Danni Yong, McGill University

Danni Yong1,2, Samara Milstein1,2, GuanQun Liu1,2,3* 1Department of Microbiology and Immunology 2The McGill Research Centre on Complex Traits 3Mark Wainberg Centre for Viral Diseases, McGill University, Montreal, Quebec, Canada

Expressing & Multiplying

Studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to demand robust molecular platforms to precisely dissect viral replication, pathogenesis, and strain-specific phenotypes. While authentic virus systems remain the gold standard, their reliance on containment level 3 (CL3) infrastructure limits accessibility, throughput, and experimental flexibility. Conversely, reductionist approaches, such as pseudoviruses or individual protein overexpression, lack the full viral genome context and fail to recapitulate coordinated replication processes. Here, we report the development of an optimized, CL2-compatible SARS-CoV-2 replicon platform that preserves viral genome integrity and markedly enhances the yield of single-cycle viral replicon particles (VRPs) when structural glycoproteins, such as spike or VSV-G, are supplied in trans. We show that the platform supports modular spike exchange across strains and recapitulates variant-specific replication phenotypes previously observed with live viruses. Collectively, our CL2-compatible VRP platform provides a versatile and scalable system for mechanistic interrogation of SARS-CoV-2 replication and host interactions.