SARS-CoV-2 nsp13 restricts episomal DNA transcription without affecting chromosomal DNA

Non-structural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication and is considered an attractive target for the development of novel antiviral drugs. We were initially interested in the interplay between nsp13 and interferon signaling, and found that nsp13 inhibited reporter signal of IFN-ß promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-ß promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted expression of foreign genes originated from plasmid transfection, but failed to inhibit them after chromosome integration. These data together with results from run-off transcription assay and RNA sequencing suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to episomal DNA transcriptional inhibition, And this restriction required direct interaction with episomal DNA. Further, we developed a high-throughput nsp13 drug screening method based on the correlation between the helicase activity and nsp13 inhibition on episomal DNA. This method evaluates the inhibitory effect of compounds on nsp13 by detecting the expression of reporter plasmids after co-transfection with nsp13 plasmids, which is economical and convenient compared with conventional methods. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. Overall design: HEK293T cells were co-transfected with pEF1a-Luc and pHA-NSP13 or empty vector for 24 h, chromosomal gene expression was determined by RNA-seq. This experiment to detect the effect of SARS-CoV-2 NSP13 on chromosomal gene expression.