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 2a (eIF2a) 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. Overall design: 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).