Assembly intermediates of orthoreovirus captured in the cell

Geoff Sutton,Geoff Sutton,Mark Boyce,Mark Boyce,Abhay Kotecha,Abhay Kotecha,Abhay Kotecha,Xiaofeng Fu,Xiaofeng Fu,Corey W Hecksel,Corey W Hecksel,Corey W Hecksel,Peijun Zhang,Peijun Zhang,Peijun Zhang,Peijun Zhang,Dapeng Sun,David I Stuart,David I Stuart,Daniel K Clare

doi:10.1038/s41467-020-18243-9

Abstract

Traditionally, molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Here, we directly visualise an unsuspected ‘single shelled’ intermediate for a mammalian orthoreovirus in cryo-preserved infected cells, by cryo-electron tomography of cellular lamellae. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2. This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. This supports the supposition that these viruses share a common ancestor, and suggests mechanisms for the assembly of viruses of the Reoviridae. Such methodology holds promise for dissecting the replication cycle of many viruses.

Highlights

Molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches
The first atomic structures for particles from the Reoviridae family were determined by X-ray crystallography[4,5,6,7], and advances in single particle cryo-electron microscopy have recently yielded structures at comparable or higher resolution for several members of the family[8,9,10,11]
Structural information is available for two types of purified orthoreovirus particles: the intact virus and partially disassembled transcriptionally competent cores[7,12]

Summary

Introduction

Molecular assembly pathways for viruses are inferred from high resolution structures of purified stable intermediates, low resolution images of cell sections and genetic approaches. Particle classification and averaging yields structures to 5.6 Å resolution, sufficient to identify secondary structural elements and produce an atomic model of the intermediate, comprising 120 copies each of protein λ1 and σ2 This λ1 shell is ‘collapsed’ compared to the mature virions, with molecules pushed inwards at the icosahedral fivefolds by ~100 Å, reminiscent of the first assembly intermediate of certain prokaryotic dsRNA viruses. Unlike most RNA viruses these particles do not completely uncoat during cell entry, instead μ1, σ3 and σ1 are stripped off, maintaining in the cytoplasm a protein shell (known as the core) secluding the dsRNA genome from cellular pathogen recognition receptors These particles are transcription-competent and, in the ribonucleoside triphosphate rich environment of the cytoplasm, they synthesise and extrude capped mRNAs, derived from the ten genome segments. We demonstrate that vitrification of infected cells followed by cryo-focussed ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) allows high resolution reconstruction of assembly intermediates of a mammalian reovirus, allowing atomic models to be constructed which throw light on some of these fundamental questions

Methods

Results

Conclusion