Polymerase trapping as the mechanism of H5 highly pathogenic avian influenza virus genesis.
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| Title: | Polymerase trapping as the mechanism of H5 highly pathogenic avian influenza virus genesis. |
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| Authors: | Funk, Mathis (AUTHOR), Spronken, Monique I. (AUTHOR), Hutchinson, Roy M. (AUTHOR), Arragain, Benoit (AUTHOR), Juyoux, Pauline (AUTHOR), Bestebroer, Theo M. (AUTHOR), de Bruin, Anja C. M. (AUTHOR), Gultyaev, Alexander P. (AUTHOR), Fouchier, Ron A. M. (AUTHOR), Cusack, Stephen (AUTHOR), te Velthuis, Aartjan J. W. (AUTHOR), Richard, Mathilde (AUTHOR) |
| Source: | Science. 3/12/2026, Vol. 391 Issue 6790, p1-15. 15p. |
| Subjects: | Polymerases, RNA, Avian influenza A virus, Influenza A virus, H5N1 subtype, Genetic mutation, Avian influenza |
| Abstract: | Highly pathogenic avian influenza viruses (HPAIVs) derive from H5 and H7 low pathogenic avian influenza viruses (LPAIVs). Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for the LPAIV-to-HPAIV transition, the mechanisms underlying the occurrence of insertion are unknown. Here, we show that transient H5 RNA structures, predicted to trap the influenza virus polymerase on purine-rich sequences, drive nucleotide insertions, providing empirical evidence of RNA structure involvement in MBCS acquisition. Introduction of H5-like sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions. Our results show that nucleotide insertions that underlie H5 HPAIV emergence result from an RNA structure–driven diversity-generating mechanism, which could also occur in other RNA viruses. Editor's summary: High-pathogenicity influenza viruses have not only been devastating the planet's wild and domestic bird populations, but they also represent a persistent threat of initiating a fatal human influenza pandemic. Funk et al. investigated how recombination of low-pathogenicity viruses is prone to incorporating sequences for the furin multibasic cleavage site in the hemagglutination gene, which promotes cell invasion by virus. The authors found that transient RNA structures in the virus replication machinery caused it to stutter on adenine-/uridine-rich sequences and allowed the nucleotide insertions that translate into the cleavage site. The authors suggest that these sorts of transient structures might also be present in other RNA viruses. —Caroline Ash INTRODUCTION: Influenza viruses pose a continuous health threat to animals and humans worldwide. Outbreaks of highly pathogenic avian influenza viruses (HPAIVs) have a devastating impact on domestic and wild bird populations as well as on wild mammals. Moreover, HPAIVs present a health risk to humans through repeated spillover events, renewing concerns about the potential for a new pandemic. HPAIVs emerge from H5 and H7 low pathogenic variants upon the introduction of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin (HA) gene. The insertion of this site enables systemic virus spread in poultry, leading to severe and often lethal disease. RATIONALE: Although MBCS acquisition was identified as the molecular hallmark of HPAIVs >40 years ago, the underlying molecular mechanisms have remained poorly understood. Recent advances in the structural and functional understanding of the influenza virus polymerase have allowed us to propose that MBCS-yielding insertions are caused by polymerase trapping by transient RNA structures formed by the entering and exiting template parts during replication of purine-rich sequences. To test this hypothesis, mutant H5 HAs with modified RNA structures or sequences were designed using an in-house bioinformatic tool predicting transient RNA template structures. A virus-free influenza replication system was used to detect all insertions, without constraints at the protein level or selection biases. The minimal replication unit of an H5 HPAIV, consisting of the polymerase, the nucleoprotein, and an H5 HA template, was reconstituted in mammalian and avian cells. A circular next-generation sequencing approach was developed to accurately determine rare insertions made by the influenza virus polymerase. RESULTS: Rare duplication insertions, analogous to those seen in H5 HPAIV in nature, were observed in mutated purine-rich HA cleavage sites. Bioinformatic analysis predicted that these insertions could be the result of a putative transient RNA template structure forming during replication of complementary RNA genome (cRNA) to viral RNA genome (vRNA). The role of this structure was shown by either disrupting or stabilizing it, which had the anticipated effect on insertion frequencies. Its role was further confirmed with unidirectional replication systems, in which HPAIV-like duplication insertions mainly occurred when using cRNA and not vRNA templates. Comparable insertion frequencies and patterns were observed in mammalian and avian cells, suggesting a lack of host factor involvement. Introduction of H5-like cleavage site sequences and structures into an H6 HA resulted in MBCS-yielding insertions. Direct biophysical evidence was provided using cryo–electron microscopy, revealing polymerase stalling through base pairing of the incoming and outgoing template parts. CONCLUSION: We provide empirical evidence of RNA structure involvement in HPAIV genesis. Our results show that MBCS acquisition is the result of the combination of insertion-prone RNA sequences and the formation of transient RNA structures. The interplay of these two factors might explain why HPAIV-yielding insertions are rare and restricted to two HA subtypes. Beyond influenza viruses, transient polymerase-trapping structures may also influence the replication or transcription of other negative-sense RNA viruses with similar polymerase entry and exit channel architecture. H5 highly pathogenic avian influenza virus genesis.: (A) Highly pathogenic viruses emerge from low pathogenic variants upon the acquisition of a multibasic HA cleavage site. (B) Our model of influenza virus polymerase trapping by transient template structures. (C) Transient template structure stabilization and purine-rich HA cleavage site sequences promote multibasic cleavage site insertions. (D) Cryo–electron microscopy structure of polymerase trapping by template RNA structure. [ABSTRACT FROM AUTHOR] |
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| Database: | Psychology and Behavioral Sciences Collection |
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