Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 117, Issue 31, Pages 18680-18691Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2006165117
Keywords
positive-strand RNA virus; replication complexes; cryotomography; replication crown; nodavirus
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Funding
- NIH [U24GM129547]
- Office of Biological and Environmental Research
- University of Wisconsin-Madison
- Advanced Computing Initiative
- Wisconsin Alumni Research Foundation
- Wisconsin Institutes for Discovery
- NSF
- US Department of Energy's Office of Science
- John W. and Jeanne M. Rowe Center for Research in Virology
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For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter crown complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 angstrom. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the similar to 19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at similar to 35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.
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