Abstract

Infections caused by coronaviruses are persistent threats to human health in recent decades, necessitating the development of innovative anti-coronaviral therapies. RNA interference (RNAi) is a conserved cell-intrinsic antiviral mechanism in diverse eukaryotic organisms, including mammals. To counteract, many viruses encode viral suppressors of RNAi (VSRs) to evade antiviral RNAi, implying that targeting VSRs could be a promising strategy to develop antiviral therapies. Here, we designed a series of peptides specifically targeting the SARS-CoV-2-encoded VSR, nucleocapsid (N)?protein. Among these peptides, one designated GL directly interacts with N protein and inactivates its VSR activity, which unlocks a potent RNAi response and effectively inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Moreover, GL exhibited RNAi-dependent antiviral effects not only against various SARS-CoV-2 variants, including Delta, Omicron BA.5, XBB, and JN.1, but also against other coronaviruses such as human coronavirus (HCoV)-229E, HCoV-OC43, and mouse hepatitis virus. The in?vivo anti-coronaviral activity of GL was also confirmed. Our findings indicate that the VSR-targeting peptide GL has the potential to be further developed as a broad-spectrum anti-coronaviral treatment, highlighting the functional importance and therapeutic potential of antiviral RNAi.