3D structural details for the initial assembly steps of poxvirus morphogenesis

Abstract

The poxvirus family is a group of DNA viruses that replicates in the cytoplasm of vertebrate and invertebrate cells. This viral family uses over 100 viral proteins for their multi-step assembly. The first unique structures detected as part of the morphogenesis of poxviruses are crescent-shaped precursor membranes derived from nascent cellular compartments. These crescents, which appear to be decorated by viral proteins on their outersurface, expand eventually to form spherical-shaped particles called immature virion (IV). Deep-etch EM analysis has determined that the crescent membranes and spherical IV particles include a single lipid bilayer stabilized by a continuous honeycomb lattice comprising a specific scaffold made up of the D13 protein. X-ray crystallography has shown the D13 protein consists of two jellyroll structures (J1 and J2) in a so-called double-barrel organization, and a head domain (H) with novel β-sandwich fold. Like many other capsid proteins of DNA viruses, D13 protein can undergo self-interaction and form polymorphic structures including trimers, filamentous forms and a large aggregates. Attachment of the D13 trimer to a membrane supports induces large, regular assemblies of D13 that are visualized by electron microscopy as a honeycomb lattice. However, D13 trimers are not able to directly bind to or intercalate into nascent viral membranes as they do not contain structures that bind to the membrane surface or cross the lipid bilayer. Previous studies show that the N-terminal domain of the integral membrane protein A17 appears to act as membrane docking signal for D13 by providing its N-terminal region. The goal of this study was to visualize the initial assembly steps of poxvirus morphogenesis. In particular, the present study sought to identify the inter-trimer contacts mediated by D13 protein and specific interaction between D13 and the N-terminal domain of the A17 protein required to induce assembly of poxviral particles. High resolution images of the D13 honeycomb lattice were obtained using a mutant D13 protein, cryo-electron microscopy as a lipid monolayer crystallization technique. Complexes of D13 protein and the A17 N-terminal domain were visualized using negative staining and single particle analysis. In conclusion, inter-trimeric interaction of D13 honeycomb lattice is primarily contributed by the loops of J1 (Jelly-roll) and head domain of trimers. In addition, D13 binding partner A17 binds a t the base domain of D13 protein to provide its N-terminus for the D13 assembly. Key Words: Poxvirus, initial assembly, crescents, D13 protein, pseudo-atomic structure honeycomb lattice, cryo-EM