The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis
Uncoupling the two steps of influenza A virus genome replication with viral promoter mutations shows that ANP32A is required for synthesis of both the complementary RNA intermediate and progeny genomic RNA, and that it acts on the actively replicating polymerase rather than the encapsidating one.
- Senior authors
- Aartjan J. W. te Velthuis
- Correspondence
- Benjamin E. Nilsson-Payant; Aartjan J. W. te Velthuis
Research areas & themes
Citation
Nilsson-Payant BE, tenOever BR, te Velthuis AJW. The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis. Journal of Virology. 2022. Volume 96, Issue 4, article e02092-21.
DOI 10.1128/jvi.02092-21. PMID 34935435. PMCID PMC8865535.
One-sentence contribution
Uncoupling the two steps of influenza A virus genome replication with viral promoter mutations shows that ANP32A is required for synthesis of both the complementary RNA intermediate and progeny genomic RNA, and that it acts on the actively replicating polymerase rather than the encapsidating one.
Executive summary
Influenza A virus replicates its segmented negative-sense genome in two steps, first copying genomic RNA into a complementary intermediate and then copying that intermediate back into progeny genomes. Each nascent product must be encapsidated by an additional polymerase, so at least two polymerase complexes participate. The host protein ANP32A is essential for replication and bridges an RNA-bound polymerase to an RNA-free one, and differences between avian and mammalian ANP32A are sufficient to restrict avian polymerases in mammalian cells. Based on work with purified protein showing enhancement of genomic RNA synthesis, it had been proposed that ANP32A is needed only for the second step. The paper notes that this does not fit recent structural evidence, since both nascent species are encapsidated and it seems unlikely that separate encapsidation complexes evolved for each.
The study resolves this by making the two steps experimentally separable. Mutations in the viral promoters block initiation on the resulting product, so a minigenome can be restricted to primary complementary RNA synthesis or to genomic RNA synthesis alone. Combined with an avian-like polymerase carrying PB2 627E and with chicken ANP32A supplied in trans, this permits each step to be tested independently.
Both steps required ANP32A. The avian-like polymerase was impaired for each and was rescued by chicken ANP32A in each case. Single-molecule FRET showed the 627 position does not affect promoter binding, and an infection experiment with pre-expressed catalytically inactive or active polymerase placed the requirement at the replicating polymerase rather than at the encapsidating one.
Scientific context
Influenza A viruses circulate mainly in wild aquatic birds and infect humans only occasionally, in part because avian viruses cannot replicate efficiently in mammalian cells without overcoming host barriers. Two adaptations are emphasized, a shift in receptor binding preference and restoration of binding between the viral polymerase and the host factor ANP32A. A single substitution in PB2, glutamic acid to lysine at position 627, is sufficient to restore avian polymerase activity in mammalian cells, and expressing avian ANP32A alone achieves the same thing, which the paper reads as indicating that the PB2 substitution compensates for an impaired interaction with mammalian ANP32A.
ANP32A has an N-terminal leucine-rich repeat domain and a C-terminal low-complexity acidic region. Avian and mammalian homologues are similar in sequence, but the avian gene carries an exon duplication that yields, through alternative splicing, isoforms with extra sequence. Restoration of avian polymerase activity by avian ANP32A depends on that duplication, and the differential interaction has been mapped to the low-complexity acidic region contacting the flexible PB2 627-domain. ANP32B supports mammalian-adapted polymerase function but does not restore avian polymerase replication.
Cryo-electron microscopy of an influenza C virus polymerase dimer showed ANP32A bridging an RNA-bound polymerase and an RNA-free polymerase, suggesting it mediates assembly of the replicase complex. ANP32A appears to be required for genome replication but not primary transcription. Work with recombinant purified protein showed human ANP32A enhancing genomic RNA synthesis by human-adapted polymerases, and from that it was proposed that ANP32A is required only for the step producing genomic RNA from the complementary intermediate and not the reverse. The paper states directly that this view does not match the molecular evidence, because both species are encapsidated and separate encapsidation machinery for each seems unlikely. Several competing explanations for avian polymerase restriction in mammalian cells also remained in play, including destabilized polymerase and nucleoprotein interactions, reduced promoter binding, differential importin-alpha interactions, and unstable complementary ribonucleoprotein structures.
Central question
At which stage or stages of influenza A virus genome replication is ANP32A required, and does it act on the polymerase that is actively synthesizing RNA or on the polymerase that encapsidates the nascent product?
