Receptor binding, capsid expansion, and conversion to A-particles are the earliest steps in enterovirus infection of a cell. The conversion of mature virus to A-particles is catalyzed by virus receptors. The goal of this work is to understand these events in order to better understand how the viruses infect cells. An allosteric model describes the kinetics for receptor-catalyzed conversion of Coxsackievirus B3 (CVB3) to inactive A- particles at 37C, and should be relevant to related enteroviruses. This model has been used to predict the effects of specific mutations that alter virus stability. Hypothesis 1: The mutation of leucine in VP1 residue 92 to valine (L1092V) increases CVB3/28 stability at 37C by shifting the conformational (?breathing?) equilibrium (Keq) in favor of the closed conformation. This hypothesis will be tested by determining Keq and k from dose response curves of soluble receptor (sCAR)-catalyzed inactivation of CVB3/28 L1092V and comparing the results to those obtained using CVB3/28. Hypothesis 2: The mutation of alanine in VP3 residue 180 to threonine (A3180T) increases virus stability at 37C by slowing the rate, k (maximum receptor-catalyzed inactivation rate), of conversion from the open conformation to the A-particle. This hypothesis will be tested as in aim 1, comparing results from CVB3/28 A3180T with those from CVB3/28. Residue 3180 is located at an interface between protomers, and the model predicts that the increased stability imparted by the mutation will diminish k, but not Keq, relative to values observed with CVB3/28. In tests of both hypotheses, estimates of Kd and Kd? (equilibrium dissociation constants for the closed and open conformations) will also result from fitting the model(s) to the data. These binding constants will also be compared among virus strains by direct analysis at temperatures <25C. The results will help refine the allosteric model, and provide new information regarding structure and function in the viruses and their interaction with receptor. The proposed experiments examine specific biological features important for infection of susceptible cells that are shared by many enteroviruses and rhinoviruses that cause human disease. New knowledge of these features may lead to new ways to impede infection by shifting the conformational equilibrium toward the stable closed form (as with some antivirals). Such knowledge may also reveal new approaches to antivirals through destabilizing the virus before it can interact with a host cell. The relationship between virus strains with different inherent stabilities and the kinetics of receptor-catalyzed conversion to A-particles have not been examined for these viruses. New information regarding virus capsid structure-function is anticipated.
The enteroviruses, all of which are structurally similar and have similar life cycles, include a number of human pathogens that cause widely-recognized diseases, including paralytic poliomyelitis (poliovirus), myocarditis and pancreatitis (Coxsackievirus), encephalitis (EV 71), and others. Knowledge gained from study of one is useful for understanding other members of the genus. New knowledge from this proposal may suggest new ways to impede infection by altering the virus conformational equilibrium.
