Fig 1.
Species-dependent support of ANP32A or ANP32B for Influenza A viral replication.
Vectors carrying 20 ng of ANP32A or ANP32B proteins, or empty vectors, were co-transfected into DKO cells, together with a minigenome reporter, a Renilla expression control, and influenza virus polymerases from either avian influenza H7N9ZJ13 (A) or H9N2ZJ12 (B); human influenza WSN (C); swine influenza H1N1NC08 (D); canine influenza H3N2GD11 (E); or equine influenza H3N8XJ07 (F). Luciferase activity was measured 24 h later. (Data are Firefly activity normalized to Renilla, Statistical differences between cells are labeled according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The results represent at least three independent experiments.) ch, chicken; hu, human; sw, swine; eq, equine; ca, canine.
Fig 2.
ANP32A proteins from different species interact with different polymerase trimeric complexes.
DKO cells were transfected with different ANP32A (0.6μg) and polymerase plasmids (0.6μg PA, 1μg PB1, and 1μg PB2) from avian influenza viruses H7N9ZJ13 (A), H9N2ZJ12 (B), human influenza virus polymerase WSN (C). The cells were lysed at 24 h post-transfection. Co-IP was performed using Anti-FLAG M2 Magnetic Beads, followed by Western blotting to detect the ANP32A and viral proteins by using specific antibodies: PA antibody (NBP2-42874, NOVUS), PB1 antibody (NBP2-42877, NOVUS), PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).
Fig 3.
Knockout of ANP32A in pig cells sharply reduced avian influenza viral RNA replication.
(A) The scheme of sgRNA used for knocking out the swANP32A gene. PK15 cells were transfected with pMJ920 vector (plasmid expressing eGFP and Cas9) and gRNAs to generate swANP32A knockout cells (PK15-AKO). (B) The endogenous proteins in wild type and knockout PK15 cells were identified by western blotting using antibodies against β-actin (sc-47778, Santa Cruz) and swANP32A (Rabbit Anti-PHAP1 polyclonal antibody, bs-6083R, Bioss). (C) Wildtype and AKO PK-15 cells were seeded in 96-well plate at 1x10^4 cells per well, 10 μL of CCK-8 reagents (meilunbio, MB4420) were added at the indicated times, and then the 96-well plate was incubated at 37°C for 1.5 h. The optical density (OD) values were measured by a microplate reader at OD450nm wavelength. (D) Wild type and knockout PK15 cells were transfected with Firefly minigenome reporter, Renilla expression control, together with polymerases from the avian influenza viruses H7N9ZJ13, or H9N2ZJ12, or the human influenza virus WSN. Luciferase activity was measured at 24 h post transfection. Data are Firefly activity normalized to Renilla. Statistical differences between cells are labeled according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, ***P < 0.001. (E and F) Wild type and knockout PK15 cells were infected with avian influenza virus H9N2ZJ12 and human influenza virus WSN at an MOI of 0.1, respectively. The supernatants were sampled at 0, 12, 24, 36, 48 h post infection and the virus titers were determined by means of endpoint titration in PK15 cells. (Statistical difference between groups were labeled, according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, ****P < 0.0001).
Fig 4.
Phylogenetic analysis and amino acid sequence alignments of ANP32A proteins from different species.
Bootstrap support values are indicated near the selected nodes. Right-hand columns display different amino acids in vertebrate ANP32A proteins.
Fig 5.
Mapping the unique sites of swANP32A responsible for the support for avian influenza viral replication.
(A) Schematic of alignment from mammalian ANP32A amino acid sequences including human ANP32A (huANP32A), canine ANP32A (caANP32A), equine ANP32A (eqANP32A) and swine ANP32A (swANP32A). Three residues were annotated as there are mutations on swANP32A, at positions 106, 156, and 228. (B, C) The mutants of swANP32A were constructed by overlapping PCR. DKO cells were co-transfected with expression plasmids carrying PB1 (40 ng), PB2 (40 ng), PA (20 ng) and NP (80 ng) from H7N9ZJ13, together with 40 ng minigenome reporter and 10 ng Renilla luciferase expression plasmids (pRL-TK, as an internal control) in the presence of swANP32A or its mutants or empty vector. Cells were then lysed using passive lysis buffer and luciferase activity was measured at 24 h post transfection. (D) The mutants of huANP32A were co-transfected into DKO cells together with polymerase plasmids from H7N9ZJ13, plus a minigenome reporter and a Renilla luciferase control. Luciferase activity was analyzed as described above. (E to G) The mutants of huANP32A or swANP32A were co-transfected together with plasmids carrying the polymerases from H7N9ZJ13 (E), H9N2ZJ12 (F), or WSN (G). Luciferase activity was analyzed as described above. (B to G, data are Firefly activity normalized to Renilla. Statistical differences between cells are labeled according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 6.
