Plant-expressed virus-like particles reveal the intricate maturation process of a eukaryotic virus

Roger Castells-Graells,Charlotte A Scarff,Tatiana Domitrovic,George P Lomonossoff,Emma L Hesketh,Jonas R S Ribeiro,John E Johnson,David M Lawson,Neil A Ranson

doi:10.1038/s42003-021-02134-w

Abstract

Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.

Highlights

Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood
When the coat protein of NωV is expressed in insect cells, it assembles into stable intermediate virus-like particles (VLPs), 48 nm in diameter, that are porous at neutral pH9
Expression of the coat protein was initiated by infiltrating leaves of N. benthamiana with suspensions of A. tumefaciens harbouring pEAQ-HT-NωV-WT followed by the collection of leaf disks various days post-infiltration

Summary

Introduction

Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. When the coat protein of NωV is expressed in insect cells, it assembles into stable intermediate virus-like particles (VLPs) (known as the procapsid), 48 nm in diameter, that are porous at neutral pH (pH 7.6)[9]. These particles undergo a maturation process when they are exposed to acidic conditions (pH 5.0) in vitro. This involves large-scale subunit reorganisation and an autocatalytic cleavage of the α capsid protein between residues Asn[570] and Phe[571] to give two polypeptides, β and γ, of 62- and 8-kDa, respectively, that remain as part of the mature particle (Fig. S1). These conformational changes are reversible at elevated pH if no more than 10% of the subunits have been cleaved or in the mutant Asn570Thr, which is incapable of effecting the cleavage[12,13]

Methods

Results

Conclusion