mapping the ph sensors critical for host cell entry by a complex nonenveloped virus bluetongue virus btv in the family reoviridae is an insectborne doublecapsid virus causing hemorrhagic disease in livestock around the world here we elucidate how outer capsid proteins vp2 and vp5 coordinate cell entry of btv to identify key functional residues we used atomiclevel structural data to guide mutagenesis of vp2 and vp5 and a series of biological and biochemical approaches including sitedirected mutagenesis reverse geneticsbased 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 virusimportance virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented to enter effectively viruses must sense the cellular environment and when appropriate initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm many viruses sense ph but how this happens and the events that follow are often poorly understood here we address this question for a large multilayered bluetongue virus we show key residues in outer capsid proteins a phsensing histidine of a zinc finger within the receptorbinding vp2 protein and certain histidine residues in the membranepenetrating vp5 protein that detect cellular ph leading to irreversible changes and propel the virus through the cell membrane our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses