Selective usage of ANP32 proteins by influenza B virus polymerase: Implications in determination of host range

Zhenyu Zhang; Haili Zhang; Ling Xu; Xing Guo; Wenfei Wang; Yujie Ji; Chaohui Lin; Yujie Wang; Xiaojun Wang

doi:10.1371/journal.ppat.1008989

. 2020 Oct 12;16(10):e1008989. doi: 10.1371/journal.ppat.1008989

Selective usage of ANP32 proteins by influenza B virus polymerase: Implications in determination of host range

Zhenyu Zhang ^1,^#, Haili Zhang ^1,^#, Ling Xu ¹, Xing Guo ¹, Wenfei Wang ², Yujie Ji ¹, Chaohui Lin ¹, Yujie Wang ¹, Xiaojun Wang ^1,^*

Editor: Martin Schwemmle³

PMCID: PMC7580981 PMID: 33045004

Abstract

The influenza B virus (IBV) causes seasonal influenza and has accounted for an increasing proportion of influenza outbreaks. IBV mainly causes human infections and has not been found to spread in poultry. The replication mechanism and the determinants of interspecies transmission of IBV are largely unknown. In this study, we found that the host ANP32 proteins are required for the function of the IBV polymerase. Human ANP32A/B strongly supports IBV replication, while ANP32E has a limited role. Unlike human ANP32A/B, chicken ANP32A has low support activity to IBV polymerase because of a unique 33-amino-acid insert, which, in contrast, exhibits species specific support to avian influenza A virus (IAV) replication. Chicken ANP32B and ANP32E have even lower activity compared with human ANP32B/E due to specific amino acid substitutions at sites 129–130. We further revealed that the sites 129–130 affect the binding ability of ANP32B/E to IBV polymerase, while the 33-amino-acid insert of chicken ANP32A reduces its binding stability and affinity. Taken together, the features of avian ANP32 proteins limited their abilities to support IBV polymerase, which could prevent efficient replication of IBV in chicken cells. Our results illustrate roles of ANP32 proteins in supporting IBV replication and may help to understand the ineffective replication of IBV in birds.

Author summary

Influenza B viruses infect humans and few other mammals, but fairly rare in birds. Here we found that IBV requires the involvement of host ANP32 proteins in the replication process, in which ANP32A and ANP32B play major roles and can fully support polymerase activity independently, while ANP32E gives only limited support to IBV polymerase because of certain substitutions compared with ANP32A/B. Chicken ANP32A has a 33-amino-acid insert not present in mammals and provides better support to avian IAV polymerase, but this insert impairs its support for IBV polymerase activity. Chicken ANP32B and ANP32E have even lower support to IBV polymerase due to specific inactive mutations at sites 129/130. Our findings reveal an important role for ANP32 proteins in IBV polymerase activity and suggest the possible molecular basis of adaptation and restriction of IBV infection in different species.

Introduction

Influenza A and Influenza B are major infectious respiratory tract diseases and cause significant morbidity and mortality in humans. The influenza A virus (IAV) and influenza B virus (IBV) belong to the group orthomyxoviridae, which are characterized by a single-stranded segmented RNA genome and enveloped spherical or filamentous particles with studded surface proteins [1, 2]. Influenza A viruses can infect a broad range of hosts including humans, other mammals, and birds. Based on antigenicity of the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) have been discovered on the influenza A viruses isolated from different hosts. Unlike influenza A viruses, all influenza B viruses have been identified as belonging to two genetic and antigenic lineages (based on the HA protein): the B/Yamagata lineage and the B/Victoria lineage, and both lineages circulate mainly in the human population [3]. IAVs of subtypes H1N1 and H3N2, and IBVs of both lineages are the main pathogens responsible for seasonal influenza [4]. It is estimated that in the US 2019–2020 season, there were at least 36 million cases of illness and 22,000 deaths from flu, of which half were caused by IBV (https://www.cdc.gov/flu/weekly/index.htm#ILIMap).

Mammalian cells have been commonly observed to have a restriction effect on infection by avian IAV and the mechanism of this interspecific infection restriction of IAV has been long studied. Some H1, H5 or H7 subtype IAV viruses can overcome the restriction by evolving certain mutations of polymerase, such as PB2 E627K or G590S and Q591R, and gain ability to replicate in mammals [5–10]. One of the main barriers to avian IAV infection of mammals is that avian viral polymerase is poorly adapted to the host acidic nuclear phosphoprotein 32 family member A (ANP32A) molecules [11–17]. Most avian ANP32A has a special 33-amino-acid insert which enhances its ability to support polymerase of IAVs from both mammals and birds. Mammalian ANP32A and ANP32B without the 33-amino-acid insert do not support avian viral polymerase [11]. The ANP32 family includes three conserved family members: ANP32A, ANP32B, and ANP32E. Previously we and other lab have identified ANP32A and ANP32B as the host molecules critical for determining the polymerase activity of influenza A viruses in different hosts [11, 13, 14], and among mammalian ANP32 proteins the swine ANP32A shows unique feather in supporting chicken AIV replication in pig cells [18]. Three splice variants of ANP32A in avian species that harbor 33, 29, or no special amino acid insertion have been identified and showed different supports to avian polymerase. In some avian species like swallow and goose the shorter variants may help to drive or maintain some mammalian-adaptive IAV polymerase mutations [12, 15].

IBVs are believed to be stably adapted to humans and are continually circulating in the human population. IBVs have also occasionally been isolated from other mammals, including dogs, pigs, and harbor seals [19–22], and there are reports from serological evidence of influenza B infection in dogs, guinea pigs, ruminants, and chimpanzees [23–26]. One early paper reported that zoo birds were infected with influenza B viruses [27], but there have been no similar reports since 1980s. This suggests that mammals are more susceptible to IBV than birds, but the mechanism or the reasons for this have never been clear.

Here we created different ANP32 protein knockout cell lines, and demonstrated that human ANP32A and ANP32B proteins are required for the polymerase activity of the influenza B virus. Unlike the polymerase of IAV, the IBV polymerase can use ANP32E, albeit with limited activity. Intriguingly, we found that chicken ANP32A has a much lower ability to support IBV polymerase activity than does mammalian ANP32A, mainly because it harbors a 33-amino-acid insert not present in mammals, which is required to support avian IAV replication. These 33 amino acids give an advantage to avian IAV, but in IBV, they become a barrier. We also found that although chicken ANP32B and ANP32E are all in “short-form” like those proteins from humans, they both have functionally inactive mutations at sites 129/130, resulting in the loss of support to the IBV polymerase. These findings reveal an important role for ANP32 proteins in IBV polymerase activity and suggest the molecular basis of restriction of IBV infection in chickens.

Results

Human ANP32A, ANP32B, and ANP32E support influenza B viral polymerase activity to different degrees

