Redefining activity in SARS-CoV-2 and its regulation by and .

RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, , along with one copy of and two copies of , forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these s and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 , , and . Molecular interactions among s and their effect on the polymerase activity were investigated, wherein alone exhibited notable activity, which was further enhanced by the presence of both and . The presence of only one cofactor, either or , completely inhibited activity and led to RNA template detachment. Computational analyses of different complexes suggested that binding of or alone to constricts the RNA entry channel, which was higher in the presence of , making it inappropriate for RNA entry/binding. We conclude that and together synergize to enhance the activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting or interactions with can be lethal to coronavirus replication.