Characterization of human poliovirus receptor (PVR) has led to a detailed description of the initial stage of the entry process, the virus-receptor interaction. However, kinetic and genetic evidence also indicate the existence of additional stages which involve membrane interactions and remain poorly understood. In this revised application, Dr. Chow at the University of Arkansas and her collaborator, Dr. M. Tosteson at Harvard, will evaluate two plausible models of RNA delivery into the cytoplasm. Both models require that the native (160S) particles interact with PVR to generate an """"""""entry-active"""""""" particle and that this altered particle interacts with host cell membranes. The simplest model proposes that, after PVR interaction, only the RNA genome is transported directly across the plasma membrane through virus-specific ion-permeable channels. The second model involves the passage of the """"""""delivery active"""""""" virus particle across the membrane into the cytoplasm and subsequent release of the RNA from the particle. To dissect these early events, the proposed studies will build on Drs. Chow and Tosteson's recent observation that entry competent viruses produce ion-permeable channels in model membranes. In the first Aim, the applicants propose to characterize conductance, kinetic characteristics and lumen size of the channels. They will also separately determine the effects on channel formation of docking the virus particles using the PVR versus using an Fc receptor with antibody coated particles. They also will monitor effects induced in the particle's channel forming properties from binding Win drugs or peptide specific antibodies.
The second Aim will characterize the capsid protein domains involved in membrane interaction and define their roles in virus entry. Existing and newly constructed mutants in VP4 or in the N-terminus of VP1 defective in entry will be examined using model membranes for any changes in channel properties; if found, these will be correlated with biological phenotypes. It is aticipated that the studies will provide the biochemical and biophysical make-up of the channel structure and allow the applicants to build a general model of the early stages of infection by nonenveloped viruses.
