicken and human ANP32B, constructed according to the known domains (separated by dotted lines: 1 to 41 amino acids, LRR1 region; 42 to 110 amino acids, LRR2,3&4 region; 111 to 161 amino acids, LRRCT region; 162 to 251 amino acids, LCAR region [as showed in panel A]). The bars indicate the origins of the genes by color as follows: white, huANP32B; and blue, chANP32B. (C and D) Chimeric clones were cotransfected with minigenome reporter, Renilla expression control, and H1N1SC09 polymerase into DKO cells. The luciferase activity was measured 24 h later. Data indicate the polymerase activity normalized to that of Renilla. The statistical differences between samples are indicated, following one-way ANOVA and subsequent Dunnett’s test (NS, not significant; ****, P < 0.0001). Error bars represent the SEM of replicates within a representative experiment.

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  FIG 6. Key amino acids in ANP32A&B determine the activity of influenza viral polymerases. (A) Amino acid sequence comparison for chicken, human, and pig ANP32B proteins (amino acids 110 to 161). The amino acid sequences are available in GenBank. The black dots indicate the different amino acid within these proteins. (B to D) huANP32B mutants were cotransfected with polymerase plasmids from H1N1SC09 (B and C) or H7N9AH13 (D), plus a minigenome reporter and a Renilla luciferase control, into DKO cells. The luciferase activity was then assayed 24 h after transfection. (E and F) huANP32A mutants were cotransfected with polymerase plasmids from H1N1SC09 (E) or H7N9AH13 (F), plus a minigenome reporter and a Renilla luciferase control, into DKO cells. (G and H) chANP32A or chANP32B mutants were cotransfected with polymerase plasmids from H7N9AH13 with either PB2 627K (G) or 627E (H), respectively. (I) Mutated muANP32A or human ANP32 proteins were cotransfected with minigenome reporter, Renilla expression control, and H1N1SC09 polymerase into DKO cells. Luciferase activity was analyzed as mentioned above. The data indicate the firefly luciferase gene activity normalized to that of the Renilla…

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  FIG 7. Amino acid N129I/D130N substitutions into ANP32A&B impaired influenza polymerase activity in 293T cells. N129I/D130N substitutions of huANP32A and ANP32B on 293T chromosome were generated by the CRISPR/Cas9 system. Positive colonies were identified by sequencing and Western blotting. (A) ANP32A stable mutated cells (ASM) were identified carrying the N129I/D130N substitutions on ANP32A but not on ANP32B. (B) ANP32B stable mutated cells (BSM) were identified harboring the N129I/D130N substitutions on ANP32B but not on ANP32A. (C) Double stable mutated cells (DSM) had N129I/D130N substitutions on both ANP32A&B. (D) The endogenous protein expressions of different cell lines were identified by Western blotting with antibodies against β-actin, huANP32A, and huANP32B. (E and F) Selected 293T cell lines were transfected with firefly minigenome reporter, Renilla expression control, and polymerase from H1N1SC09 (E) or H7N9AH13 (F). Cells were assayed for luciferase activity. The data indicate the firefly luciferase gene activity normalized to that of the Renilla luciferase activity. Statistical differences between cells are given, following a one-way ANOVA and subsequent Dunnett’s test…

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  FIG 8. The 129-130 site in ANP32B contributes to the interaction with influenza polymerase. (A) Schematic showing the structure of ANP32B. LRR, leucine-rich repeat, including four LRR repeats and one C-terminal LRR domain (LRRCT); LCAR, low-complexity acidic region; NLS, nuclear localization signal. (B and C) 293T cells were transfected with different ANP32-Flag constructs, together with the viral polymerase subunits PA, PB1, and PB2. The coimmunoprecipitation of the anti-Flag antibodies and the proteins was assessed by Western blotting.