Generation and Characterization of a Tagged Recombinant SARS‐CoV‐2 for Functional Replicase Studies

Coronavirus macrodomains have been described as important virulence factors which compromise type-I interferon (IFN) signaling, but their precise mechanism of action remains unclear and robust tools for interactome studies are still missing. Using reverse genetics, we generated and characterized recombinant SARS-CoV-2 (rALFA) encoding an ALFA-tag upstream of Mac1 to enable targeted analysis during viral replication. Infection assays and immunofluorescence staining (IF) were performed in comparison to recombinant SARS-CoV-2 without replicase modifications (rWT) and a variant lacking Mac1 (rΔMac1). All recombinant variants proved replication competent. Preserved lytic infection was confirmed for rALFA with identical plaque morphology compared to rWT and rΔMac1. On Vero E6 cells the recombinant viruses reached comparably high titres (mean PFU/ml rWT: 3.11 x106, rΔMac1: 1.20 x107, rALFA: 7.55 x106), on Calu-3 cells titres reached at 72 hpi (mean PFU/ml rWT: 8.89 x107, rΔMac1: 9.33 x105, rALFA: 2.28 x107) were comparable for rWT and rALFA yet significantly lower for rΔMac1. Immunofluorescence staining confirmed robust expression and cytoplasmic localization of the tagged nsp3. ALFA-tagging allowed for specific, super resolved IF images of Mac1 by anti-ALFA nanobodies in rALFA infected A549 cells. In conclusion, the tagged SARS-CoV-2 represents a versatile tool for functional studies of nsp3 interactions and dynamics of subcellular trafficking during infection, prospectively enabling validation of new therapeutic approaches.