Viral capsid proteins are important regulators of viral processes from translation, to RNA replication, to RNA packaging, and the modulation of cellular innate immune responses. In viruses with segmented genomes that are packaged into separate virions, the capsid protein must coordinate the activities for each of the RNAs. The overall goal of this project is to better understand the early steps in an RNA virus infection. That is, how does the capsid interact with the viral RNAs within the virion and facilitate entry into cells? Brome mosaic virus (BMV) will be the model virus for this study. BMV is a nonenveloped segmented positive-strand RNA virus that has served as a model system with notable contributions to the cis- and trans-acting factors, the formation of the replicase complex, and the mechanism of viral RNA-dependent RNA synthesis. Preliminary results showed that an arginine-rich motif of the BMV capsid contributes to the selective encapsidation of BMV RNAs and acts as a cell-penetration signal. The latter set of observations led to the hypothesis that plant viruses gain entry into cells through a protein transduction mechanism. This project contains two aims, the first will seek to understand the architecture of the virion through a combination of cryoelectron microscopy and single particle reconstruction, biochemical mapping of RNA conformation and RNA-capsid interaction, and a molecular genetic analysis of how the interactions will influence the infection process.
The second aim seeks to elucidate the mechanism of cell entry of the cell-penetration peptides and the viral capsid using fluorescently-labeled peptides and viruses, confocal microscopy and electron microscopy. The work will lead to improved understanding of the viral infection process.

Public Health Relevance

Viral capsid proteins have many essential roles during a virus infection, from the regulation of translation to the protection of the viral genome against cellular innate immune detection. This proposal contains a comprehensive set of experiments to examine how the capsid protein of a plant-infecting RNA virus interacts with the viral RNA, reconfigures the shape of the RNA during encapsidation, and mediates entry into cells. The information gained from the proposed studies will contribute new insights to virus architecture, protein-RNA interactions, and how viruses gain entry into the cell.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI090280-01
Application #
7976300
Study Section
Virology - A Study Section (VIRA)
Program Officer
Park, Eun-Chung
Project Start
2010-05-01
Project End
2014-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
1
Fiscal Year
2010
Total Cost
$341,754
Indirect Cost
Name
Indiana University Bloomington
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
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Vaughan, Robert C; Kao, C Cheng (2015) Mapping protein-RNA interactions by RCAP, RNA-cross-linking and peptide fingerprinting. Methods Mol Biol 1297:225-36
Kao, C Cheng; Chuang, Ella; Ford, James et al. (2014) Mapping RNA Sequences that Contact Viral Capsid Proteins in Virions. Bio Protoc 7:
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Ni, Peng; Vaughan, Robert C; Tragesser, Brady et al. (2014) The plant host can affect the encapsidation of brome mosaic virus (BMV) RNA: BMV virions are surprisingly heterogeneous. J Mol Biol 426:1061-76

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