bCoxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin ␣ v family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for ␣ v  6 , which recognizes the arginine-glycine-aspartic acid (RGD) loop present on the C terminus of viral capsid protein, VP1. As the atomic model of CVA9 lacks the RGD loop, we used surface plasmon resonance, electron cryo-microscopy, and image reconstruction to characterize the capsid-integrin interactions and the conformational changes on genome release. We show that the integrin binds to the capsid with nanomolar affinity and that the binding of integrin to the virion does not induce uncoating, thereby implying that further steps are required for release of the genome. Electron cryo-tomography and single-particle image reconstruction revealed variation in the number and conformation of the integrins bound to the capsid, with the integrin footprint mapping close to the predicted site for the exposed RGD loop on VP1. Comparison of empty and RNA-filled capsid reconstructions showed that the capsid undergoes conformational changes when the genome is released, so that the RNA-capsid interactions in the N termini of VP1 and VP4 are lost, VP4 is removed, and the capsid becomes more porous, as has been reported for poliovirus 1, human rhinovirus 2, enterovirus 71, and coxsackievirus A7. These results are important for understanding the structural basis of integrin binding to CVA9 and the molecular events leading to CVA9 cell entry and uncoating.
Picornaviruses that belong to the Human enterovirus B species (HEV-B) in the family Picornaviridae are the major cause of aseptic meningitis (1, 2). HEV-B types, including coxsackievirus A9 (CVA9), also exhibit a wide range of other clinical manifestations of acute disease, including respiratory infections, myocarditis, encephalitis, paralysis, rash, and severe generalized infections in newborns (3-7). Moreover, there is evidence that suggests that HEV-B, specifically, CVA9, CVB1, CVB3, and CVB5, may be involved in the pathogenesis of childhood diabetes, the rate of incidence of which is increasing in Western countries (8-11).The CVA9 particle is about 28 nm in diameter and consists of a nonenveloped capsid with icosahedral symmetry, surrounding a positive-sense RNA genome of 7,452 nucleotides (12, 13). The CVA9 Tϭ1 (pseudo-Tϭ3) capsid is made up of 60 copies each of the four viral capsid proteins (VP1 to VP4) as revealed by its atomic model (12). Each of VP1, VP2, and VP3 possesses an eightstranded -barrel structure. VP1 mainly forms the vertices of the capsid, with VP2 and VP3 alternating on the 3-fold and 2-fold axes of symmetry (12). These are common characteristics that are shared by most picornaviruses (12,14). Depressions of about 15 Å form the canyon region and are also seen around the 2-fold axes (12). CVA9 interacts with cell surface ␣ v integrins during the early stages of infection via an arginine-g...