IBV has similar replication mechanism to that of IAV. Our previous work and results from other labs showed that ANP32A&B proteins play a crucial role in supporting the polymerase activity of IAV [14, 28], and we wanted to investigate whether ANP32 proteins were also indispensable for IBV replication. We had previously constructed 293T knockout cell lines, including ANP32A knockout (AKO), ANP32B knockout (BKO), and ANP32A and ANP32B double knockout (DKO) cell lines, and identified that the polymerases from human seasonal H1N1 influenza viruses and human adapted H7N9 virus have similar dependence on the ANP32 proteins [14]. Here we confirmed that the polymerase of IAV H7N9_AH13 had similar levels of activity in AKO cells, BKO cells, and wild type 293T cells. However, in DKO cells, the H7N9_AH13 viral polymerase activity decreased sharply by about 5000-fold, which was comparable to the background value of polymerase activity (293T del PB2) (Fig 1A). The activity of the IBV polymerase reduced by 100-fold in DKO cells, but was still much higher than the background value (293T del PB2) (Fig 1A). We speculated therefore that the conserved ANP32 family member, ANP32E, may contribute to the support of IBV polymerase activity. ANP32E has a similar sequence to those of both ANP32A and ANP32B, but is not able to support IAV replication [14, 29]. Therefore, based on our DKO cell line, we knocked out ANP32E to make an ANP32A, ANP32B, and ANP32E triple knockout (TKO) cell line (Fig 1B). The viability of TKO cells is as good as the 293T cells (S1 Fig). We found that IAV polymerase activity showed no significant differences between DKO and TKO cells, while the polymerase activity of IBV decreased significantly (10-fold) in TKO cells compared with that in DKO cells (Fig 1C). These results suggested that ANP32E is able to give certain support for IBV polymerase activity, although the support is mild. To investigate the contribution of ANP32 proteins in supporting viral replication, an IBV strain B/Yamagata/PJ/2018, which showed comparable replication efficiency in 293T cells with that in MDCK cells (S2 Fig), was used to infect 293T cells and the ANP32 protein knock out cells. We found that B/Yamagata/PJ/2018 could replicate well in wild-type 293T, AKO, and BKO cells, but not in DKO or TKO cells (Fig 1D). Reconstitution of either ANP32A or ANP32B in TKO cells completely restored the IBV polymerase activity, but reconstitution of ANP32E was able only partially to restore the viral polymerase activity (Fig 1E). Dose-dependent experiments showed that transfection of 10 nanograms of pCAGGS-ANP32A or ANP32B was enough to recover IBV polymerase activity (S3A and S3B Fig), while even high doses of ANP32E were not able to restore polymerase activity completely (S3C Fig). These results confirm that ANP32A and ANP32B play a major role in the support of IBV replication through promoting polymerase activity; however, ANP32E shows limited IBV polymerase support, which is not enough to support IBV replication at its natural expression level in DKO cells.

Fig 1 — (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 H7N9_AH13 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 H7N9_AH13 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.

Species-specific support of influenza B viral polymerase activity by ANP32A proteins from different animals

IBV mainly infects humans and a few other mammals, and there is no strong evidence of infection in birds, indicating a species specific evolution pattern of IBV. Recently, ANP32 proteins have been reported as key host factors limiting the spread of IAV from birds to mammals. Whether ANP32 proteins from different species give differing supports to IBV, and whether they can act as a barrier limiting the spread of IBV between species, are unclear. We compared the ability of human and chicken ANP32 proteins to support IBV polymerase activity in TKO cells, and we found that human ANP32A (huANP32A) and ANP32B (huANP32B) gave strong support to IBV polymerase, but chicken ANP32A (chANP32A), chicken ANP32B (chANP32B), and chicken ANP32E (chANP32E) gave only weak support to IBV replication, levels which were comparable to human ANP32E (huANP32E) (Fig 2A). Similarly, in wild-type chicken DF-1 cells, overexpression of huANP32A or huANP32B dramatically increased IBV polymerase activity, while chANP32A and huANP32E exhibited weak support. Surprisingly, the chANP32B or chANP32E had a negative effect on IBV polymerase activity, which could be a negative effect caused by over expression of a non-functional ANP32 protein (Fig 2B). Furthermore, we observed overexpression of huANP32B in DF1 cells could enhance IBV infectivity (Fig 2C). The above results confirmed that huANP32A&B give strong but species specific support to polymerase of IBV compared with the chicken proteins chANP32A&B.

Fig 2 — (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.

Because ANP32A proteins from humans and other mammals are in “short form” compared with chANP32A, we next investigated the support given by ANP32A proteins from different species to the polymerase activity of the two IBV strains B/Yamagata/1/73 and B/Victoria/Brisbane/60/2008. We found that ANP32A from mammals (human, pig, horse, and dog) and ostrich, that are all in “short form” without 33-amino-acid insert (S4 Fig), supported IBV polymerase activity significantly more than the ANP32As from poultry (duck, turkey, and chicken) or finch, which all have a 33-amino-acid insert (Fig 3A and 3B). Sequence alignment of mammalian with avian revealed that avian ANP32A contains an additional 33-amino-acid insert comprising a predicted SUMO interaction motif-like sequence (SIM) and a 27 amino acid repeat sequence [12, 13] (Fig 3C). We next investigated whether the 33 additional amino acids were responsible for the difference in activity between the ANP32A proteins. Insertion of the avian-specific 33 amino acids into human ANP32A (huANP32A₊₃₃) reduced the polymerase activity in TKO cells, giving it an activity similar to chicken ANP32A. Conversely, deletion of the 33 amino acids from chicken ANP32A (chANP32A_Δ33) increased the polymerase activity to a level similar to that of huANP32A (Fig 3D and 3E). This suggests that the 33 amino acids difference between mammals and most birds is indeed an important domain in determining the different activities of IBV polymerase. Interestingly, this phenomenon reversed in IAV, where avian ANP32A proteins with the 33-amino-acid insert support the replication of avian-sourced IAV as well as human strains.

Fig 3 — (**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; chANP32A_Δ33, chANP32A without the 33-amino-acid insert missing in huANP32A; chANP32A_ΔSIM, chANP32A without the SIM(VLSL) sequence which is missing in huANP32A.

The 33 extra amino acids comprise 27 amino acids identical to those in the section neighboring the insert, together with 6 specific amino acids. Previous research has shown that 4 of these 6 amino acids (VLSL) make up a SUMO-interaction motif, and substitution or deletion of these four amino acids can weaken the replication of the avian influenza virus [12, 13]. Chickens have an ANP32A isoform that lacks these four hydrophobic residues, which slightly reduces its support to avian IAV polymerase [12, 13]. We found that deletion of this SUMO interaction motif (SIM)-like sequence from chANP32A (chANP32AΔSIM) could not alter IBV polymerase activity (Fig 3F). In conclusion, the ability of chANP32A to support IBV polymerase activity is much lower than that of mammalian ANP32A, because of a 33 or 29 amino acid insert in chANP32A. This result provides evidence of differential usage of ANP32A by IAV and IBV and indicates a potential role of the 33-amino-acid insert in species specific host-virus selection and evolution pattern.

Avian ANP32B has low support of influenza B viral polymerase

All ANP32B proteins from different mammalian species lack the 33-amino-acid insert compared with chANP32A, although the chANP32B has a longer C-terminal of LCAR region than that of huANP32B. We showed that huANP32B gives strong support to IBV replication, while chANP32B gives only limited support to the IBV polymerase (Fig 4A and 4B). Our previous studies have demonstrated that chANP32B is naturally non-functional and cannot support the activity of IAV polymerase at all, because it lacks the 129N/130D functional signature found in other ANP32Bs, having instead 129I/130N, but not the longer LCAR tail [14]. Murine ANP32B (muANP32B), which encodes 129S/130D different from the more common 129N/130D found in huANP32B, huANP32A, and chANP32A, had been reported to support both IAV and IBV polymerase [28], could support IBV polymerase at similar level as huANP32B (Fig 4A and 4B). In order to verify whether this phenomenon was caused by the difference in 129/130 sites, huANP32B_N129I/D130N and chANP32B_I129N/N130D mutants were constructed and tested. The results show that the activity of the viral polymerase supported by huANP32B_N129I/D130N is significantly decreased compared with huANP32B, to the same extent as chANP32B. However, chANP32B I129N/N130D had a significantly increased viral polymerase activity compared with chANP32B, which was similar to the activity of huANP32B (Fig 4C and 4D). These results show that chicken ANP32B has limited support for IBV polymerase activity compared with that from mammals, and that this effect is caused by the different amino acids at positions 129/130.

Fig 4 — 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.

