Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography

DOI: 10.1016/j.chom.2023.03.004

Authors: Pranav N. M. Shah (The Wellcome Centre for Human Genetics, University of Oxford) , James B. Gilchrist (Diamond Light Source) , Björn O. Forsberg (The Wellcome Centre for Human Genetics, University of Oxford; Karolinska Institute) , Alister Burt (Francis Crick Avenue) , Andrew Howe (Diamond Light Source) , Shyamal Mosalaganti (University of Michigan) , William Wan (Vanderbilt University Center for Structural Biology) , Julika Radecke (Diamond Light Source) , Yuriy Chaban (Diamond Light Source) , Geoff Sutton (The Wellcome Centre for Human Genetics, University of Oxford) , David I. Stuart (The Wellcome Centre for Human Genetics, University of Oxford; Diamond Light Source) , Mark Boyce (The Wellcome Centre for Human Genetics, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Cell Host & Microbe , VOL 190

State: Published (Approved)
Published: March 2023
Diamond Proposal Number(s): 21004

Abstract: Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.

Journal Keywords: virus assembly; dsRNA viruses; rotavirus; Reoviridae; in situ; cryoelectron tomography; subtomogram averaging; virus structure; electron microscopy; cellular structural biology

Diamond Keywords: Viruses

Subject Areas: Biology and Bio-materials

Diamond Offline Facilities: Electron Bio-Imaging Centre (eBIC)
Instruments: Krios III-Titan Krios III at Diamond

Added On: 03/04/2023 08:49

Documents:
1-s2.0-S1931312823001087-main.pdf

Discipline Tags:

Health & Wellbeing Structural biology Life Sciences & Biotech

Technical Tags:

Imaging Tomography Cryo Electron Tomography (Cryo ET)