A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates 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 common sequences shared by all 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 that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.

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A promising approach to combat SARS-CoV-2 constitute small interfering (si)RNAs. We systematically analyzed which SARS-CoV-2 replication steps are accessible for RNAi. We found that siRNAs can target the genome of incoming SARS-CoV-2 terminating replication after cell entry and preventing cytopathy. Suprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting sequences shared by different viral transcripts allowed simultaneous suppression of genomic and subgenomic viral RNAs by a single siRNA. The most effective suppression of viral replication and spread was achieved by siRNAs targeting open reading frame 1 (ORF1) which is solely part of genomic RNA. We propose that an improved accessibility of translational-active ORF1 during early replication, as well as the outcompetition of RNAi factors by common sequences of transcripts are responsible for this. Our work encourages efforts to develop siRNA-based therapies for COVID-19. Targeting ORF1, together with early treatment start or prophylactic use may be key for high antiviral efficiency.