Avian ANP32E has a limited ability to support influenza B viral polymerase activity

HuANP32E is an important member of the ANP32 family and has been shown to have histone chaperone activity [30, 31]. We found that huANP32E has no impact on IAV polymerase activity but gives limited support to IBV polymerase activity (Fig 1 and Fig 2). However, whether ANP32E proteins from other animals, especially those from birds, can support IBV replication is largely unknown. Alignment of ANP32E sequences from different species indicated that there were two major differences in the C-terminus of ANP32E between mammals and birds. The first one was located upstream of the 200^th amino acid, presenting a consecutive ten acidic amino acids deletion in birds; the second one was located downstream of the 200^th amino acid, with eight consecutive amino acids differences between mammals and birds (Fig 5A). We first compared the support of ANP32Es from various species for IBV polymerase activity. The results showed that the ability of ANP32Es from mammals (human, pig, horse, dog, and mouse) to support IBV polymerase activity was nearly 10 times higher than that of ANP32Es from birds (zebra finch, duck, turkey, and chicken) (Fig 5B and 5C). To map the critical residues that determine the differences between mammalian and avian ANP32Es, we generated and tested certain chimeric clones between huANP32E and chANP32E (Fig 5D). We found that replacement of the 140 C-terminal amino acids of huANP32E with those of chANP32E (hu-chANP32E) reduces the activity of huANP32E to a level to that of chANP32E, and conversely, replacement of the C-terminus of chANP32E with that of huANP32E (ch-huANP32E) increases the level of activity chANP32E to that of huANP32E (Fig 5E). Exchanging of the N-terminal 200 amino acids between chANP32E and huANP32E revealed that the key region determining the differences in ability to support IBV polymerase activity was located between the 140^th and the 200^th amino acid, in the consecutive acidic amino acid (D & E amino acid) insertion region. To verify this, we conducted our transfection experiments using four mutants: 10-amino-acid deletion in huANP32E (huANP32E_Δ10aa), 10-amino-acid insert in chANP32E (chANP32E_10aa+), and an 8-amino-acid replacement between huANP32E and chANP32E (huANP32E_8aamut and chANP32E_8aamut). The results showed that huANP32E_Δ10aa had similar ability to support IBV polymerase to that of chANP32E; chANP32E_10aa+ had similar activity to huANP32E; and that the 8-amino-acid replacement did not change the activity of huANP32E and chANP32E at all (Fig 5E). These results suggested that the deletion of the consecutive acidic amino acid (D & E amino acid) region in chANP32E was the main cause for the lower activity. We also noticed murine ANP32E (muANP32E) lacks the 10-amino-acid insert, which is different from other mammalian ANP32E. While muANP32E has a double aspartic acid insert at position 162–163, which is absent in other mammalian and avian ANP32E. We found that muANP32E has lower support to IBV polymerase then that of the other mammal (Fig 5A–5C), and the reason need to be further studied.

Fig 5 — (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.

A single amino acid at position 129 determines the support of ANP32Es to IBV polymerase

The above results showed that the activity of huANP32E in supporting IBV polymerase activity was 10 times lower than that of huANP32A or huANP32B (Fig 2A), and the avian ANP32Es have even lower support for IBV than huANP32A&B or those from other mammals (Fig 5B and 5C). Previous work demonstrated that chicken ANP32B provided no support for IAV polymerase because it harbors a 129I/130N signature which is a natural mutation away from the functional 129N/130D signature in other ANP32B molecules [14, 32]. By comparing the sequences, we found that huANP32E and chANP32E both have a Glutamic Acid (E) at amino acid position 129, which is conserved in ANP32E in most mammalian and avian species, while there is an Asparagine (N) in that position in ANP32A and ANP32B in mammals (Fig 6A). To investigate the impact of this 129E residue in ANP32E on its support of IBV polymerase, we generated two mutants, huANP32E_E129N and chANP32E_E129N. We found that in our co-transfection experiments, the support of huANP32E_E129N for IBV polymerase was significantly higher than that of huANP32E, and reached a level comparable to that of huANP32A; while the support of chANP32E_E129N for IBV polymerase activity was significantly higher than that of either chANP32E or chANP32A, and was not different from that of huANP32A (Fig 6B). These results suggest that the 129E is responsible for the low ability of chANP32E and huANP32E to support the viral polymerase.

Fig 6 — (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.

Different abilities of chicken and human ANP32A and ANP32E to bind to IBV polymerase

ANP32A and ANP32B are proposed to only bind to the complete IAV viral polymerase heterotrimeric complex, but do not bind the single subunit of polymerase to promote polymerase activity [12, 14, 15]. It has been suggested that the C-terminal acidic tail (LCAR) and amino acids 129–130 are important in the maintenance of the interaction between ANP32B and the influenza viral polymerase [14]. Whether either ANP32A or ANP32B can bind to the IBV polymerase is unknown. We performed a co-immunoprecipitation assay between IBV polymerase and ANP32 proteins, and found that huANP32B co-immunoprecipitated with IBV polymerase, but that the huANP32B C-terminal deletion mutants huANP32B_165T and huANP32B_190T cannot bind with the viral polymerase; and the huANP32B_165T does not have the ability to support IBV polymerase activity, while huANP32B_190T has weak support (Fig 7A and 7B). However, huANP32B_216T, in which 35 amino acids were deleted, retained the ability to bind and support IBV viral polymerase (Fig 7A and 7B). When we compared the abilities of huANP32B, chANP32B, and a huANP32B with the functionally inactive mutations N129I/D130N, to bind and support the IBV polymerase, we found that chANP32B and huANP32B_N129I/D130N showed weaker binding ability to the IBV polymerase compared with that of huANP32B (Fig 7C), which is consistent with the polymerase activity assay. HuANP32A also showed strong binding to the IBV polymerase (Fig 7D). Inconsistent with the polymerase assay results, in which chANP32A gave lower support to the IBV polymerase than did huANP32A (Fig 3D and 3E), chANP32A and huANP32A₊₃₃ showed similar binding to IBV polymerase to that of huANP32A (Fig 7D), suggesting that the immunoprecipitation results are not correlated with the viral polymerase activity.

Fig 7 — (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).

We further confirmed that huANP32E, chANP32B, and chANP32E have lower binding ability to IBV polymerase compared that of huANP32A, huANP32B, and chANP32A. The E129N mutations in huANP32E and chANP32E enhance the binding to IBV polymerase, while with or without the 10-amino-acid insert in huANP32E or chANP32E could not affect their interaction with IBV polymerase (Fig 7E). These data are consistent with the supporting activity of different ANP32Es to IBV polymerase.

To investigate and characterize the interaction dynamics of huANP32A and chANP32A with the viral polymerase, we next carried out a surface plasmon resonance (SPR) assay to evaluate the binding kinetics and affinity between polymerase and ANP32 proteins. HuANP32A, chANP32A, huANP32A+33, and huANP32A_165T were fusion expressed at downstream of GST-HRV3C peptide in a pCAGGS vector and purified using Glutathione Sepharose 4B and then digested by PreScission Protease. The purity of ANP32 proteins was checked using SDS-PAGE analysis and western blotting (S5 Fig). IBV polymerase PB1, PB2 and PA-His were expressed in 293T cells and the expression was checked by Ni Sepharose purification followed by SDS-PAGE analysis and mass spectrometry identification (S5 Fig).

Consistent with our co-IP results, all these three ANP32A proteins (huANP32A, chANP32A and huANP32A₊₃₃) at concentrations of 0.15625–5 μM had valid binding stability to the viral polymerase immobilized on the CM5 chip in a dose-dependent manner (Fig 8A–8C). In contrast, huANP32A_165T has almost no binding to the polymerase (Fig 8D). In another control experiment, in which only two polymerase subunits (PA and PB1) were immobilized on the CM5 chip, no specific affinity was detected between huANP32A and the viral polymerase subunits (Fig 8E). However, the calculated dissociation constants (KD) for huANP32A, chANP32A and huANP32A₊₃₃ from the SPR assay showed significant differences. In the SPR assay, the binding stability is highly related to the KD value, with the smaller value, the more stable the interaction. We found that the huANP32A binds to the IBV viral polymerase with a KD = 0.05418 μM, almost 3-fold lower than that of chANP32A. The KD value for huANP32A and polymerase binding also increased from 0.05418 μM to 0.1013 μM for huANP32A with the 33-amino-acid insert (Fig 8F). Although huANP32A, chANP32A and huANP32A₊₃₃ have similar dissociation rate constants (kd), the association rate constants (ka) were distinctly different. The ka of huANP32A was twice as high as those from the other two proteins. This result indicated that huANP32A has a stronger binding affinity to IBV polymerase than chANP32A, and that the 33-amino-acid insert in chANP32A reduces the binding affinity of ANP32A to IBV polymerase. These results may help to explain why chANP32A gives only low support to the IBV polymerase.

Fig 8 — (**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).

