Conformations of the monomeric hepatitis C virus RNA-dependent RNA polymerase

Sreedhar Chinnaswamy^1,2,3, Ayaluru Murali^1,3, Hui Cai1, Guanghui Yi¹, Satheesh Palaninathan², C Cheng Kao¹

¹Interdisciplinary Biochemistry Program, Indiana University, Bloomington, Indiana, USA; ²Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas, USA; ³These authors contributed equally to this work and can be considered co-equal first authors

Abstract: The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) changes its conformation and oligomerization state in association with the steps in RNA synthesis. Using a human right hand as an analogy, the crystal structure of the HCV RdRp has extensive interactions between the “finger” and “thumb” domains which result in a closed conformation. However, the RdRp must form a partially open conformation to accommodate the nascent and template RNA duplex during RNA synthesis, and to interact with the retinoblastoma protein through residues in the “palm” domain. A motif named the Δ1 loop has been previously proposed to regulate the transition from the closed to the open conformation. We used negative-stain electron microscopy and single particle reconstruction to identify several conformations of the HCV RdRp monomer, from closed to open. An RdRp with five amino acids deleted in the tip of the Δ1 loop resulted in an open conformation, confirming the importance of this loop in regulating RdRp conformations. Bioinformatics analysis of HCV strains focusing on the Δ1 loop and its interacting surfaces further defined the requirements for this gating mechanism in the HCV RdRp. These results provide glimpses into the dynamic conformations of the HCV RdRp and should provide insights into alternative conformations of the HCV polymerase that could serve as targets for antiviral development against HCV.

Keywords: hepatitis C virus, RNA-dependent RNA polymerase, single particle reconstruction, conformational change