STRUCTURAL STUDIES OF POLIOVIRUS ASSEMBLY AND CELL-ENTRY INTERMEDIATES. James M. Hogle, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115

The high resolution structures of polio and related picornaviruses have provided considerable insights into those properties that are relevant to the extracellular form of the virus. It is known, however, that the virus undergoes significant conformational rearrangements during assembly and cell entry. We have recently described the structure of the empty capsid assembly intermediate of poliovirus (1). This form of the virus lacks the viral RNA and has yet to undergo the maturation cleavage of the immature capsid protein precursor VP0 to yield VP4 and VP2. The structure demonstrates that the cleavage of VP0 is required for the correct formation of a network of interactions of VP4 and the amino terminal extension of VP1 on the inside surface of the virion. Interestingly, those portions of the network which form only after VP0 cleavage involve normally internal portions of the capsid protein which are externalized during conformational changes which are induced by binding to the poliovirus receptor. These results, together with analysis a wide variety of poliovirus variants with altered stability and/or receptor interactions, have given rise to a picture in which the virus is a metastable intermediate which links virus assembly and cell-entry. In this model the cleavage of VP0 and subsequent rearrangements of VP4 and the aminoterminal extension of VP1 trap the virus in the metastable form and prime it for subsequent structural rearrangements required for cell-entry. The receptor "catalyzes" these transitions by utilizing some of the energy which is released upon its binding to receptor to lower the activation barrier which traps the virus in this metastable state (2).

In order to further characterize this process we have undertaken a program of structural studies of intermediates which are thought to be important in the cell entry process using cryoelectron microscopy (in collaboration with Alasdair Stevens, Frank Booy, Benes Trus and David Belnap at NIH) and x-ray crystallography. Low resolution models for two such intermediates will be presented.