Discussion

Both IAV and IBV belong to the family of Orthomyxoviruses and are the two main types of influenza virus that cause epidemical infection in humans every year. IAV has been investigated extensively because it can cause both seasonal infection and pandemics. However, although accumulating evidence shows that IBV is also an important pathogen that causes high morbidity and mortality in human populations, the characters and replication mechanism of IBV remain largely unknown. In this study, we demonstrate that the polymerase activity of IBV depends on the human cellular ANP32 proteins, of which ANP32A and ANP32B gave strong support to IBV polymerase activity. ANP32E, a member of ANP32 family that was shown no function in supporting IAV polymerase, gave mild support to IBV polymerase activity. Avian ANP32 proteins gave weak support to IBV replication, which we demonstrate to be because they harbor certain mutations compared with these proteins in mammals. These results revealed the importance of ANP32 proteins in IBV replication and the distinct species-specific usage of ANP32 proteins of IAV and IBV polymerase.

The ANP32 family comprises several members, including ANP32A, ANP32B, ANP32C, ANP32D, and ANP32E. ANP32A, ANP32B, and ANP32E are conserved in vertebrates, but the ANP32C and ANP32D are predicted to be intronless-gene-coded proteins, therefore, their genes are considered to be pseudogenes or retrogenes [29]. In a previous study, we found that ANP32A and ANP32B provide fundamental support to influenza A virus replication, and that double knockout of ANP23A and ANP32B (DKO) aborted polymerase activity of IAV [14]. Here, we show that a single knockout of ANP32A, ANP32B, or ANP32E does not affect IBV polymerase activity. Interestingly, double knockout of ANP32A and ANP32B reduces IBV polymerase activity by 100-fold, which is not as strong an effect as that on the IAV polymerase (which reduced more than 1000 folds in DKO cells), suggesting that another factor may contribute to IBV polymerase activity. As expected, triple knockout of ANP32A, ANP32B, and ANP32E (TKO) further reduces IBV polymerase activity by 10-fold (Fig 1). Reconstitution of ANP32A or ANP32B can fully restore IBV polymerase activity, but reconstitution of ANP32E can only partially restore IBV polymerase activity, suggesting that ANP32E has only limited ability to support IBV replication (Fig 2). We confirm that ANP32E does not support IAV polymerase activity.

All our evidence supports the argument that, similar to IAV, IBV may rely on ANP32A or ANP32B in its replication, with the exception that IBV can also use ANP32E to a certain extent. It is very interesting to see that the amino acid at position 129 of ANP232E is Glutamine acid (129E), while that in the corresponding position of ANP32A or ANP32B is Asparagine (129N). An E129N mutation in ANP32E can completely restore support of IBV polymerase activity (Fig 6), indicating that the 129E of ANP32E is a dominant impact on its ability to support IBV. We also observed that the ability of avian ANP32E in supporting IBV polymerase is more than 10 times lower than that of huANP32E, which was due to a 10-amino-acid deletion in the LCAR domain. This species-specific difference was due to a 10-amino-acid deletion in the LCAR domain of avian ANP32E.

Unlike IAV, which comprises a heterogeneous group of different subtypes, IBV forms a more homogeneous cluster with two main lineages, Victoria and Yamagata. IAV can infect many animal species but IBV can only be identified clinically in humans, seals, and pigs. Infection by IBV in avian species has not been confirmed. Taking into consideration that IBVs have the ability to bind to α2,6-linked or α2,3-linked sialyl-glycans [33], it is proposed that there are interspecies barriers existing, protecting the avian hosts against IBV infection. To further illustrate the contribution of ANP32 proteins to IBV replication and possible interspecies restriction, we compared the abilities to support IBV replication by ANP32A, ANP32B, and ANP32E proteins from different species. Chicken ANP32A, ANP32B, or ANP32E showed related low activity to support IBV polymerase in TKO or chicken DF-1 cells compared with human ANP32A and ANP32B. It is intriguing to find out that chicken ANP32A gives only weak support to IBV polymerase because of the extra 33- or 29-amino-acid inserts (Fig 3). The 33-amino-acid insert is required for chANP32A to support chicken IAV polymerase activity, while proteins with this insert are unable to support IBV replication. This result indicates different evolutionary patterns of IAV and IBV in the use of host ANP32 proteins. It is worth to note that avian ANP32A has three isoforms due to differential splicing, including a long isoform with 33-amino-acid insert in exon 4, a shorter isoform with 29-amino-acid insert, and a mammalian-like isoform without insertion [12, 15]. The express ratios of the different isoforms in avian cells are varied. The abundance of the mammalian-like isoform of chANP32A in DF-1 cells is about 9% in all expressed isoforms, and the 33 insert and 29 insert isoforms are 66% and 25% respectively [15]. We identified that overexpressing of huANP32A or huANP32B in DF1 cells enhances IBV polymerase activity and viral replication, indicating a limited support to IBV polymerase exists in DF-1 cells. We also found that when we use a high MOI of B/Yamagata/PJ/2018 virus to inflect DF1 cells, the virus can replicate to a certain level (Fig 2C). It remains unknown whether the avian species with high ratio of mammalian-like isoform of chANP32A expression can support better IBV replication.

It is known that the IAV polymerase complex binds to ANP32A or ANP32B for its normal function. The chANP32A shows stronger binding ability to avian IAV strains than do mammal ANP32As or ANP32Bs, which is consistent with its ability to support chicken IAV replication. The 33-amino-acid insert is responsible for this stronger binding ability [13]. Surprisingly, we found that chANP32A and huANP32A showed a similar ability to bind IBV polymerase, despite the fact that they showed dramatically different abilities to support the activity of IBV polymerase. It is not known to date how the ANP32 proteins interact with the polymerase, or how this interaction between ANP32A proteins and viral polymerase can support viral polymerase replication. Our experiments show that although the huANP32A and chANP32A have similar binding ability to IBV polymerase in co-IP assay, the SPR assay shows that the association rate constant (ka) of huANP32A is higher than that of chANP32A, therefore, the calculated dissociation constant (KD) of huANP32A to the polymerase is significantly lower than that of chANP32A, indicating that huANP32A has a higher affinity to the polymerase and thus may lead to a stronger ability to support the activity of the polymerase of IBV. Given the fact that the viral polymerase have highly compact structure and interact with many host factors during replication [17, 34], it is remain unclear that the detailed interaction mechanism between ANP32A and polymerase of IBV and how much this interaction contributes to the host range selection.

Taking the roles of ANP32 proteins in IAV and IBV together, we summarized the current understanding of interaction between ANP32 proteins and influenza viral polymerase. HuANP32A and huANP32B play major role in supporting both human IAV and IBV replication, but huANP32E has mild support to IBV and no support to human IAV. None of the human ANP32A, ANP32B, or ANP32E supports avian IAV because they do not have a 33-amino-acid insert as chANP32A. Chicken ANP32A harbors a 33-amino-acid insert which enables supporting to both avian and mammal IAV, but not IBV. Chicken ANP32B is a nature inactive molecule to IAV and IBV polymerase because of 129I/130N substitution. Chicken ANP32E has no function in supporting IAV polymerase, but shows a weak support to IBV. The low level support to IBV of ANP32E is due to a 129E mutation. Thus, none of chicken ANP32 proteins show good support to IBV (Fig 9). The differences in using of the ANP32 proteins by IAV and IBV are correlated to the species-specific restriction of influenza virus replication. In conclusion, our functional investigation of ANP32 proteins in supporting IBV replication and the species-specific restriction of IBV polymerase may provide new insight to understand viral adaptation and evolution.

Fig 9 — 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.

