Unravelling the Maturation Pathway of a Eukaryotic Virus through Cryo-EM

Abstract

ABSTRACTThe importance of virus maturation has been appreciated for nearly 70 years1as it provides models for large-scale protein reorganization resulting in functional activation as well as being a target for antiviral therapies2. However, a detailed description of the pathway from the initial assembly product (procapsid) to the mature, infectious particle (virion) has been elusive. This is due to the “in cell” nature of the natural process, the 2- state behavior of maturation (no detectable intermediates) in some virusesin vitro3and heterogeneous populations of particle intermediates that are only partially matured in other systems4. The non-enveloped, T=4, ssRNA-containing,Nudaurelia capensisomega virus (NωV), is a highly accessible model system that exemplifies the maturation process of a eukaryotic virus. During maturation the particle shrinks in outer diameter from 482 Å (pH 7.5) to 428 Å (pH 5.0). It is possible to mimic the maturation processin vitroby lowering the pH of a population of procapsids produced in heterologous systems5. Indeed, by controlling the pHin vitroit is possible to produce homogenous populations of intermediate NωV virus-like particles (VLPs) that occur too fleetingly to be observedin vivo6.Here we report structural models, based on cryo-electron microscopy (cryo-EM), of five intermediates in the NωV maturation process. The structures of the intermediate particles reveal unique, quaternary position-dependent trajectories and refolding of subunit N and C-terminal regions, including the formation of the autocatalytic cleavage site at N570. The detailed structures reported here, coupled with previously determined structures of the procapsids and mature particles, allows the maturation pathway to be described in detail for the first time for a eukaryotic virus.