Experimental strategy
The central difficulty is that in an infection the two replication steps are obligately coupled, since complementary RNA must be made before genomic RNA can be made from it. The strategy is to break that coupling genetically at the level of the promoter.
Two promoter manipulations do the work. A G5U change in the 5-prime genomic promoter prevents internal initiation on the resulting complementary RNA product, permitting primary messenger RNA and primary complementary RNA synthesis while blocking subsequent genomic RNA synthesis. A G2C and C9G pair in the 5-prime complementary promoter prevents 3-prime terminal initiation on the resulting genomic product, abolishing further complementary RNA synthesis without blocking transcription. Each isolates one step. A separate G3A and C8U pair in the 3-prime genomic promoter, previously reported to improve avian polymerase activity, is included to test whether promoter strength alone explains the restriction.
Avian-like polymerase is produced not by using an avian virus but by introducing a single PB2 K627E substitution into a mammalian-adapted background, which keeps every other viral component constant. Chicken ANP32A supplied in trans is the rescue arm, with human ANP32A and green fluorescent protein as controls, so the species difference in one host protein is the only variable.
Several alternative explanations are tested and excluded rather than argued away. Cycloheximide restricts analysis to primary transcription of incoming ribonucleoproteins, separating a transcription defect from a downstream consequence of impaired replication. Knockdown of both human ANP32A and ANP32B addresses the possibility that mammalian ANP32A contributes to transcription of mammalian-adapted polymerases, which the avian rescue experiments cannot address. Immunoprecipitation of a catalytically inactive polymerase and single-molecule FRET with labeled promoters test whether the 627 position affects RNA binding. Short minigenome templates with and without nucleoprotein test whether the effect depends on nucleoprotein or on template length.
Finally, to place the requirement on one of the two polymerases in the replicase, catalytically inactive or active polymerase is pre-expressed before infection under actinomycin D, so that replication depends entirely on the pre-expressed components and the encapsidation function can be examined with the replication function disabled.
Key findings
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In human HEK-293T cells the avian-like WSN-K627E virus was significantly restricted in both replication and transcription relative to wild type, while in chicken DF-1 cells it showed slightly increased levels (Figure 2A, Figure 2B). Transient expression of chicken ANP32A restored WSN-K627E growth in human cells to near wild-type levels (Figure 2C, Figure 2D), and restored viral RNA accumulation, whereas human ANP32A did not, with neither affecting wild-type virus (Figure 2E).
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Reconstituting the minimal replication machinery in cells reproduced the restriction at the level of the polymerase, with PB2-627E ribonucleoproteins showing reduced production of all three viral RNA species and restoration by chicken ANP32A (Figure 2F), which localizes the effect to the polymerase rather than to other viral factors.
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Under cycloheximide, which permits primary transcription but blocks genome replication, wild-type and K627E viruses showed no difference in messenger RNA synthesis (Figure 2G). The observation is equivalence in primary transcription. The interpretation is that the reduced messenger RNA seen otherwise is a downstream consequence of impaired replication and reduced template.
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Knocking down both human ANP32A and ANP32B in A549 cells left primary transcription unchanged early in infection while strongly reducing genome replication (Figure 2H, Figure 2I), extending the transcription-independence conclusion to mammalian-adapted polymerase and to the mammalian proteins.
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Deleting the avian exon duplication from chicken ANP32A abolished rescue of the 627E polymerase, while a C-terminal or internal deletion of 31 amino acids in the low-complexity acidic region remained compatible with activity and longer deletions of that region abolished it (Figure 3A, Figure 3B, Figure 3C). The authors conclude that the low-complexity acidic region, and specifically its N-terminal portion, is pivotal.
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Immunoprecipitation of a TAP-tagged, catalytically inactive polymerase showed that genomic RNA binding by a 627E polymerase was not impaired relative to 627K, and that chicken ANP32A had no effect on binding (Figure 4A).
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Single-molecule FRET with fluorescently labeled promoters and recombinant catalytically inactive polymerases found comparable bound populations for genomic promoter binding whether the polymerase carried 627K, 627E, or lacked the entire 627-domain (Figure 4D), and comparable shifts from the unbound state for the complementary promoter (Figure 4E). The conclusion drawn is that the 627-domain plays no role in promoter binding, which contradicts earlier reports proposing weaker promoter binding by avian polymerases. The authors note that small amounts of endogenous human ANP32A and ANP32B may have co-purified with the recombinant polymerase, and argue this does not affect the outcome because the substitution had no significant effect on binding.