Materials and methods

Human 293T (ATCC CRL-3216) and MDCK (CCL-34) cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS; Clark) and 1% penicillin and streptomycin (Gibco). The polymerase plasmids of human influenza A virus H7N9 A/Anhui/01/2013 (H7N9_AH13) were kind gifts from Dr. Hualan Chen. The plasmids carrying the genes of influenza Bvirus B/Yamagata/1/73 were kindly provided by Dr Yoshihiro Kawoaka. B/Yamagata/PJ/2018 (available in our lab, GenBank accession numbers: NP: MN700018, PB1: MN700019, PB2: MN700020, PA: MN700021) was used to infect 293T and knockout cells. pCAGGS plasmids containing ANP32A, ANP32B from different species are kept in our lab [18]. The pCAGGS plasmids containing full length ANP32E isoforms of several species and influenza B virus B/Victoria/Brisbane/60/2008 were generated by gene synthesis (Synbio technologies, China) according to the sequences deposited in GenBank, including human ANP32E (huANP32E, NM_030920.5, NP_112182.1), pig ANP32E (pgANP32E, XM_021089919, XP_020945578.1), equine ANP32E (eqANP32E, XM_001917235.4, XP_001917270.1), dog ANP32E (dgANP32E, XM_003639621.4, XP_003639669.1), mouse ANP32E (muANP32E, NM_023210.4, NP_075699.3), zebra finch ANP32E (zfANP32E, XM_012570886.1, XP_012426340.1), duck ANP32E (dkANP32E, XM_005030153.3, XP_005030210.2), turkey ANP32E (ty ANP32E, XM_003212772.3, XP_003212820.2), chicken ANP32E (chANP32E, NM_001006564.2, NP_001006564.2), huANP32E_with_chTail, chANP32E_with_huTail, huANP32E_8aamut, chANP32E_8aamut, huANP32E_Δ10, chANP32E₊₁₀, B/Victoria/Brisbane/60/2008 PB1 (CY115157.1, AFH57918.1), B/Victoria/Brisbane/60/2008 PB2 (CY115158.1, AFH57919.1), B/Victoria/Brisbane/60/2008 PA (CY115156.1, AFH57917.1), B/Victoria/Brisbane/60/2008 NP (CY115154.1, AFH57914.1). To obtain pCAGGS- Yamagata-PB2-Flag and pCAGGS-Victoria-PB2-Flag plasmids, pHH21-Yamagata-PB2 and pCAGGS-Victoria-PB2 was used as the template to amplify the PB2-Flag sequences, and then fused with pCAGGS vector according to the online In-Fusion HD Cloning Kit User Manual (http://www.clontech.com/CN/Products/Cloning_and_Competent_Cells/Cloning_Kits/xxclt_searchResults.jsp). Using the same method, we changed the promoter of the firefly minigenome reporter from human to chicken to generate pchPOL1-vluc. To create the pCAGGS-huANP32A₊₃₃ plasmid, pCAGGS-chANP32A was used as the template to amplify the 33 amino acids, and then fused with the pCAGGS-huANP32A. Site-directed mutants of these sequences were generated using overlapping PCR and identified using DNA sequencing.

Knockout cell lines

The generation of the 293T AKO, BKO and DKO knockout cell lines were described in our previous report [14]. EKO and TKO knockout cell lines were generated using the same approach. Briefly, 293T or DKO cells cultured in 6-well plates were transfected with 0.5 μg pMJ920 (Addgene plasmid # 42234) plasmids and 0.5 μg gRNA expression plasmids in Polyet Transfection Reagent (Signagen, SL100688) using the recommended protocols. GFP-positive cells were sorting by flow cytometry (MoFlo XDP, Backman) at 24 h post-transfection, then monoclonal knockout cell lines were screened using western blotting and/or DNA sequencing.

Polymerase assay

HEK293T or DF1 cells were transfected with plasmids for the expression of the viral proteins PB1, PB2, PA, NP and pPol1-WSN-HAutr-vluc/pPol1-B/Yamagata/1/73-NSutr-vluc or pchPol1-B/Yamagata/1/73-NSutr-vluc. Renilla luciferase expression plasmids (pRL-TK, kindly provide by Dr. Luban) were used as an internal control for the dual-luciferase assay. To determine the effect of ANP32 proteins on viral polymerase activity, 293T or DF1 cells in 24-well plates were transfected with plasmids of PB1 (20 ng), PB2 (20ng), PA (10 ng) and NP (40 ng), together with 40 ng vluc and 5 ng Renilla luciferase expression plasmids, using Polyjet Transfection Reagent (Signagen, SL100688) according to the manufacturers’ instructions. As a negative control, cells were transfected with the same plasmids, with the exception of the PB2 or ANP32 expression plasmid. After transfection, the cells were incubated at 37°C for 24 h, and then luciferase activity was measured with a dual-luciferase reporter system (Promega, E1960) on a Centro XS LB 960 luminometer (Berthold technologies) according to the manufacturer's instructions. The expression levels of polymerase proteins in different cell lines were detected by western blotting, using specific antibodies (Genetex, GTX128538) for NP and anti-Flag tag antibody (Sigma, F1804) for PB2-Flag protein.

Influenza virus infection and infectivity

HEK293T cells, huANP32A knockout 293T cells (AKO cells), huANP32B knockout 293T cells (BKO cells), huANP32A &B double knocked out 293T cells (DKO), huANP32A, huANP32B, and huANP32E triple knocked out 293T cells (TKO), huANP32B or empty vector transfected DF1 cells were infected with B/Yamagata/PJ/2018 virus at a multiplicity of infection (MOI) of 0.1 for 2 h, washed twice with PBS, and then cultured at 37°C in Opti-MEM containing tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-trypsin (Sigma) at 0.5 μg/ml. At the indicated time points, the culture supernatant was harvested and a Focus Formation Units Assay (FFU) was run as previously described [35].

Immunoprecipitation and western blotting

For immunoprecipitation and western blotting, transfected cells were lysed using an ice-cold lysis buffer (50 mM Hepes-NaOH [pH 7.9], 100 mM NaCl, 50 mM KCl, 0.25% NP-40, and 1 mM DTT), and centrifuged at 13,000× g and 4°C for 10 min. After centrifugation, the crude lysates were incubated with Anti-FLAG M2 Magnetic Beads (SIGMA-ALDRICH, M8823) at 4°C for 2 h. After incubation, the resins were collected by magnetic separator and washed three times with PBS. The resin-bound materials were eluted by 3X Flag peptide (150 ng/ul) and subjected to SDS-PAGE, then transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk powder in Tris-buffered saline (TBS) for 2 h. Incubation with the first antibody (Anti-Flag antibody from SIGMA (F1804), Anti-NP antibody from Genetex (GTX128538), Anti-β-actin from Sigma(A1978)) was performed for 2 h at room temperature (RT), followed by washing three times with TBST. The secondary antibody (KPL, 1:10,000) was then applied and samples were incubated at RT for 1 h. Subsequently, membranes were washed three times for 10 min with TBST. Signals were detected using a LI-COR Odyssey Imaging System (LI-COR, Lincoln, NE, USA).

Surface plasmon resonance (SPR) measurement

The binding activity of different ANP32 proteins to the IBV polymerase was measured using a Biacore T200 instrument (GE Healthcare). Anti-His antibody was immobilized on the CM5 chip surface (flow cells 1 and 2) via the amine coupling method. 293T cells transfected with PA-His, PB1, with or without PB2 were lysed with cell lysis buffer (50 mM Hepes-NaOH [pH 7.9], 100 mM NaCl, 50 mM KCl, 0.25% NP-40, and 1 mM DTT). Cells lysates were diluted with running buffer (HBS-EP+) to the corresponding concentration and allowed to flow through the immobilized chip for 90 s 5 ul/min. ANP32 proteins were individually fusion expressed at downstream of GST-HRV3C peptide in a pCAGGS vector backbone in DKO cells. Proteins were purified using Glutathione Sepharose 4B and then digested by PreScission Protease (Beyotime, P2303). The purified proteins were diluted with running buffer to different indicated concentrations and control flow cells at a flow rate of 30 μl/min for 90s. After 120s dissociation, the chip surface was regenerated with 10mM Glycine-HCL pH 1.5 at 30 μl/min for 45 s. Data were analyzed using Biacore Evaluation 3.1 software with a 1:1 fit model.

Statistics

Statistical analyses were performed using GraphPad Prism, version 7.04 (Graph Pad Software, USA). Statistical differences between groups were assessed using One-way ANOVA followed by a Dunnett’s post-test. Error bars represent the SD (standard deviation) of the replicates within one representative experiment. NS, not significant (p>0.05), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All the experiments were performed independently at least three times.

