Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication

Proceedings of the National Academy of Sciences of the United States of America
Nuruddin UnchwaniwalaP Ahlquist

Abstract

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 o...Continue Reading

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Citations

Sep 12, 2020·Science·Nuruddin Unchwaniwala, Paul Ahlquist
Feb 3, 2021·Viruses·Georg Wolff, Montserrat Bárcena
Feb 22, 2021·Journal of Structural Biology·Jie E YangElizabeth R Wright
Apr 3, 2021·Cell Chemical Biology·Yang ZhangZhigang Yi
Apr 8, 2021·Acta Crystallographica. Section D, Structural Biology·Francisco J Barrantes
Jan 1, 2021·Current Opinion in Virology·Masaki Nishikiori, Paul Ahlquist
May 22, 2021·Molecular Cell·Manish Kumar, Nihal Altan-Bonnet
Jun 3, 2021·Viruses·Kaïn van den ElsenDahai Luo
Jul 14, 2021·Annual Review of Virology·Vasiliya KrilAli Amara

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Methods Mentioned

BETA
electron tomography
electron microscopy
X-ray
fluorescence microscopy
PCR
transfection

Software Mentioned

Segment Map Fit to Segment Chimera
Elements
IMOD
iTasser
Fiji ( ImageJ )
ImageJ
NIS
Pred
Chimera
PEET

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