Fig 1.
ANP32A, ANP32B, and ANP32E support influenza B viral polymerase activity to different degrees.
(A) 293T, huANP32A knockout cells (AKO), huANP32B knockout cells (BKO), and huANP32A&B double knockout cells (DKO) were transfected with firefly minigenome reporter, Renilla expression control, and B/Yamagata/1/73 or H7N9AH13 polymerase. As a negative control, 293T cells were transfected with the same plasmids, with the exception of the PB2 expression plasmid. (B) Schematic diagram of gene analysis of human ANP32A, ANP32B and ANP32E sgRNA target positions in the chromosomes. (C) 293T, huANP32E knockout cells (EKO), huANP32A&B double knockout cells (DKO), and huANP32A&B&E triple knockout cells (TKO) were transfected with firefly minigenome reporter, Renilla expression control, and B/Yamagata/1/73 or H7N9AH13 polymerase. As a negative control, 293T cells were transfected with the same plasmids, with the exception of the PB2 expression plasmid. (D) 293T, AKO, BKO, DKO, and TKO cells were infected with B/Yamagata/1/73 virus at a MOI of 0.1. The supernatants were sampled at 12, 24, 36, 48, 60, 72 h post infection and the viral titers were determined using Fluorescence Focus Units (FFU) assay on MDCK cells. The result is shown as average of n = 3 ± SD. (E) TKO cells were co-transfected with B/Yamagata/1/73 polymerase, minigenome reporter, and Renilla expression control together with 10 ng huANP32A, 10 ng huANP32B, 10 ng huANP32E, or 10 ng empty vector, and luciferase activity was assayed at 24 h after transfection. The expression of ANP32 proteins and polymerase was assessed using western blotting. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment.
Fig 2.
Species-specific support of influenza B viral polymerase activity by ANP32 proteins from different animals.
(A) TKO cells were co-transfected with B/Yamagata/1/73 polymerase, minigenome reporter, Renilla expression control and 10 ng of one of the following: huANP32A, huANP32B, huANP32E, chANP32A, chANP32B, chANP32E or 10 ng empty vector. Luciferase activity was assayed at 24 h after transfection. The expression of ANP32 proteins and polymerase was assessed using western blotting. (B) DF1 cells were co-transfected with B/Yamagata/1/73 polymerase, minigenome reporter with chicken polI promoter, Renilla expression control and 10 ng of one of the following: huANP32A-flag, huANP32B-flag, huANP32E-flag, chANP32A-flag, chANP32B-flag, chANP32E-flag or 10 ng empty vectors. Luciferase activity was assayed at 24 h after transfection. The protein expression was determined by western blotting using different antibodies: anti-flag antibody for ANP32 proteins, and specific antibodies to polymerase and β-actin. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment. (C) DF1 cells were transfected with 1 μg huANP32B-flag or empty vector in 6 well plate. Twenty-four hours post transfection DF1 cells were infected with B/Yamagata/PJ/2018 virus at a MOI of 0.1 and cultured at 33°C or 37°C. The supernatants were sampled at 12, 24, 36, 48 h post infection and the viral titers were determined using Fluorescence Focus Units (FFU) assay on MDCK cells. The expression of huANP32B was assessed by western blotting using anti-flag antibody. The result is shown as average of n = 3 ± SD.
Fig 3.
Support of influenza B viral replication by ANP32A from different species and the key amino acids responsible for the support.
(A and B) TKO cells were co-transfected with 10 ng of ANP32A from different species or empty vector with minigenome reporter, Renilla expression control, influenza B virus polymerase from B/Yamagata/1/73 (A) or B/Victoria/Brisbane/60/2008 (B). (C) ANP32A amino acid sequences from humans and chicken were aligned using the Geneious R10 software. Gaps are marked with dashes. (D to F) TKO cells were co-transfected with 10 ng of huANP32A or chANP32A or the indicated mutations with minigenome reporter, Renilla expression control, and influenza B virus polymerase of either B/Yamagata/1/73 (D and F), or B/Victoria/Brisbane/60/2008 (E). The expression of ANP32 proteins and polymerase was assessed using western blotting. Luciferase activity was measured 24 h following transfection. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment. pg, pig; eq, equine; dg, dog; os, ostrich; zf, zebra finch; dk, duck; ty, turkey; huANP32A+33, huANP32A with the 33-amino-acid insert from chANP32A; chANP32AΔ33, chANP32A without the 33-amino-acid insert missing in huANP32A; chANP32AΔSIM, chANP32A without the SIM(VLSL) sequence which is missing in huANP32A.
Fig 4.
The 129/130 site of ANP32B determines the supporting of influenza B viral polymerase.
TKO cells were co-transfected with 10 ng of either ANP32B from different species or empty vector was co-transfected with minigenome reporter, Renilla expression control, and influenza B virus polymerase from B/Yamagata/1/73 (A), or B/Victoria/Brisbane/60/2008 (B). HuANP32B or chANP32B or the indicated mutations was co-transfected with minigenome reporter, Renilla expression control, influenza B virus polymerase of B/Yamagata/1/73 (C), B/Victoria/Brisbane/60/2008 (D). The expression of ANP32 proteins and polymerase was assessed using western blotting. Luciferase activity was measured 24 h after transfection. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment.