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In minigenome assays using a 76 nucleotide segment 5 template that does not require nucleoprotein, the K627E substitution still caused significant loss of activity and chicken ANP32A still restored it, with nucleoprotein presence or absence making no difference (Figure 5A). The same held on 47 nucleotide and 30 nucleotide templates (Figure 5B, Figure 5C), so the restriction and the ANP32A requirement are independent of nucleoprotein and of template length. The authors contrast this with an earlier report that restriction was diminished or abolished on short templates.
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The 3A8U genomic promoter mutation greatly enhanced replication overall, but the 627E polymerase remained significantly, if less markedly, restricted, and chicken ANP32A fully restored it (Figure 6B). The interpretation is that improved promoter strength alone does not overcome the restriction.
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With the G5U mutation limiting the template to primary complementary RNA synthesis, the 627E polymerase was unable to synthesize complementary RNA efficiently, and chicken ANP32A restored it, on both wild-type and 3A8U backgrounds (Figure 6C, Figure 6D, Figure 6E). This is the finding that contradicts the proposal that ANP32A acts only on the second step.
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With the 2C9G complementary promoter mutation preventing further complementary RNA synthesis, no genomic RNA synthesis was observed with the 627E polymerase, and chicken ANP32A significantly increased activity (Figure 6F, Figure 6G). Together with the preceding point, both directions of replication require ANP32A.
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Pre-expressing polymerase, nucleoprotein and chicken ANP32A before infection under actinomycin D showed that no complementary RNA was stabilized without polymerase, that 627K and 627E polymerases stabilized nascent complementary RNA equally well when their own catalytic activity was disabled, and that when active replication was permitted the 627E polymerase produced reduced genomic RNA, which chicken ANP32A overcame (Figure 6H). The observation is that the encapsidation function is unaffected by the 627 identity while the replication function is. The conclusion drawn is that ANP32A is needed for the actively replicating polymerase and not for the encapsidating one.
Mechanistic model
The model the data support is that ANP32A is required at both stages of genome replication and acts on the polymerase performing synthesis rather than on the polymerase capturing the nascent product. Species-specific differences in ANP32A therefore restrict avian-adapted polymerases at both steps in mammalian cells, and the PB2 627 substitution or supply of avian ANP32A restores the interaction.
Several elements are directly demonstrated. The requirement for ANP32A at each step separately is demonstrated by the promoter-uncoupling minigenome experiments, which is the core new result. The independence of the requirement from nucleoprotein, from template length, and from promoter binding is demonstrated by the nucleoprotein-free templates, the short templates, the co-immunoprecipitation and the single-molecule FRET measurements. The dissociation between an intact encapsidation function and an impaired replication function is demonstrated by the pre-expression experiment with a catalytically inactive polymerase.
The bridging mechanism itself is not established here. The idea that ANP32A mediates assembly of a replicase complex by bridging an RNA-bound and an RNA-free polymerase comes from published cryo-electron microscopy of an influenza C virus polymerase dimer, and the authors present their data as consistent with that structure rather than as a demonstration of it. No structure, no dimer measurement and no direct assembly assay is presented in this study.
The evolutionary account in the discussion is explicitly marked as speculation by the authors, who write that it is tempting to speculate that influenza A viruses evolved this dependency in avian species and that the shorter mammalian ANP32A cannot efficiently support replicase assembly at both stages, thereby requiring adaptive mutations in avian strains.
Conceptual or technical advance
The methodological contribution is the use of single-step promoter mutations to separate two steps of genome replication that are obligately coupled during infection. The G5U and 2C9G mutations restrict a minigenome to one direction of synthesis, which makes it possible to ask where a host factor is required rather than only whether it is required. That approach is reusable for other factors and other steps in the replication cycle.
Conceptually, the result removes a model. The proposal that ANP32A is required only for genomic RNA synthesis from the complementary intermediate is inconsistent with a requirement demonstrated in both directions, and the further placement of the requirement on the replicating rather than the encapsidating polymerase constrains how the bridging role seen in structural work must operate functionally. The promoter binding measurements also weigh against the competing explanation that avian polymerase restriction reflects weaker template binding.
Relationship to the broader research program
This paper is led by the te Velthuis laboratory at Princeton, with the first author based in the tenOever laboratory at New York University, and the tenOever contribution is one component of a collaborative study rather than the direction of the work. The mechanistic questions addressed, host factor requirements and promoter behavior in influenza polymerase function, sit alongside the tenOever laboratory's own long-running interest in the influenza polymerase and the transcription-to-replication switch, including the earlier work on small viral RNAs as enhancers of polymerase activity. Both lines converge on the question of what converts the polymerase between its activities and what supplies the missing component in each mode, but relating them formally is category 3 synthesis and depends on the corpus records for those papers rather than on anything asserted here.
