Elucidation of the mechanism generating insertions in the MBCS sequence of influenza A viruses.
High pathogenicity avian influenza viruses (HPAIVs) emerge from H5 and H7 low pathogenicity avian influenza virus (LPAIV) progenitors through mutations that introduce a multibasic cleavage site (MBCS) in haemagglutinin (HA). Although nucleotide insertions are a recurrent route to MBCS acquisition, the molecular determinants of this process, and whether specific H5 and H7 HA sequences are genetically predisposed to evolve towards HPAIV, remain unknown. Here, combining experimental evolution, ultra-deep sequencing, analysis of natural emergence events and thermodynamic modelling, we show that insertion risk is primarily determined by the local RNA sequence within the viral polymerase catalytic site. Predicted stationary or transient RNA secondary structures are neither required nor sufficient for high-frequency insertions and, at most, modestly modulate insertion rates. Insertions instead arise through polymerase slippage, in which destabilization of the product–template duplex and sequence-dependent reannealing generate template duplications. We formalize this mechanism in RNApredict, a thermodynamic model that captures key features of insertion frequency and composition and identifies H5 and H7 sequence intermediates with elevated potential to acquire a functional MBCS. These findings establish a primary sequence–driven mechanism for insertion-driven HPAIV emergence and provide a framework for identifying HA sequences at increased risk of evolving towards high pathogenicity
High pathogenicity avian influenza viruses (HPAIVs) emerge from H5 and H7 low pathogenicity avian influenza virus (LPAIV) progenitors through mutations that introduce a multibasic cleavage site (MBCS) in haemagglutinin (HA). Although nucleotide insertions are a recurrent route to MBCS acquisition, the molecular determinants of this process, and whether specific H5 and H7 HA sequences are genetically predisposed to evolve towards HPAIV, remain unknown. Here, combining experimental evolution, ultra-deep sequencing, analysis of natural emergence events and thermodynamic modelling, we show that insertion risk is primarily determined by the local RNA sequence within the viral polymerase catalytic site. Predicted stationary or transient RNA secondary structures are neither required nor sufficient for high-frequency insertions and, at most, modestly modulate insertion rates. Insertions instead arise through polymerase slippage, in which destabilization of the product–template duplex and sequence-dependent reannealing generate template duplications. We formalize this mechanism in RNApredict, a thermodynamic model that captures key features of insertion frequency and composition and identifies H5 and H7 sequence intermediates with elevated potential to acquire a functional MBCS. These findings establish a primary sequence–driven mechanism for insertion-driven HPAIV emergence and provide a framework for identifying HA sequences at increased risk of evolving towards high pathogenicity
Ecole Nationale Veterinaire Toulouse - Institut National de Recherche pour l'Agriculture, l'alimentation et l'Environnement