The trapped-RdRp model

For archived published version, see:

Trapping of the influenza virus RNA-dependent RNA polymerase (RdRp) as the molecular mechanism underlying recurrent insertions in influenza virus genomes was proposed in Gultyaev et al 2019¹. The model states that insertion hotspots can occur when transient RNA structures form around the influenza virus RdRp during replication of insertion-prone sequences. The physical constraint on the RdRp leads to an increase in error rates. Insertion hotspots therefore result from a synergistic combination of error-prone RNA sequences and error-enhancing RNA structures.

This repository is intended to regroup scripts written to support study of the formation of RdRp-trapping RNA structures and their impact on RdRp errors during influenza genome replication in the workgroup of Mathilde Richard.

A brief overview of the scripts and their purpose:

Slidingfold

The slidingfold script is used to predict the formation of transient RdRp-trapping RNA structures, based on the sliding window approach proposed in French and Pitré et al 2022². We slide the RdRp nucleotide-by-nucleotide along the template and use the ViennaRNA package to predict folding of small upstream and downstream sequence parts.

Results can be output as simple text files, pdf images, or interactive graphs thanks to Plotly and Jinja.

Cirseq-analysis

In Funk et al 2024³, we use a CirSeq-based approach to investigate the impact of RNA sequence and structure on insertion frequencies at the HA cleavage site. The NGS data is first processed using a slightly modified version of the original CirSeq⁴ script and is then further analyzed using a custom script to

• Identify UMIs and remove PCR duplicates (thanks to Edlib)
• Identify all possible alignments of insertions/deletions
• Realign all insertion/deletions to the most stable RdRp-trapping structure possible (thanks to ViennaRNA)
• Calculate position-by-position coverage of the reference

References and notes

Footnotes

1. A. P. Gultyaev, M. Richard, M. I. Spronken, R. C. L. Olsthoorn, R. A. M. Fouchier, Conserved structural RNA domains in regions coding for cleavage site motifs in hemagglutinin genes of influenza viruses. Virus Evol. 5, 1–11 (2019). ↩

2. H. French, E. Pitré, M. S. Oade, E. Elshina, K. Bisht, A. King, D. L. V. Bauer, A. J. W. te Velthuis, Transient RNA structures cause aberrant influenza virus replication and innate immune activation. Sci. Adv. 8, 1–11 (2022). ↩

3. M. Funk, M. I. Spronken, T. M. Bestebroer, A. C. M. de Bruin, A. P. Gultyaev, R. A. M. Fouchier, A. J. W. te Velthuis, M. Richard, Transient RNA structures underlie highly pathogenic avian influenza virus genesis. ↩

4. A. Acevedo, R. Andino, Library preparation for highly accurate population sequencing of RNA viruses. Nat. Protoc. 9, 1760–1769 (2014). ↩