Systematic analysis of RNAi-accessible SARS-CoV-2 replication steps identifies ORF1 as promising target

Abstract A promising approach to tackle the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. The proof of concept that SARS-CoV-2 can be inhibited with siRNAs, however, is missing. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, terminating replication before start of transcription and preventing cytopathic effects. Coronaviruses replicate via negative sense intermediate transcripts using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5’ and 3’ end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting sequences shared by different viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs targeting open reading frame 1 (ORF1) which is solely part of gRNA. We propose two independent mechanisms for this: An increased accessibility of translational-active ORF1 before the start of transcription, as well as highly abundant sgRNAs out-competing siRNAs that target common sequences of transcripts. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that an early therapy start, together with targeting ORF1, might be key for high antiviral efficacy.