The Integrated Stress Response Suppresses Antiviral RNA Interference by Autophagy-mediated Degradation of RNA-induced Silencing Complex

The small interfering RNA (siRNA) pathway is a highly conserved antiviral defense mechanism in vertebrates and invertebrates. Although the core components of this pathway are well characterized, its upstream regulatory networks remain poorly understood. Here, we identify the integrated stress response (ISR) as a negative regulator of the siRNA pathway, and demonstrate that the picorna-like virus CrPV (Cricket Paralysis Virus) exploits this mechanism for immune evasion. Mechanistically, the picorna-like virus triggers the ISR through transcriptional suppression of ppp1r15, a key regulator of eukaryotic initiation factor 2α (eIF2α) dephosphorylation. ISR activation subsequently induces the autophagy-lysosomal pathway by up-regulating Atg1 transcription in an ATF4-dependent manner. This process leads to selective degradation of Argonaute 2 (Ago2) and other core components of the RNA-induced silencing complex (RISC), thereby suppressing the host RNA interference (RNAi) machinery and enhancing viral replication. Our findings uncover an unconventional immune evasion strategy employed by a picorna-like virus and establish a previously unrecognized crosstalk between the ISR and siRNA pathways. This RNA-sequencing aims to analyze the effect of ppp1r15 gene knockdown on transcription in Drosophila S2 cells. We transfected Drosophila S2 cells with dsRNA targeting GFP (as a control,labeled as dsGFP) and two dsRNAs targeting the ppp1r15 gene (labeled as dsppp1r15#1 and dsppp1r15#2), respectively. Each experiment was performed with three biological replicates (labeled as _1, _2, and _3). Total RNA was extracted from cells using the RNA isolater Total RNA Extraction Reagent (Vazyme, Cat #R401-01). The mRNA was then enriched and reverse-transcribed to construct a cDNA library. The cDNA libraries were sequenced using the Illumina NovaSeq 6000 (Illumina, San Diego, USA). mRNA enrichment, reverse transcription, cDNA library construction, quality control, and sequencing were performed by Biomarker Technologies (Biomarker Technologies Ltd, Beijing, China). The analysis of transcriptomic sequencing data was performed on the Secevo HPC cluster of the School of Ecology, Sun Yat-sen University (Shenzhen, China).

小干扰RNA（small interfering RNA, siRNA）通路是脊椎动物与无脊椎动物中高度保守的抗病毒防御机制。尽管该通路的核心组分已得到充分表征，但其上游调控网络仍有待深入解析。本研究鉴定出整合应激反应（integrated stress response, ISR）作为siRNA通路的负调控因子，并证实类小RNA病毒蟋蟀麻痹病毒（Cricket Paralysis Virus, CrPV）可利用该机制实现免疫逃逸。从机制层面而言，该类病毒通过转录抑制ppp1r15——真核起始因子2α（eukaryotic initiation factor 2α, eIF2α）去磷酸化的关键调控因子——来触发整合应激反应。随后，整合应激反应的激活会以ATF4依赖的方式上调Atg1的转录水平，进而诱导自噬-溶酶体通路。该过程会导致Argonaute 2（Ago2）以及RNA诱导沉默复合体（RNA-induced silencing complex, RISC）的其他核心组分发生选择性降解，从而抑制宿主的RNA干扰（RNA interference, RNAi）系统，增强病毒复制。本研究的发现揭示了一类类小RNA病毒所采用的非经典免疫逃逸策略，并确立了整合应激反应通路与siRNA通路之间此前未被认知的交叉调控关系。本次RNA测序实验旨在探究ppp1r15基因敲低对果蝇S2细胞转录组的影响。我们分别将靶向GFP的双链RNA（double-stranded RNA, dsRNA，作为对照，标记为dsGFP）与两种靶向ppp1r15基因的双链RNA（分别标记为dsppp1r15#1与dsppp1r15#2）转染至果蝇S2细胞中。每组实验均设置3次生物学重复（分别标记为_1、_2与_3）。使用RNA isolater总RNA提取试剂（Vazyme，货号Cat #R401-01）从细胞中提取总RNA。随后富集mRNA并进行逆转录以构建cDNA文库。使用Illumina NovaSeq 6000测序平台（Illumina, 美国圣地亚哥）对cDNA文库进行测序。mRNA富集、逆转录、cDNA文库构建、质量控制及测序工作均由Biomarker Technologies Ltd（中国北京）完成。转录组测序数据的分析工作在中山大学深圳校区生态学院的Secevo高性能计算集群上完成。