RR:C19 Evidence Scale rating by reviewer:
Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.
In this article, the authors described the creation of a non-infectious replicon of SARSCoV-2 into a bacterial artificial chromosome (BAC). They constructed the BAC by using overlapping PCR and gene fusion to piece together the SARS-CoV-2 sequence from RNA derived from cells infected with the nCoV-SH01 strain. They removed all structural genes from the replicon except the Nucleocapsid gene, which is known to be required for CoV RNA replication. The BAC contains a T7 promoter so that the viral RNA can be transcribed and then transfected into a variety of cell lines. The authors clearly showed, using a luciferase reporter, that the replicon was active in multiple cell lines, and it was inhibited by known RNA pol inhibitors and IFN. Interestingly they also showed that the replication was inhibited by overexpressing ZAP (PARP13). The replicon could be useful for identifying the function of non-structural proteins or screening antiviral compounds outside of BSL-3 conditions.
Overall this is a reliable study that is well-written and presented. The conclusions are well supported by their data and appropriate controls are included. However, it’s unclear how much this reagent will ultimately benefit the research community. While replicons have been developed for SARS-CoV and MERS-CoV, they are not widely used. Replicons have distinct differences from real virus infections, which limits their usefulness. There are a few things I would suggest they need to add. First, there are no statistics included, so it’s difficult to tell which results are statistically significant. Similarly, it’s not stated how many times each experiment was performed. There is a significant increase in the background of the assay between 0 and 8 hpi, which is not explained or discussed. Finally, the authors should use a method besides luminescence as evidence for replication, like quantitative real-time PCR, which could measure both sub-genomic and genomic RNA replication