Transcriptome-wide identification of the influenza A virus endoribonuclease PA-X cut sites by 5' RACE-Seq

Influenza A virus induces host shutoff mainly through its endoribonuclease PA-X. Like many host shutoff nucleases, PA-X activity affects different host RNAs to varying degrees. To understand how PA-X discriminates between different mRNAs, we took a high-throughput approach to directly identify PA-X cut sites transcriptome-wide and examine PA-X cleavage characteristics. To do this, we compared RNA fragments from mock infected cells to cells infected with the wild-type (WT) A/PuertoRico/8/1934 H1N1 (PR8) influenza virus strain, or the same strain engineered to lack PA-X, referred to as PR8-PA(∆X). Xrn1 knock-out human lung carcinoma epithelial A549 cells were used to limit degradation of the fragments created by PA-X and identify the specific location of the initial cut (Liu and Moss, 2016). These cells were infected in triplicate with wild-type (WT) A/PuertoRico/8/1934 H1N1 (PR8) influenza virus strain, or the same strain engineered to lack PA-X referred to as PR8-PA(∆X), or mock infected. Taking advantage of the fact that RNAs cleaved by PA-X have a free 5’ phosphate, we ligated an RNA adapter at the 5’ end of every cut RNAs, then added a 3’ adapter during reverse transcription, and finally added Illumina sequencing adapters on each end through a few rounds of PCR and performed RNAseq to sequence RNA fragments.