106V/156S sites of swANP32A are crucial for the interaction with avian polymerase trimeric complexes but not with mammalian polymerase.
DKO cells were transfected with different ANP32A or mutants or empty vector (0.6μg), and plasmids carrying components of the viral polymerase (0.6μg PA, 1μg PB1, and 1μg PB2) from avian influenza virus H7N9ZJ13 (A), H9N2ZJ12 (B), or human influenza virus WSN (C). The cells were lysed at 24 h post-transfection. Co-IP was performed using Anti-FLAG M2 Magnetic Beads, followed by Western blotting to detect the ANP32A and viral proteins by using specific antibodies, PA antibody (NBP2-42874, NOVUS), PB1 antibody (NBP2-42877, NOVUS), PB2 antibody (NBP2-42879, NOVUS), Anti-Flag antibody (F1804, SIGMA).
Fig 7.
Effect of the ANP32A 106/156 sites on avian influenza viral polymerase transcription and replication.
DKO cells were transfected with the minireplicon system of avian influenza virus H7N9ZJ13, and a minigenome reporter pHH21-huPolI-vLuc, together with different ANP32A mutants or empty vector. Total RNA of DKO cells was extracted at 24 h post-transfection and reverse transcription was performed, followed by quantitative PCR (qRT–PCR) for vRNA (A), cRNA (B), and mRNA (C) from the luciferase gene. Luciferase RNA levels were normalized to the β-actin RNA level (means SD from three independent experiments. NS = not significant, ****P < 0.0001; all by one-way ANOVA followed by a Dunnett’s test). The expression of different ANP32A mutants were evaluated by Western blotting.
Fig 8.
106V/156S sites of swANP32A are responsible for the infectivity of avian influenza virus in PK15 cells.
Plasmids (200ng) carrying ANP32A or mutants, or empty vectors were co-transfected with plasmids carrying polymerase from H7N9ZJ13 (A) or H9N2ZJ12 (B). Luciferase activity was assayed at 36 h after transfection. (Data are Firefly activity normalized to Renilla. Statistical differences between cells are labeled according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, ***P < 0.001, ****P < 0.0001). (C) PK15-AKO cells were transfected with 1μg ANP32A or mutants or empty vector. After 24 h, the cells were infected with avian influenza virus H9N2ZJ12 at an MOI of 0.1. The supernatants were collected at 0, 12, 24, 36, 48 h post infection and the virus titers in these supernatants were determined as above. The expressions of different ANP32A mutants were evaluated by Western blotting. (Statistical difference between cells were labeled, according to a one-way ANOVA followed by a Dunnett’s test; NS = not significant, ****P < 0.0001).
Fig 9.
Proposed model of ANP32 proteins as “the key” for the inter-species transmission of avian influenza A virus in different species.
AIVs can replicate and transmit naturally in birds. Pigs are thought to play an important role in influenza virus transmission and triple-reassortant virus generation, because they are susceptible to both avian and mammalian influenza viruses, and different species of influenza viruses can therefore genetically recombine in pigs, producing new strains that can transmit to humans. Avian ANP32A can support the replication of the avian influenza virus due to an additional 33-aa insertion; huANP32A only can support the polymerase activity of mammalian or mammal-adapted (e.g. PB2 E627K, or D701N) viruses, but not that of avian viruses); as swANP32A contains a unique active site 106V/156S, it can support the replication of not only all mammalian influenza viruses, but also avian influenza viruses. The co-infection of two or more virus strains in pig providing more chances to produce new recombinant strains, which can spread to other mammals such as humans.