Cryo-electron tomography reveals novel features of a viral RNA replication compartment.

Kenneth J Ertel,Daniel Castaño-Diez,Paul Ahlquist,Janice G Pennington,Marisa S Otegui,Desirée Benefield,Johan A Den Boon,Mark Horswill

doi:10.7554/elife.25940

Kenneth J Ertel, Daniel Castaño-Diez + Show 6 more

Open Access

https://doi.org/10.7554/elife.25940

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Abstract

Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule's necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control.

Highlights

Positive-strand RNA viruses, whose virions contain mRNA-sense single stranded RNA, represent the largest genetic class of viruses (King et al, 2012), including many human, animal, and plant pathogens
Our group showed that mitochondria isolated from Flock house nodavirus (FHV)-infected Drosophila cells retain RNA replication compartments that are active in viral RNA synthesis with a specific activity approaching that in infected cells (Kopek et al, 2007)
In chemically-fixed samples, FHV RNA replication complexes had oblong shapes significantly elongated perpendicular to the outer mitochondrial membrane (Kopek et al, 2007; Miller et al, 2001), in plunge-frozen samples they were much more spherical (Figure 2B)

Summary

Introduction

Positive-strand RNA viruses, whose virions contain mRNA-sense single stranded RNA, represent the largest genetic class of viruses (King et al, 2012), including many human, animal, and plant pathogens These viruses complete their entire replication cycle in the host cell cytoplasm. To generate sites for their genomic RNA replication, positive-strand RNA viruses expand and rearrange host cellular membranes (Fernandez de Castro et al, 2017; den Boon et al, 2010; Kovalev et al, 2016; Paul and Bartenschlager, 2013; Paul et al, 2014; Romero-Brey and Bartenschlager, 2014) Such membrane-associated RNA replication sites are thought to concentrate essential viral and host factors, and to organize successive steps for efficient viral RNA synthesis (Atasheva et al, 2010; Neuvonen et al, 2011).

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