Interaction of SCoV-2NSP7orNSP8alone withNSP12causes constriction of the RNA entry channel: Implications for novel RdRp inhibitor drug discovery

Abstract

RNA-dependent RNA polymerase (RdRP) is a critical component of the RNA virus life cycle, including SCoV-2. Among the Coronavirus-encoded proteins, non-structural protein 12 (NSP12) exhibits polymerase activity in collaboration with one unit ofNSP7and two units ofNSP8, constituting the RdRp holoenzyme. While there is abundant information on SCoV-2 RdRp-mediated RNA replication, the influence of interplay amongNSP12,NSP7, andNSP8on template RNA binding and primer extension activity remains relatively unexplored and poorly understood. Here, we recreated a functional RdRp holoenzyme in vitro using recombinant SCoV-2NSP12,NSP7, andNSP8, and established its functional activity. Subsequently, molecular interactions among theNSPsin the presence of a variety of templates and their effects on polymerase activity were studied, wherein we found thatNSP12alone exhibited notable polymerase activity that increased significantly in the presence ofNSP7andNSP8. However, this activity was completely shut down, and the template RNA primer complex was detached fromNSP12when one of the to cofactors was present. Through computational analysis, we found that the template RNA entry channel was more constricted in the presence of one of the two cofactors, which was relatively more constricted in the presence ofNSP8compared to that in the presence ofNSP7. In conclusion, we report thatNSP7andNSP8together synergise to enhance the activity ofNSP12, but antagonise when present alone. Our findings have implications for novel drug development, and compounds that obstruct the binding ofNSP7orNSP8toNSP12can have lethal effects on viral RNA replication.