Joseph Luna & Inna Ricardo-Lax - May 12, 2021

Joseph Luna, PhD, Postdoctoral Fellow, Inna Ricardo-Lax, PhD, Postdoctoral Fellow, Laboratory of Charles M. Rice, PhD Rockefeller University, "Efficient replication and trans-packaging of SARS-CoV-2 replicons"

They made a Spike-deleted SARS-Cov-2 replicon. Self-replication RNA (replicon) provides advantages of antiviral drug discovery, flexible and selectable reporter readouts and no infection (safe to use at BSL2). The SARS-CoV-2 virus is consists of 2 large ORFs (1a and 1b) formed viral replicase and structural protein and accessory proteins. Initially, the spike protein region was replaced with a reporter gene cassette. Yeast base reverse genetic system was used which produced 13 pieces of overlapping PCR fragments and engineered one of the fragments to replace. They are transformed into yeast and assembled replicon plasmid then sequenced to confirm. However, the replicon plasmid was contaminated with yeast DNA. DNA polymerase from Phi29 bacterial phage was applied to amplify circular DNA and boost transcription reaction. Full length of banding pattern of replicon was obtained without contamination from E.coli and yeast. RNA was made from the DNA and introduced into cells with a higher yield of replicon (10~20% positive). These reporter replicons were tested for compound testing, host factor screening and viral variant characterization. Nsp1 loss of function mutants was less cytotoxic and hyper-sensitive to IFNs. However, a challenge of this system is the electroporation of RNA is artificial and an accessible amount of transfection could be toxic to cells.

To overcome this limitation, they made a trans-packaged SARS-CoV-2 replicon (TPR) by providing glycoprotein spike protein. This glycoprotein trans-complemented replicon system has the advantages that it avoids the harsh electroporation gene delivery and provides an isogenic platform and enables to make the rapid generation of newly occurring spike protein variants for Neutralization assay. It is a VSV-G pseudotyping system so, target cells are not necessary to express ACE2 to get infected. The replicon also can be delivered to a various range of cell types and culture systems (Huh7.5, Vero, Caco2, Calu3, A549, NHBE and NHLF). Minimal replicons lacking gene products (ex; deletion of accessory genes) can be packaged. The key point is the virions are single-cycle. The recipient cell (P1) contains TRPs but no TPRs were found in recipient cells (P2).