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). Overall design: 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.

甲型流感病毒（Influenza A virus）主要通过其核糖核酸内切酶（endoribonuclease）PA-X介导宿主关闭程序（host shutoff）。与多数宿主关闭核酸酶类似，PA-X的酶活对不同宿主RNA的影响程度存在显著差异。为解析PA-X区分不同信使RNA（mRNA）的机制，本研究采用高通量策略在全转录组范围内直接鉴定PA-X的切割位点，并系统考察其切割特征。

为达成这一研究目标，我们将模拟感染细胞的RNA片段，与分别感染野生型（WT）A/PuertoRico/8/1934 H1N1（PR8）甲型流感病毒毒株，或经基因改造缺失PA-X的同毒株（命名为PR8-PA(ΔX)）的细胞的RNA片段进行比对分析。

实验设计概述：本研究选用Xrn1敲除的人肺癌上皮A549细胞，以抑制PA-X切割产生的RNA片段的降解，从而精准定位初始切割位点（Liu及Moss，2016）。将上述细胞分为三组，分别以野生型（WT）A/PuertoRico/8/1934 H1N1（PR8）甲型流感病毒毒株、经改造缺失PA-X的同毒株（PR8-PA(ΔX)）进行三次生物学重复感染，同时设置模拟感染对照组。

利用PA-X切割的RNA带有游离5'磷酸基团这一特性，我们在所有被切割RNA的5'端连接RNA接头，随后在逆转录过程中添加3'接头；最后通过数轮PCR在片段两端添加Illumina测序（Illumina sequencing）接头，并开展RNA测序（RNAseq）以获取RNA片段的序列信息。