Coronavirus Replicase-Reporter Fusions Provide Quantitative Analysis of Replication and Replication Complex Formation

Abstract

Coronaviruses are a family of enveloped, positive-sense RNA viruses that are important agents of disease, including severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Replication is associated with multiple virus-induced membrane structures that evolve during infection; however, the dynamics of this process remain poorly understood. In this study, we tested whether reporter molecules expressed from native locations within the replicase polyprotein of murine hepatitis virus as fusions with nonstructural proteins could define the expression and targeting of replicase proteins during infection in live cells. We demonstrate that the replicase gene tolerates the introduction of green fluorescent protein or firefly luciferase as fusions with replicase proteins. These viruses allow early quantitation of virus replication as well as real-time measurement of replication complexes.