DOI: 10.5061/dryad.x69p8czs4
title: Polymerase trapping as mechanism of H5 highly pathogenic avian influenza virus genesis

description: 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 the first strong 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 a previously unknown RNA-structure-driven
 diversity-generating mechanism, which could be shared with other RNA
 viruses.
description: # Polymerase trapping as mechanism of H5 highly pathogenic avian influenza
 virus genesis Authors: Mathis Funk, Monique I. Spronken, Roy M.
 Hutchinson, Benoit Arragain, Pauline Juyoux, Theo M. Bestebroer, Anja C.M.
 de Bruin, Alexander P. Gultyaev, Ron A.M. Fouchier, Stephen Cusack,
 Aartjan J.W. te Velthuis, Mathilde Richard This README file describes the
 data package accompanying the above publication. #### Context of the study
 Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease
 and high fatality in poultry. HPAIVs spontaneously emerge from low
 pathogenic avian influenza viruses (LPAIV). Although insertion of a
 furin-cleavable multibasic cleavage site in the hemagglutinin (HA) gene
 was identified decades ago as the genetic basis for LPAIV-to-HPAIV
 transition, the exact mechanisms underlying said insertion have remained
 unknown. We have suggested previously that these insertions are driven by
 transient RNA template structures forming around the influenza polymerase
 as it replicates this region. Here we performed circular sequencing of the
 cleavage site in wild-type HAs and HAs in which putative
 polymerase-trapping RNA structures were altered by nucleotide subsitutions
 in the vicinity of the cleavage site. #### Experimental design A
 virus-free influenza replication system was used to detect atons without
 constraints at the protein level or selection biases. Mammalian cells
 (293T cells) or avian cells (DF1 cells) were transfected with plasmids
 coding for the influenza virus RNA-dependent RNA poRNA-dependentp), the
 nucleoprotein, and the hemagglutinin, in viral RNA (vRNA) or complementary
 RNA (cRNA) to reconstitute viral ribonucleoproteins, the replication unit
 of influenza viruses. Two days after transfection, cells were lysed, and
 total RNA was extracted. The hemagglutinin cleavage site was sequenced
 using a circular next-generation sequencing approach to accurately
 discriminate insertions by the influenza RdRp from background insertions
 due to plasmid transcription by the human polymerase I, allowing detection
 of rare insertions with high confidence. Briefly, total RNA was
 circularized, and DNA was produced through reverse transcription and
 second synthesis. Second-strand cons were then generated with polymerase
 chain reaction and sequenced using the Illumina plateform. Type of
 splatformtant hemagglutinins with modified RNA structures or sequences
 were designed using an in-house bioinformatic tool bioinformaticnsient RNA
 structures, and their impact on insertion pattern and frequency was
 assessed. Each hemagglutinin construct was tested in two independent
 transfection experiments (replicate experiments 1 and 2). Hemagglutinins
 of three avian influenza viruses were studied: IN (A/Indonesia/5/2005,
 H5N1), NL (A/mallard/Netherlands/3/1999, H5N1) and SW (A/mallard/S,
 Sweden/81/2002, H6N1). Hemagglutinin cleavage sites were mutated from the
 low pathogenic avian influenza virus H5 or H6 consensus sequence (RETR or
 IETR, respectively) to REKR or RKKR. In addition, substitutions were
 introduced in parts flanking the hemagglutinin cleavage site to either
 disrupt or stabilize predicted RNA structures. To understand whether
 insertion patterns insertionfic to the hemagglutinin cleavage site, two
 other regions in the hemagglutinin were studied in the NL hemagglutinin
 (NL-556 and NL-1275). To confirm the role of a putative transient cRNA
 structure, unidirectional replication experiments were performed, in which
 either cRNA or vRNA were produced by the wasluenza virus RdRp (IN-cv (cRNA
 to vRNA) or IN-vc (vRNA to cRNA)). Each sample was paired with a negative
 control to assess background insertions introduced during initial plasmid
 transcription by the human polymerase 1. In these samples, cells were
 transfected only with plasmids coding for the hemagglutinin v or cRNA and
 the nucleoprotein (np samples, without influenza virus RdRp). #### Type of
 data Uploaded reads are single merged reads after paired-end sequencing.
 Reads were merged using Adapter Removal (v. 2.3.2) and the following
 command: AdapterRemoval --file1 reads_1.fq --file2 reads_2.fq --basename
 output_paired --collapse --threads Merged reads were further unprocessed
 beyond the removal of contaminating sequences as stated in the Methods
 section of the associated article. #### Organization of the data Reads are
 in the "fastq.zip" folder. Metadata is found in the file named
 "sample_information.tsv". The .tsv file is organized as follows:
 1. Column 1: sample number, corresponding to the number indicated at the
 beginning of each fastq file for sample identification, organized under
 categories corresponding to presentation of the data in the main text or
 supplementary data of the article. 2. Column 2: sample name, corresponding
 to the name of each sample as described in the article. 3. Column 3:
 next-generation sequencing plateform used to sequence the sample. 4.
 Column 4: name of the fastq file. 5. Column 5: sample description as
 described in the article. 6. Column 6: replicate number.