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Virus-Cell Interactions

Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification

Erik M. Lenarcic, Dori M. Landry, Todd M. Greco, Ileana M. Cristea, Sunnie R. Thompson
Erik M. Lenarcic
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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Dori M. Landry
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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Todd M. Greco
bDepartment of Molecular Biology, Princeton University, Princeton, New Jersey, USA
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Ileana M. Cristea
bDepartment of Molecular Biology, Princeton University, Princeton, New Jersey, USA
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Sunnie R. Thompson
aDepartment of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA
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DOI: 10.1128/JVI.00950-13
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ABSTRACT

Eukaryotic RNA viruses are known to utilize host factors; however, the identity of these factors and their role in the virus life cycle remain largely undefined. Here, we report a method to identify proteins bound to the viral RNA during amplification in cell culture: thiouracil cross-linking mass spectrometry (TUX-MS). TUX-MS relies on incorporation of a zero-distance cross-linker into the viral RNA during infection. Proteins bound to viral RNA are cross-linked prior to cell lysis, purified, and identified using mass spectrometry. Using the TUX-MS method, an unbiased screen for poliovirus (PV) host factors was conducted. All host and viral proteins that are known to interact with the poliovirus RNA were identified. In addition, TUX-MS identified an additional 66 host proteins that have not been previously described in poliovirus amplification. From these candidates, eight were selected and validated. Furthermore, we demonstrate that small interfering RNA (siRNA)-mediated knockdown of two of these uncharacterized host factors results in either a decrease in copy number of positive-stranded RNA or a decrease in PV translation. These data demonstrate that TUX-MS is a robust, unbiased method to identify previously unknown host cell factors that influence virus growth. This method is broadly applicable to a range of RNA viruses, such as flaviviruses, alphaviruses, picornaviruses, bunyaviruses, and coronaviruses.

FOOTNOTES

    • Received 7 April 2013.
    • Accepted 24 May 2013.
    • Accepted manuscript posted online 5 June 2013.
  • Address correspondence to Sunnie R. Thompson, Sunnie{at}uab.edu.
  • ↵* Present address: Erik M. Lenarcic, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.

  • D.M.L. and T.M.G. contributed equally to this article.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.00950-13.

  • Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification
Erik M. Lenarcic, Dori M. Landry, Todd M. Greco, Ileana M. Cristea, Sunnie R. Thompson
Journal of Virology Jul 2013, 87 (15) 8697-8712; DOI: 10.1128/JVI.00950-13

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Thiouracil Cross-Linking Mass Spectrometry: a Cell-Based Method To Identify Host Factors Involved in Viral Amplification
Erik M. Lenarcic, Dori M. Landry, Todd M. Greco, Ileana M. Cristea, Sunnie R. Thompson
Journal of Virology Jul 2013, 87 (15) 8697-8712; DOI: 10.1128/JVI.00950-13
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