Coronavirus endoribonuclease nsp15 suppresses host protein synthesis and evades PKR-eIF2α-mediated translation shutoff to ensure viral protein synthesis

The endoribonuclease (EndoU) nsp15 of coronaviruses plays a crucial role in evading host innate immune responses by reducing the abundance of viral double-stranded RNA (dsRNA). However, our understanding of its interactions with host cellular targets remains limited. In this study, we demonstrate that overexpression of nsp15 from four coronavirus genera inhibits cellular protein synthesis and causes nuclear retention of PABPC1. Mutation analysis confirms the essential role of EndoU activity in these processes. Fluorescence in situ hybridization (FISH) analysis shows that cellular mRNA co-localizes with nsp15 in certain cells. Real time RT-PCR indicates that the mRNA levels of several antiviral genes decrease in cells expressing nsp15, and this reduction depends on the EndoU activity of nsp15. Using infectious bronchitis virus (IBV) as a model, we investigate the inhibitory effect of nsp15 on protein translation during infection. We find that infection with IBV with functional nsp15 suppresses protein synthesis in a PKR-eIF2α independent manner, with PABPC1 mainly located in the cytoplasm. However, infection with EndoU activity-deficiency mutant virus rIBV-nsp15-H238A results in the accumulation of viral dsRNA, triggering a PKR-eIF2α-dependent shutdown of protein synthesis and leading to the nuclear relocation of PABPC1. In the absence of the PKR-eIF2α pathway, IBV is still able to suppress host protein synthesis, while the inhibitory effect of rIBV-nsp15-H238A on protein synthesis was significantly reduced. Although nsp15 locates to replication-transcription complex (RTC) during infection, RNA immunoprecipitation (RIP)-Seq analysis confirms that IBV nsp15 binds to six viral RNAs and 237 cellular RNAs. The proteins encoded by the nsp15-associated cellular RNAs predominantly involved in translation. Additionally, proteomic analysis of the nsp15 interactome identifies 809 cellular proteins, which are significantly enriched in pathways related to ribosome biogenesis, RNA processing, and translation. Therefore, nsp15 helps virus circumvent the detrimental PKR-eIF2α pathway by reducing viral dsRNA accumulation and suppresses host protein synthesis by targeting host RNAs and proteins. This study reveals unique yet conserved mechanisms of protein synthesis shutdown by catalytically active nsp15 EndoU, shedding light on how coronaviruses regulate host protein expression.