Supporting information

S1 Fig. Viability of TKO cells measured by CCK-8 assay.

The cell viability of 293T and TKO cells were measured at 24, 48, and 72 h by the CCK-8 reagent in accordance with the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China).

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S2 Fig. Replication of influenza B virus in MDCK and 293T cells.

MDCK and 293T cells were infected with B/Yamagata/PJ/2018 virus at a MOI of 0.1. The supernatants were sampled at 12, 24, 36, 48, 60, and 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.

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S3 Fig. ANP32 proteins supported the IBV viral polymerase activity in a dose-dependent manner.

Increasing doses of huANP32A(A), huANP32B(B) or huANP32E(C) were co-transfected with minigenome reporter, Renilla expression control, influenza B virus polymerase of B/Yamagata/1/73 in TKO cells. The expression of ANP32 proteins and polymerase was assessed by western blotting. Luciferase activity was measured 24 h later. (Data are firefly activity normalized to Renilla, Statistical difference between cells were 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.)

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S4 Fig. Sequence alignment of ANP32A and ANP32B proteins from different species.

The protein sequences of ANP32A for human (huANP32A), pig (pgANP32A), equine (eqANP32A), dog (dgANP32A), ostrich(osANP32A), zebra finch (zbANP32A), duck (dkANP32A), turkey (tyANP32A), and chicken (chANP32A) were aligned using the Geneious R10 software. huANP32A was set as the reference sequence. The 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 huANP32A.

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S5 Fig. Purification and identification of ANP32 proteins and viral polymerase.

(A) ANP32 proteins were fusion expressed at downstream of GST-HRV3C peptide in a pCAGGS vector and purified using Glutathione Sepharose 4B and then digested by PreScission Protease. Purified ANP32 proteins were diluted to 100ug/ml and 1ug of the purified protein was checked using SDS-PAGE analysis and western blotting. (B) IBV polymerase PB1, PB2 and PA-His were expressed in 293T cells and purified with Ni Sepharose (GE). The purified protein was checked using SDS-PAGE analysis. (C) The proteins of the purified band in (B) were identified using the mass spectrometry.

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Click here for additional data file.^{(1.1MB, pdf)}

Acknowledgments

We thank Dr. Hualan Chen and Dr. Yoshihiro Kawaoka for providing plasmids. We thank Dr. Ervin Fodor for helpful discussions. We thank the Core Facility of the Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences for providing the technic support.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the grants from the Natural Science Foundation of China (www.nsfc.gov.cn) to X.W. (as a part of grant to Dr. Hualan Chen’s grant: 31521005) and Z.Z. (31702269) and the Natural Science Foundation of Heilongjiang Province (http://jj.hljkj.cn/zr/) to X.W. (No. JC2018010).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Pathog. doi: 10.1371/journal.ppat.1008989.r001

Decision Letter 0

Ron A M Fouchier, Martin Schwemmle

10 May 2020

Dear Dr Wang,

Thank you very much for submitting your manuscript "Selective usage of ANP32 proteins by Influenza B Virus Polymerase: implications in determination of host range" (PPATHOGENS-D-20-00687) for consideration at PLOS Pathogens. As with all papers peer reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent peer reviewers. Based on the reports, we regret to inform you that we will not be pursuing this manuscript for publication at PLOS Pathogens

The reviews are attached below this email, and we hope you will find them helpful if you decide to revise the manuscript for submission elsewhere. We are sorry that we cannot be more positive on this occasion. We very much appreciate your wish to present your work in one of PLOS's Open Access publications.

Thank you for your support, and we hope that you will consider PLOS Pathogens for other submissions in the future.

Sincerely,

Ron Fouchier

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

**************************************

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: The authors dissect species-specific functional interactions between ANP32 proteins and influenza viruses, with a focus on influenza B virus. They find that avian ANP32 molecules are generally unable to efficiently support influenza B virus polymerase activity, which is the opposite to avian influenza A virus. Using mutagenesis, binding assays and KO technologies, they map the specific determinants in different ANP32 molecules that are responsible for this effect. The work is notable for its use of constructs from many different species, and its fine dissection of different ANP32 genes (A/B/E) and isoforms. The finding that ANP32E plays a role in influenza B virus replication, but not ANP32A is a highlight. The data are of very high quality and clearly support the conclusion that avian cells may not be permissive for influenza B virus replication due to incompatibility with ANP32s. Taken together with the work on influenza A viruses, this new work is therefore very important as it suggests interesting species-specific evolution of influenza viruses with host ANP32 molecules.

Reviewer #2: Influenza A viruses (IAV) circulate in birds and infect many mammalian species. Although influenza A and B viruses are closely related, IBVs do not infect birds, and the cause of this host range restriction is unknown. ANP32A was shown to be the major species barrier preventing avian IAV polymerases from functioning in mammalian cells. Species-specific differences in ANP32A, ANP32B and ANP32E have since been shown to impact the function of the viral polymerase in different hosts. Here, Zhang, Zhang, et al. thoroughly evaluate the role of ANP32s in supporting influenza B virus polymerase activity. Using a clean genetic background (ANP32A, ANP32B, ANP32E triple knockout; TKO), the authors show that human ANP32A and B support IBV polymerase and that either protein is sufficient for viral replication. ANP32E does not support IAV polymerase, but the authors make the interesting observation that ANP32E provides low levels of activity for the IBV polymerase. None of the avian ANP32 proteins support IBV. Mutant and chimeras reveal that an insertion of acidic residues in human ANP32E contributes to its ability to support IBV polymerase. However, this activity is restricted by the glutamate encoded at amino acid 129; converting this to the asparagine found in ANP32A confers a high degree of activity to both human and avian ANP32E. Thus, ANP32E functions poorly due to variation at aa129, and the species-specific absence of an acidic stretch further eliminates activity in birds, suggesting ANP32E is a major factor restricting IBV host range. Physical interactions between IBV and ANP32A and B are demonstrated by coIP and SPR. The functional assays and dissection of species-specific activities are well-controlled and compelling. However, the authors over-interpret their SPR data, especially in light of how ANP32A interactions do not correlate with support of polymerase activity. Nonetheless, this submission offers strong evidence to explain why influenza B viruses do not circulate in infect birds.

Reviewer #3: Influenza B virus is the virus type that together with influenza A viruses (IAV) causes seasonal outbreaks of influenza. A distinctive feature of influenza B virus is the restriction to humans as the main host species, whereas only IAV has established phylogenetic stable lineages in other host species including birds and pigs. The molecular basis for the species specificity of influenza viruses is currently in the focus of intense research and pre-pandemic risc assessment. Previous work has identified the functional interaction of host proteins of the ANP32 family with the viral polymerase complex as a major determinant of host range. While human ANP32-A and -B proteins are essential co-factors of human-adapted polymerases, e.g. chicken ANP32A supports only avian polymerase activity. Several recent papers have unravelled these interactions, the contribution of different ANP32 proteins and the detailed molecular requirements on both viral and host proteins. In this manuscript, Zhang et al describe the requirement of influenza B virus for human ANP32A or B proteins to support polymerase activity and replication in human cells, while chicken orthologues fail to fully support these human-specific viruses. The authors then expand these analyses on a set of ANP32 proteins from other species and demonstrate that the molecular basis for this species specific interaction resides in a 33 amino acid stretch that is exclusive to chicken ANP32A. Further, they investigate the role of ANP32E, which is shown to only play a minor role in supporting IBV polymerase activity. Finally, binding of polymerase and ANP32 proteins is investigated.

The work is well-structured, follows a clear line of thought and is presented in an understandable way, although language would benefit from some enhancements. The conclusions are valid and the data are of some interest to the community. However, much of the work is rather confirmatory with incremental advance in our understanding of the role of ANP proteins in influenza B virus infection at this stage of analysis. This is in part also due to the strong focus of the work on the use of ectopically expressed genes and less on the situation in virus-infected cells.

