Nanoscale organization of rotavirus replication machineries
- Universidad Nacional Autónoma de México, Mexico
- Universidad Autónoma del Estado de Morelos, Mexico
- Harvard Medical School, United States
Abstract
Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describes the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as 5 concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Juan Manuel Rendon-Mancha
Funding
DGAPA-PAPIIT-UNAM (IG200317)
- Susana López
- Carlos F Arias
DGAPA-PAPIIT-UNAM (IA202417)
- Adán Guerrero
DGTIC-UNAM (SC15-1-IR-89)
- Adán Guerrero
Conacyt-Mexico (252213)
- Adán Guerrero
DGAPA-PAPIIT-UNAM (IN202312)
- Haydee Olinca Hernández
DGTIC-UNAM (SC16-1-IR-102)
- Adán Guerrero
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Arup K Chakraborty, Massachusetts Institute of Technology, United States
Version history
- Received: October 17, 2018
- Accepted: July 22, 2019
- Accepted Manuscript published: July 25, 2019 (version 1)
- Version of Record published: August 13, 2019 (version 2)
- Version of Record updated: August 22, 2019 (version 3)
- Version of Record updated: September 17, 2019 (version 4)
Copyright
© 2019, Garcés Suárez et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Nanoscale organization of rotavirus replication machineries
eLife 8:e42906.
https://doi.org/10.7554/eLife.42906
Further reading
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The relative positions of viral DNA genomes to the host intranuclear environment play critical roles in determining virus fate. Recent advances in the application of chromosome conformation capture-based sequencing analysis (3 C technologies) have revealed valuable aspects of the spatiotemporal interplay of viral genomes with host chromosomes. However, to elucidate the causal relationship between the subnuclear localization of viral genomes and the pathogenic outcome of an infection, manipulative tools are needed. Rapid repositioning of viral DNAs to specific subnuclear compartments amid infection is a powerful approach to synchronize and interrogate this dynamically changing process in space and time. Herein, we report an inducible CRISPR-based two-component platform that relocates extrachromosomal DNA pieces (5 kb to 170 kb) to the nuclear periphery in minutes (CRISPR-nuPin). Based on this strategy, investigations of herpes simplex virus 1 (HSV-1), a prototypical member of the human herpesvirus family, revealed unprecedently reported insights into the early intranuclear life of the pathogen: (I) Viral genomes tethered to the nuclear periphery upon entry, compared with those freely infecting the nucleus, were wrapped around histones with increased suppressive modifications and subjected to stronger transcriptional silencing and prominent growth inhibition. (II) Relocating HSV-1 genomes at 1 hr post infection significantly promoted the transcription of viral genes, termed an ‘Escaping’ effect. (III) Early accumulation of ICP0 was a sufficient but not necessary condition for ‘Escaping’. (IV) Subnuclear localization was only critical during early infection. Importantly, the CRISPR-nuPin tactic, in principle, is applicable to many other DNA viruses.
