Polymerase trapping as the mechanism of H5 highly pathogenic avian influenza virus genesis

The emergence of highly pathogenic avian influenza viruses, such as the H5 subtype, is driven by the insertion of basic amino acids at the hemagglutinin cleavage site. A leading hypothesis suggests these nucleotide insertions result from «polymerase trapping» by a transient RNA pseudoknot (t-loop) formed between the ingoing and outgoing template during viral replication. To demonstrate this mechanism, we determined the cryo-EM structure of a pseudoknot-stalled influenza A polymerase.

Because the wild-type t-loop is highly transient, we engineered a functionally optimized 70 nucleotide mini-vRNA genome template designed to precisely stop ongoing RNA synthesis upon formation of the stabilised pseudoknot. In vitro RNA synthesis assays confirmed that the polymerase specifically stalls as intended. Cryo-EM analysis of the stalled state revealed the formation of the predicted double-stranded RNA t-loop distally formed outside the polymerase core, accommodated by the outward swinging of the PB1 beta-ribbon. Furthermore, the structural data captured a two-nucleotide backtracking of the product-template duplex. This backtracking provides direct insight into the insertion mechanism: in a wild-type A:U-rich context, strain likely induces inter-strand slippage, analogous to viral polyadenylation, resulting in the multi-nucleotide insertions as observed in virological studies.

These findings provide the first direct visual proof of the t-loop concept, providing mechanistic insights into H5 highly pathogenic avian influenza virus nucleotide insertion.