Generation of a fully infectious SARS-CoV-2 encoding an internally tagged nsp3

The analysis of coronavirus replicase proteins during authentic infection is experimentally challenging, particularly for large and essential components embedded within virus-induced replication organelles. Non-structural protein 3 (nsp3), the largest coronavirus replicase protein, plays a central role in replication organelle formation, but has remained difficult to genetically manipulate. Here, we generated a fully infectious recombinant SARS-CoV-2 encoding an internal ALFA epitope tag upstream of the Mac1 domain within nsp3 (rALFA). Growth kinetics analyses demonstrated that rALFA replicated with kinetics and peak titers comparable to those of untagged recombinant SARS-CoV-2, and retained lytic infection. Tagged nsp3 displayed subcellular localization patterns equal to those observed in infections with untagged virus, and nanobody-based detection enabled robust visualization of nsp3 in infected cells. This internally tagged SARS-CoV-2 establishes a useful experimental platform for future analyses of nsp3 localization and replication organelle dynamics under authentic infection conditions, and provides a foundation for subsequent functional studies. IMPORTANCE The manuscript presents a practical tool that advances coronavirus methodology.