Structural basis for the formation of a poliovirus RNA replication complex
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Poliovirus (PV) replication occurs in a complex associated with rearranged intracellular membranes. Oligomerization of PV RNA-dependent RNA polymerase (3Dpol) is expected to optimize RNA replication. However, the mechanism of membrane remodeling by PV and the structure of oligomeric 3Dpol remain elusive. In this study, cryo-electron microscopy is employed to specify the role of the PV protein 3AB in the generation of double-membrane vesicles and to explore the interfaces required for 3Dpol self-assembly. Examination of the ultrastructure of HeLa cells containing 3AB protein reveals severe cytopathic effects, implying a physiological role of 3AB in membrane remodeling. Addition of 3AB protein to liposomes generates double-membrane vesicles. Liposome transformation by 3AB proceeds by membrane invagination induced by 3AB adsorption, as demonstrated by a topological analysis of 3AB-proteoliposomes. In both planar and tubular arrays of 3Dpol, subunits interact across interface-I with quasi-21 symmetry, as revealed by image processing of electron microscopy data. Parallel association of interface-I filaments mediated by interface-II interactions is proposed, consistent with previous results from site-directed mutagenesis. Ghost reflections from diffraction patterns of 3Dpol helical arrays indicate lateral bundling of interface-I filaments using non-equivalent subunit-subunit contacts for interface-II. Studies of membrane remodeling by 3AB provide insight into cellular membrane remodeling processes. Structures of 3Dpol lattices described in this thesis are consistent with the hypothesis that heterogeneous 3Dpol oligomerization facilitates RNA replication.
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