1.) A major result of this manuscript, the dependence of influenza B virus propagation on ANP32A and -B proteins, has already been demonstrated (e.g. Staller et al., J.Virol. 2019). Due to its depth of analysis, the current manuscript extends this finding, but is rather confirmatory in character. The second novel aspect is the analysis of the role of ANP32E. As the authors show and state themselves, however, the contribution of this isoform to polymerase function is smaller compared to the previously identified major host range determinants ANP32A and B.

2.) The major body of evidence gained in this manuscript relies on standard polymerase activity assays. These are performed with skill and the immunoblots showing control expressions are of high quality. However on top of that, only one panel (Fig.1D) investigates the impact of ANP32 proteins on virus replication. Also, little significant novel insight is gained here, since deletion of ANP32E in cells already lacking ANP32A and B does not have an impact on virus replication. The interaction analyses in figures 7 and 8 again only marginally increase knowledge.

--------------------

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: No major issues identified

Reviewer #2: 1. Experiments studying interactions between IBV polymerase and ANP32s are underdeveloped and the claims need to be softened

a. The authors use results from SPR to calculate Kd and claim avian ANP32A binds IBV polymerase ~2.5 fold less well than human ANP32A. However, these data have no statistics associated with Kd calculations. The curve in Fig 8F does not correlate with the individual data in 8A-C. If anything, data in 8C suggest higher response rates for huANP32A+33 than for ANP32A.

b. Minor differences in Kd determined by SPR can be caused by differences in protein quality or concentration. Data showing the purity and equivalent concentration of ANP32s is needed. It would also be useful to understand how well IBV polymerase proteins are expressed, as the methods say these proteins are captured on the chip directly from cell lysate.

c. While a major new claim is that ANP32E support IBV polymerases, ANP32E was not included in any of the binding assays.

d. IBV polymerase interacts by coIP with both huANP32A and chANP32A, yet only huANP32A supports function. If the authors are able to confirm minor differences in binding proposed in Fig 8, would these even be relevant to IBV pol function in cells?

e. Given the limited support of Fig 8, lines 388-399 in the Discussion are too speculative. In addition, SPR was done in the absence of viral RNAs, thus different functional states of the polymerase cannot be assumed.

2. The authors identify a 10aa insertion of acidic residues present in mammalian ANP32E that they nicely demonstrate is important for the low levels of huANP32E activity. While murine ANP32E is tested in cells, it is conspicuously absent from their alignment.

a. Alignments show that muANP32E lacks this insert. This should be addressed in the text.

b. Fig 4A,B; it is not clear why muANP32B is used as a control here. Staller et al. 2019 showed that muANP32B actually encode 129S 130D, which is different than the more common 129N 130D found in huANP32B, huANP32A, and chANP32A. Fig 6 here goes on to show that the identify of aa129 is important for ANP32E function. This difference in muANP32E should also be addressed in the text.

c. care should also be taken to make clear that the 10aa insertion confers species-specific function, while residue 129 is the dominant change controlling activity regardless of the host.

3. The authors claim that ANP32s are a major barrier preventing IBV replication in birds. However, this was never tested. Does expression of huANP32A or B (or perhaps chANP32E_E129N) allow IBV replication in birds, or do other barriers still exist?

Reviewer #3: (No Response)

--------------------

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: Lines 163-167. Please check this statement. I thought that mammalian ANP32s were ‘short’, while avian ones were ‘long’? Statement seems contrary to line 160.

Line 185. Please clarify what is meant by chickens have an ANP32A isoform that lacks the 4 hydrophobic amino-acids – are the authors referring to multiple isoforms of ANP32A? Please make this clearer – perhaps discuss different isoforms earlier in the introduction to make it obvious to the reader that they exist. Also, chickens are reported to have a short isoform lacking the entire ‘insertion’ – eg like del33. Please comment.

Line 188 – please specific what ‘alter’ means – enhance, reduce? Not clear what meant here.

Line 193 – other than chickens, some birds have higher ratios of the ‘short’ ANP32A isoform to the ‘long’ isoform (Baker et al, Domingues et al) – can the authors make a definitive statement whether these birds could support IBV replication? In the discussion, perhaps comment on the amino-acid compositions in ANP32B and ANP32E of the bird species that are dominant in expressing ANP32Adel33 isoforms. Would their ANP32B/E molecules also suggest a support for IBV polymerase?

Figure 4 – what is muANP32B? Murine? Why is its apparent size larger than huANP32B? Doesn't seem to be referred to anywhere in the text of Figure legend.

Reviewer #2: 1. Fig legends 3, 4, 5B,C, do not indicate which cell lines are being used. This reviewer assumes the experiments are in TKO. If not, I will have to reconsider my interpretation of the data.

2. A model or table in the Discussion indicating the functionality of avian and human ANP32s with huIAV, chIAV or huIBV would be a useful addition to help solidify current and previous findings.

3. Table 1 was not present in the manuscript received for revue. However, given the concerns about SPR above, it is not clear how much Table 1 contributes.

4. Please indicate that Staller et al. 2019 showed that ANP32B enhances influenza B polymerase activity.

5. Statistics throughout. Reporting SEM for technical replicates is not accurate, please use SD to illustrate error bars and to run statistical tests on. Number of replicates are not indicated.

6. Fig 3 misrepresents where the duplication event occurred – chANP32A duplication was downstream of SIM sequence, so “repeat 1” should have matching alignment of ch and hu. See Long 2015 Extended Data Fig 8 or Domingues 2017 Fig 2. Fig S3 alignment is inaccurate for the same reason. Figure S3 Legend indicates numbering with respect to huANP32A but this is not how the alignment is presented.

7. ANP32B and ANP32E were independently shown to be essential (in 1 of 5-7 screens each; http://ogee.medgenius.info/browse/). Please discuss the health of TKO cells.

8. Line 292 – these results are not “suprising” as they have already been reported for IAV:ANP3A interactions by others.

Reviewer #3: (No Response)

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS Pathog. 2020 Oct 12;16(10):e1008989. doi: 10.1371/journal.ppat.1008989.r002

Author response to Decision Letter 0

15 Jul 2020

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PLoS Pathog. doi: 10.1371/journal.ppat.1008989.r003

Decision Letter 1

Ron A M Fouchier, Martin Schwemmle

12 Aug 2020

Dear Dr Wang,

Thank you very much for submitting your manuscript "Selective usage of ANP32 proteins by Influenza B Virus Polymerase: implications in determination of host range" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Martin Schwemmle

Guest Editor

PLOS Pathogens

Ron Fouchier

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: I am still very positive about this manuscript, and believe that it adds significantly to the field and our understanding of species-specific functional interactions between ANP32 proteins and influenza viruses. The data are novel, experiments well-executed, and the manuscript provides new detailed insights into the molecular basis of ANP32E usage, as well as avian versus mammalian ANP32A/B interplay with the polymerases of both influenza A and influenza B viruses. The findings have clear implications for trying to understand host restriction. Importantly, all of my previous comments, which were very minor, have been addressed satisfactorily.

Reviewer #2: The authors have addressed my major concerns. I appreciate their attention to each of the points raised and thorough responses.

Reviewer #4: The results presented in this manuscript advance our understanding of the important role of ANP32 proteins in the regulation and adaptation of the polymerase activity of Influenza A but most importantly on Influenza B viruses. It provides compelling and solid evidence, that ANP32 proteins promote IBV polymerase activity in human cells in a protein specific manner, which is in some parts divergent from the effects seen on the IAV polymerase.

Many of the primary research questions in this manuscript have been addressed before by Staller et al. 2019, Journal of Virology using similar methodology. Thus the originality and novelty of the subject is limited. In contrast to Staller et al. who reporter a certain degree of functional diversity between ANP32A and B proteins on the IBV Polymerase activity, the results in this manuscript show functional redundancy of both proteins in human cells. These differences may be attributed to the choice of IBV strain, cell type and method of generating KO clones and are in general interesting but not ground breaking.

The investigation of ANP32E, which shows limited support for the IBV polymerase is new and interesting, however, compared to ANP32A/B it appears to be of minor importance and its role in the species restriction of IBV to humans remains uncertain. The chosen methods mostly assess protein functions under non-natural (overexpression, purified) conditions and only very few assays verify results in the context of a viral infection, which overall is a weak point.

