Mapping the pH sensors critical for host cell entry by a complex Nonenveloped Virus

Bluetongue virus (BTV), in family Reoviridae, is an insect-borne, double capsid virus causing haemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. The recently solved high-resolution structures reveal unique features of BTV VP2 and VP5. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.