Skip to main content

Structure / Function Analysis of Positive-Strand RNA Virus Replication Complexes


EMSL Project ID
50505

Abstract

Positive-strand RNA ((+)RNA) viruses, the largest genetic class of viruses, include numerous major pathogens such as the Zika, MERS, SARS and hepatitis C viruses. A key target for understanding and controlling these viruses is their RNA genome replication, which occurs inside vesicular invaginations of intracellular membranes. Using an advanced Nodavirus model system, we published the first and to date only cryo-electron microscopy imaging of such replication complexes, providing revolutionary initial views of the viral RNA and protein components (Ertel et al. 2017 eLife).

Our cryo-electron tomography (cryo-ET), sub-tomogram averaging, and immunogold labeling show the necked aperture of each replication vesicle to be crowned by a striking, 12-fold symmetric ring of viral protein A, which provides polymerase and other functions for RNA replication. These crowns appear to be the major enzymatic engine of RNA synthesis and are origins for cytoplasmic fibrils implicated as newly synthesized RNA. In this project, we will use cryo-EM to markedly illuminate (+)RNA virus replication complex structure, assembly, function and control by:

1) Mapping the key polymerase, RNA capping and other domains of viral replication protein A within the crown structure by labeling internal epitope tags in our engineered protein A derivatives with Ni-NTA-Nanogold and similar approaches.

2) Advancing protein A domain mapping and modeling, membrane interaction modeling, and other structural analyses to near atomic levels by using improved cryo-ET image acquisition and sub-tomogram averaging to increase resolution of the critical crown structure.

3) Defining the effects of other viral components on crown formation and function.

Project Details

Start Date
2018-10-30
End Date
2021-01-31
Status
Closed

Team

Principal Investigator

Paul Ahlquist
Institution
University of Wisconsin, Madison

Team Members

Masaki Nishikiori
Institution
University of Wisconsin, Madison

Nancy Meyer
Institution
Oregon Health & Science University

Janice Pennington
Institution
University of Wisconsin, Madison

Nuruddin Unchwaniwala
Institution
University of Wisconsin, Madison

Hong Zhan
Institution
University of Wisconsin, Madison

Irina El Khoury
Institution
Environmental Molecular Sciences Laboratory

Related Publications

Unchwaniwala, N., Zhan, H., Pennington, J., Horswill, M., Boon, J. A., & Ahlquist, P. (2020). 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, 117(31), 18680-18691. doi:10.1073/pnas.2006165117