In the light of these shortcomings I would suggest submission of this manuscript to a more specialized journal.

In addition, I would like to pronounce my concerns about the response of the authors to reviewer #3, which is in parts rude and inappropriate. In a peer review process authors and reviewers should adopt a professional and supportive attitude even if the outcome is not as preferred.

To avoid any misinterpretation: My evaluation of the presented scientific work is by no means affected by this statement.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Reviewer #1: None

Reviewer #2: (No Response)

Reviewer #4: (No Response)

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: None

Reviewer #2: (No Response)

Reviewer #4: General remarks:

1. What is the rational of using a H7N9 IAV derived polymerase? This subtype has only recently jumped to humans but is not stably adapted (not circulating) to the human population in comparison to the H1N1 and H3N2 subtypes. Please include some information on why this strain was chosen as a comparison to IBV, which is a seasonal strain and much more stable adapted to humans.

2.The labeling of the y-axis in all figures displaying IAV polymerase activity using the polymerase reconstitution assay is wrong and needs to be changed to:

Firefly Luciferase activity instead of Firefly Polymerase.

Specific remarks:

1. lines 76-78: What means “the ANP32 family MAINLY includes ANP32A, ANP32B and ANP32E.”? Does this refer to in most species? Or expression levels? Are there other ANP32 proteins present? Please specify this statement.

2. lines 78: This sentence is misleading. Please acknowledge that also other groups have contributed to this finding.

3. Figure 1: Please indicate the subtype of the FluA polymerase as this is a relevant information for this assay.

4. Lane 123: what means: the last “functional member”? What function does this refer to? Please describe this in more detail. Also lane 124: what means “considered to be an important member of the ANP32 family”? Are there other non-important members? What is this hypothesis based on? Please provide more details.

5. Line 149: what means ANP32As? Is that s a typo? Please correct.

6. Figure 2: The title of this figure is misleading as it states that only ANP32As from different animals are investigated which is not true

7. Figure 2B: why is there no ANP32 protein band in the empty vector control lane? These are wild type cells and should express detectable levels of endogenous chicken ANP32 proteins. Please explain and interpret this result.

8. Figure 2C: Why were virus growth curves performed at 37°C? For IBV 33°C is commonly used as it also support much higher titers. I wonder whether overexpression of human ANP32B also has a positive effect at 33°C? Can you provide data on this?

9. lane 174: Please correct the sentence to …”that ARE all in short form…” and remove the are in lane 176.

10. Are there any other aa mutations between the different mammalian and avian type ANP32A proteins beside the 33 aa insertion? Please state so clearly in the primary text (in the respective section) as these mutations can also affect the protein function on the polymerase.

11. Which cells were used in figure 3d? Please also include this information in the text and legend.

12. lane 187: correct IVB to IBV

13. lane 204: I understand that chicken ANP32B also lacks the 33 aa insert. Is that correct? But there is a size shift in the westernblot bands compared to human ANP32B indicating addition amino acids. Please add this information to the beginning of this paragraph.

14. line 306: please change mutant to mutations

15. figure 7D: What happens if the LCAR region of ANP32A is removed? Is binding to IBV polymerase then abolished? Is that a common mechanism for all ANP32 proteins? Why was the ANP32A_165T mutant not tested in the co-IP assay but in the SPR?

16. line 360: I assume that the authors meant write morbidity instead of mobility? Please correct.

17: line 362: “polymerase replication” is a confusing description. Please substitute with “polymerase activity” Please substitute

18. overall the grammar, spelling and wording in the discussion part is of lower quality than the previous parts of the manuscript and needs major improvement (e.g. lines 417-425)

19. lines 420-425: The authors claim that the amount of mammalian-like ANP32A isoform in DF1 cells is not enough to support IBV replication. But in Figure 2C a growth kinetic of B/Yamagata/PJ/2018 is presented. So in principle, IBV replication seems to be supported in theses cells. Please rephrase this part of the discussion accordingly.

20. line 427: “chicken IAV”? does this mean avian IAV strains or do you really mean IAV from chicken? Please be precise.

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PLoS Pathog. 2020 Oct 12;16(10):e1008989. doi: 10.1371/journal.ppat.1008989.r004

Author response to Decision Letter 1

12 Sep 2020

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PLoS Pathog. doi: 10.1371/journal.ppat.1008989.r005

Decision Letter 2

Ron A M Fouchier, Martin Schwemmle

17 Sep 2020

Dear Dr Wang,

We are pleased to inform you that your manuscript 'Selective usage of ANP32 proteins by Influenza B Virus Polymerase: implications in determination of host range' has been provisionally accepted for publication in PLOS Pathogens.

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Reviewer Comments (if any, and for reference):

PLoS Pathog. doi: 10.1371/journal.ppat.1008989.r006

Acceptance letter

Ron A M Fouchier, Martin Schwemmle

6 Oct 2020

Dear Dr Wang,

We are delighted to inform you that your manuscript, "Selective usage of ANP32 proteins by Influenza B Virus Polymerase: implications in determination of host range," has been formally accepted for publication in PLOS Pathogens.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Viability of TKO cells measured by CCK-8 assay.

The cell viability of 293T and TKO cells were measured at 24, 48, and 72 h by the CCK-8 reagent in accordance with the manufacturer’s instructions (Beyotime Biotechnology, Shanghai, China).

(PDF)

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S2 Fig. Replication of influenza B virus in MDCK and 293T cells.

(PDF)

Click here for additional data file.^{(220.9KB, pdf)}

S3 Fig. ANP32 proteins supported the IBV viral polymerase activity in a dose-dependent manner.

(PDF)

Click here for additional data file.^{(651.5KB, pdf)}

S4 Fig. Sequence alignment of ANP32A and ANP32B proteins from different species.

(PDF)

Click here for additional data file.^{(1.1MB, pdf)}

S5 Fig. Purification and identification of ANP32 proteins and viral polymerase.

(PDF)

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Selective usage of ANP32 proteins by influenza B virus polymerase: Implications in determination of host range

Zhenyu Zhang

Haili Zhang

Ling Xu

Xing Guo

Wenfei Wang

Yujie Ji

Chaohui Lin

Yujie Wang

Xiaojun Wang

Roles

Abstract

Author summary

Introduction

Results

Human ANP32A, ANP32B, and ANP32E support influenza B viral polymerase activity to different degrees

Fig 1. ANP32A, ANP32B, and ANP32E support influenza B viral polymerase activity to different degrees.

Species-specific support of influenza B viral polymerase activity by ANP32A proteins from different animals

Fig 2. Species-specific support of influenza B viral polymerase activity by ANP32 proteins from different animals.

Fig 3. Support of influenza B viral replication by ANP32A from different species and the key amino acids responsible for the support.

Avian ANP32B has low support of influenza B viral polymerase

Fig 4. The 129/130 site of ANP32B determines the supporting of influenza B viral polymerase.

Avian ANP32E has a limited ability to support influenza B viral polymerase activity

Fig 5. Support of influenza B viral replication by ANP32E from different species and the key amino acids responsible for the support.

A single amino acid at position 129 determines the support of ANP32Es to IBV polymerase

Fig 6. A single amino acid at position 129 determines support of huANP32E and chANP32E for IBV polymerase.

Different abilities of chicken and human ANP32A and ANP32E to bind to IBV polymerase

Fig 7. Different binding abilities of chicken and human ANP32A and ANP32E for IBV polymerase.

Fig 8. The 33-amino-acid insert impacts the interaction dynamics between ANP32A proteins and viral polymerase.

Discussion

Fig 9. Selective usage of ANP32 proteins by IAV and IBV polymerase and their molecular basis.

Materials and methods

Knockout cell lines

Polymerase assay

Influenza virus infection and infectivity

Immunoprecipitation and western blotting

Surface plasmon resonance (SPR) measurement

Statistics

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Martin Schwemmle

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Martin Schwemmle

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Martin Schwemmle

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Acceptance letter

Ron A M Fouchier

Martin Schwemmle

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Associated Data

Supplementary Materials

Data Availability Statement

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