The host adaptation framing, in which a single host protein difference sets the species barrier for an avian virus in mammalian cells, also connects to the broader recurring theme of species-specific constraints on influenza replication.
Related publications
- Perez and colleagues 2012, Journal of Virology, a small-RNA enhancer of viral polymerase activity. Relationship conceptual extension, from the tenOever laboratory. Not cited in this paper, and the connection is at the level of shared subject matter, namely control of the influenza polymerase transition between transcription and replication, rather than a direct lineage.
- Carrique and colleagues, cryo-electron microscopy of an influenza C virus polymerase dimer bridged by ANP32A. Relationship predecessor from another laboratory. This is the structural result the present functional data are presented as consistent with, cited as reference 7.
- Long and colleagues, on avian ANP32A and its exon duplication restoring avian polymerase activity in mammalian cells. Relationship predecessor from another laboratory, cited as reference 20, and the source of the framing that the study sets out to test systematically.
- Sugiyama and colleagues, on human ANP32A as an enhancer of genomic RNA synthesis with purified protein. Relationship predecessor from another laboratory, cited as reference 27, and the source of the single-step model that this study contradicts.
- te Velthuis and colleagues, previous work from the senior author's group on the PB2 627-domain and basic polymerase function, cited as reference 37, and on promoter mutants and minigenome constructs. Relationship methodological foundation.
Limitations and boundaries
Avian polymerase is modeled by a single PB2 K627E substitution in the mammalian-adapted A/WSN/33 background rather than by an authentic avian isolate, so the conclusions apply to that polymorphism and not necessarily to every determinant of avian polymerase restriction. Likewise, avian ANP32A is represented by chicken ANP32A supplied by transient transfection, which is an overexpression condition and not endogenous avian expression.
Much of the mechanistic work uses minigenome assays with short internally truncated templates of 76, 47 and 30 nucleotides. These are deliberately chosen to remove nucleoprotein and template length as variables, but they do not reproduce a full-length ribonucleoprotein, and the paper itself notes that the promoter-uncoupling question can only be addressed in minigenomes because both steps necessarily occur during infection.
The single-molecule FRET experiments use recombinant catalytically inactive polymerases, and the authors acknowledge that small amounts of endogenous human ANP32A and ANP32B may have co-purified. Their argument that this does not matter rests on the absence of a 627-dependent difference rather than on demonstrating that the preparations were free of these proteins.
The knockdown experiment removes both ANP32A and ANP32B by small interfering RNA, so residual protein and the contributions of the two family members are not separated, and the transcription conclusion rests on an early time point at high multiplicity.
The encapsidation conclusion comes from one experimental configuration, pre-expression of catalytically inactive polymerase under actinomycin D, and rests on complementary RNA stabilization as the readout for encapsidation rather than on a direct measurement of complex composition.
No structural or biophysical evidence for replicase assembly is generated in this study, so the bridging model remains imported from published structural work. No animal experiments are included, and no claim is made about transmission or pathogenesis. The cell systems are human, chicken and canine lines, with no primary human airway tissue.
Audience summaries
25 words
Influenza copies its genome in two steps. Blocking each step separately with promoter mutations shows the host protein ANP32A is needed for both, not just one.
75 words
Influenza A virus replicates its genome by first making a complementary copy and then copying that back into genomes, and the host protein ANP32A is essential for this and sets a barrier to avian viruses in mammalian cells. It had been proposed that ANP32A acts only at the second step. Using viral promoter mutations that permit one step at a time, this study shows both steps require ANP32A, acting on the actively synthesizing polymerase.
150 words
Influenza A virus genome replication proceeds through a complementary RNA intermediate, and each nascent product is encapsidated by an additional polymerase, so more than one polymerase participates. The host factor ANP32A is essential for replication, bridges an RNA-bound and an RNA-free polymerase in published structures, and differs between birds and mammals in ways that restrict avian polymerases. Work with purified protein had suggested ANP32A is needed only for synthesis of genomic RNA from the intermediate. Using mutations in the viral promoters that block initiation on the resulting product, this study restricts minigenomes to one replication step at a time and finds that an avian-like PB2 627E polymerase is impaired at both steps and rescued by chicken ANP32A at both. Single-molecule FRET shows the 627 position does not affect promoter binding, and pre-expression experiments place the requirement on the replicating rather than the encapsidating polymerase.
Documented publication relationships
- A Small-RNA Enhancer of Viral Polymerase Activity — conceptual extension.