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

Research output: Contribution to journalArticle

26 Scopus citations

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 unchar-acterized 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, bun-yaviruses, and coronaviruses.

Original languageEnglish (US)
Pages (from-to)8697-8712
Number of pages16
JournalJournal of virology
Volume87
Issue number15
DOIs
StatePublished - 2013

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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