Fig 5.
Support of influenza B viral replication by ANP32E from different species and the key amino acids responsible for the support.
(A). The protein sequences of ANP32E for human (huANP32E), pig (pgANP32E), equine (eqANP32E), dog (dgANP32E), mouse (muANP32E), zebra finch (zbANP32E), duck (dkANP32E), turkey (tyANP32E) and chicken (chANP32E) were aligned using the Geneious R10 software. huANP32E was set as the reference sequence, and colors represent similarity of amino acid identity (Black = 100%, dark grey = 80–100%, light grey = 60–80%, white = <60%). Gaps are represented by dashes. Residue numbers correspond to huANP32E. TKO cells were co-transfected with 10 ng of ANP32E from different species or empty vector was co-transfected with minigenome reporter, Renilla expression control, and influenza B virus polymerase from B/Yamagata/1/73 (B), or B/Victoria/Brisbane/60/2008 (C). (D) Schematic diagram of the chimeric clones constructed between chicken and human ANP32E. The colors of the bars show the origins of the genes as follows: grey, huANP32E; blue, chANP32E. (E) Human or chicken ANP32E, or one of the chimeric clones were co-transfected with minigenome reporter, Renilla expression control, and B/Yamagata/1/73 polymerase into TKO cells. The expression of ANP32 proteins and polymerase was assessed using western blotting. Luciferase activity was measured 24 h later. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment.
Fig 6.
A single amino acid at position 129 determines support of huANP32E and chANP32E for IBV polymerase.
(A) 129–130 site alignment of human ANP32A (huANP32A), chicken ANP32A (chANP32A), human ANP32E (huANP32E) and chicken ANP32E (chANP32E) protein sequences by Geneious R10 software. (B) Human and chicken ANP32 plasmids and different chimeric clones were co-transfected with minigenome reporter, Renilla expression control, and B/Yamagata/1/73 polymerase into TKO cells. The expression of ANP32 proteins and polymerase was assessed using western blotting. Luciferase activity was measured 24 h following transfection. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment.
Fig 7.
Different binding abilities of chicken and human ANP32A and ANP32E for IBV polymerase.
(A) Detection of the interactions of differently truncated human ANP32B proteins with IBV polymerases. 293T cells were transfected with different truncated human ANP32B-Flag constructs, together with the viral polymerase subunits PA, PB1, and PB2. The coimmunoprecipitation of the anti-Flag antibodies and the proteins was assessed using western blotting. (B) Human ANP32B or its differently truncated clones were co-transfected with minigenome reporter, Renilla expression control, and B/Yamagata/1/73 polymerase into TKO cells. The expression of ANP32 proteins and polymerase was assessed using western blotting. Luciferase activity was measured 24 h following transfection. The data indicate the 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 < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent the SD of the replicates within one representative experiment. (C to E) 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 using western blotting. Detection of the interactions of human ANP32B (huANP32B), chicken ANP32B (chANP32B) and huANP32B with N129I/D130N mutations (huANP32B_N129I/D130N) proteins with IBV polymerases (C). Detection of the interactions of human ANP32A (huANP32A), chicken ANP32A (chANP32A), and human ANP32A with 33-amino-acid insert (huANP32A+33) proteins with IBV polymerases (D). Detection of the interactions of human ANP32A (huANP32A), human ANP32B (huANP32B), human ANP32E (huANP32E), chicken ANP32A (chANP32A), chicken ANP32B (chANP32B), chicken ANP32E (chANP32E), human ANP32E with E129N mutation (huANP32E_E129N), chicken ANP32E with E129N mutation (chANP32E_E129N), human ANP32E with 10-amino-acid delete (huANP32E_△10aa) and chicken ANP32E with 10-amino-acid insert (chANP32E_10aa+) proteins with IBV polymerases (E).
Fig 8.
The 33-amino-acid insert impacts the interaction dynamics between ANP32A proteins and viral polymerase.
(A to E) Surface plasmon resonance (SPR) measurements of the binding between IBV polymerase trimeric complex (Pol3) and the ANP32 proteins or their mutants purified from 293T cells. human ANP32A (huANP32A) (A). chicken ANP32A (chANP32A) (B). human ANP32A with the 33-amino-acid insert (huANP32A+33) (C). human ANP32A C terminal truncated (huANP32A_165T) (D). human ANP32A (huANP32A) with 2 polymerase subunits (Pol2) as negative control (E). Different concentrations of ANP32 proteins were capture by the chips and shown are the corresponding sensor grams expressed in RU (response unit) versus time after subtracting the control signal. (F) Response units plotted against protein concentrations. Orange, huANP32A; green, huANP32A+33; blue, chANP32A. The binding affinity (KD) values were calculated using a 1:1 fit model produced with Biacore T200 analysis software (Biacore T200 Evaluation Software Version 3.1).
Fig 9.
Selective usage of ANP32 proteins by IAV and IBV polymerase and their molecular basis.
Schematic model of interaction between ANP32 proteins and influenza viral polymerase. ANP32A, ANP32B, ANP32E from human and chicken interact with polymerase (Pol3) of IBV, avian-origin IAV (with PB2 627E), and mammal adapted IAV (with PB2 627K). The specific amino acid residues are indicated.