1. Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication
- Author
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Janice Pennington, Nuruddin Unchwaniwala, Johan A. den Boon, Hong Zhan, Paul Ahlquist, and Mark Horswill
- Subjects
Models, Molecular ,Cryo-electron microscopy ,viruses ,Genome, Viral ,Virus Replication ,Microbiology ,replication crown ,Viral Proteins ,Nodaviridae ,Replication protein A ,Polymerase ,positive-strand RNA virus ,Multidisciplinary ,replication complexes ,biology ,Chemistry ,C-terminus ,Vesicle ,Cryoelectron Microscopy ,RNA ,Biological Sciences ,Cell biology ,Cytoplasm ,nodavirus ,Mitochondrial Membranes ,biology.protein ,RNA, Viral ,cryotomography ,Protein A - Abstract
Significance Positive-strand RNA [(+)RNA] viruses constitute the largest genetic class of viruses and include many high-impact pathogens, such as SARS–CoV-2 (COVID-19 pandemic coronavirus), MERS CoV, Zika, chikungunya, dengue, and hepatitis C viruses. (+)RNA virus genome replication invariably occurs on virus-induced, membrane-bound organelles called RNA replication complexes, an attractive potential target for broadly active antivirals. To better understand, control and beneficially employ (+)RNA viruses, urgent needs exist to define RNA replication complex structure and function at a molecular level. This study uses cryoelectron microscopy and complementary approaches to provide previously inaccessible native, near atomic-resolution views of the well-characterized nodavirus RNA replication complex, advancing fundamental understanding of (+)RNA virus genome replication complex structure, organization, stability, and function., 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 Å. 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 ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼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.
- Published
- 2020
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