Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase

Abstract

Adaptation of viruses to their hosts can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently identified as a cellular co-factor affecting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation among birds differentially affects viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

Keywords: ANP32A; influenza virus; splicing; viral host range; viral polymerase.

Figure 1.

Natural Variation in ANP32A Splicing Patterns in Aves Duplication and insertion of ANP32A exon 4 results in three major splice isoforms in birds. Schematic of chANP32A protein and exonic (ex) mRNA organization indicating duplicated domains. C, C-cap; N, N-cap; R1–R5, leucine-rich regions 1–5. Exon numbering is based on chANP32A₃₃. All human ANP32A transcripts lack the exon duplication (light gray). Schematics of chicken and goose transcripts show splicing upstream to capture codingsequence for the SIM (blue), splicing downstream to omit the SIM (red), or in some cases skipping the repeated exon to create a mammalian-like transcript (light gray). The relative abundance of each splice isoform in RNA-seq data is indicated by the pie charts. SIM, SUMO-interacting motif. Sashimi plots of ANP32A corresponding to examples in (B) and colored similarly. The abundance of each splice variant is indicated on the lines corresponding to the intron-spanning reads. ANP32A splice patterns in diverse bird species overlaid on the Aves consensus phylogeny (dashed lines represent branches that were in two of three consensus trees from Reddy et al., 2017). Pie charts represent relative transcript abundance from RNA-seq datasets (listed in Table S1).

Figure 2.

Avian ANP32A₂₉ Is Sufficient to Restore SpeciesRestricted Avian Polymerase Activity and Replication Activity of human-style (PB2 K627) and avian-style (PB2 E627) polymerases was measured in the presence of increasing concentrations of the indicated ANP32A proteins. Protein expression was assessed via western blot. ANP32A₃₃ selectively rescues restricted avian polymerase activity in human 293T cells. ANP32A₂₉ is sufficient to restore polymerase activity in human cells (left) but does not significantly affect human viral polymerase in human cells oreither polymerase in chicken LMH cells (right). Insertion of the avian 29 amino repeat into huANP32A (huANP32A₊₂₉) enhances activity, whereas deletion of the repeat in chANP32A (chANP32A_D) disables its function. chANP32A₃₃ is the most potent enhancer of avian polymerase activity in human cells compared with chANP32A₂₉ or chANP32A_33mut. For all assays, polymerase activity was normalized to an internal control and compared with PB2 K627 polymerase in the absence of ANP32A. Data are shown as mean (columns) of n = 3 technical replicates (dots) ± SD derived from representative results of at least three independent biological replicates. C, empty vector control. In (A)–(D), pairwise comparisons between PB2 K627 and E627 at each condition were significant (p < 0.05, Student’s t test) except where indicated as not significant (ns). Replication kinetics of influenza virus encoding the avian S009 RNP (WT) ora human human-adapted mutant (SRK), WSN, or B/Brisbane. WT A549 cells or those stably expressing chANP32A were infected (MOI = 0.1), and viral titers were determined at the indicated time points. Data are shown as average of n = 3 ± SD. *p < 0.05, one-way ANOVA with post hoc Tukey honestly significant difference (HSD) test compared with WT A549 cells. See also Figures S1 and S2.

Figure 3.

ANP32A Binds Directly to the PB2 627 Domain in a Species-Independent Fashion (A) ANP32A does not stably interact with the PB2 subunit when it is expressed alone. PB2 K627 or E627 was immunoprecipitated (IP) from cells co-expressing ANP32A or the positive control NP. (B) Both chANP32A₂₉ and the humanized chANP32A_D interact with the polymerase trimer. Immunoprecipitations were performed from cells expressing ANP32A and the trimeric polymerase containing PB2 K627 or E627. (C) ANP32A interacts with the viral polymerase during infection. WT A549 cells and those stably expressing ANP32A were infected, and PB2 was immunoprecipitated. (D and E) Genomic RNA increases PB2-ANP32A interactions. Immunoprecipitations were performed from cells expressing ANP32A, a vRNA (v) or cRNA (c) genomic segment, and the trimeric polymerase containing PB2 K627 or E627. Binding was measured with catalytically active polymerase (D) or an inactive PB1a mutant polymerase (E). (F) ANP32A binds directly to the PB2 627 domain. In vitro binding was measured between chANP32A variants and GST-tagged PB2 627 domain (top) or PB2 627-NLS (bottom) and visualized by Coomassie staining. GST alone (+) or an irrelevant GST fusion (C) were included as specificity controls.

Figure 4.

Molecular Defects of a Restricted Avian Polymerase Are Rescued by chANP32A₂₉ (A) chANP32A₂₉ enhances RNP formation for an avian-style polymerase. RNPs were reconstituted in human cells by co-expressing the indicated polymerase, NP, a genomic RNA, and chANP32A variants. NP was immunoprecipitated, and co-precipitating PB2 was measured as a proxy for RNP formation. RNP assembly was quantified from two independent assays, normalized to controls, and represented as the mean ± SD. chANP32A₂₉ functions redundantly with enhancing promoter mutations. Polymerase activity assays were conducted with the indicated ANP32A proteins and either WT or 3–8 mutant vRNA reporters. Proteins were detected by western blot. C, empty vector control. Data are shown as mean (column) of technical triplicates (dots) ± SD. *p < 0.05 (Student’s t test) between PB2 K627 and E627 at each condition. chANP32A increases the enzymatic activity of a restricted polymerase independent of RNP formation. NP-independent polymerase activity on a micro-gene vRNA template was measured by primer extension. chANP32A variants were co-expressed where indicated. mRNA and vRNA products were quantified by phosphorimaging from three independent experiments, normalized to PB2 K627 in the absence of ANP32A, and presented as mean ± SD. *p < 0.05, one-way ANOVA with post hoc Tukey HSD test. Tests were performed separately for vRNA and mRNA levels, and comparisons were made to PB2 K627 or E627 polymerase activity in the absence of ANP32A.