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Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

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.

SUBMITTER: Lenarcic EM

PROVIDER: S-EPMC3719794 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

Lenarcic Erik M EM Landry Dori M DM Greco Todd M TM Cristea Ileana M IM Thompson Sunnie R SR

Journal of virology 20130605 15

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, an ...[more]

PMID: 23740976

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Project description:Public health threats posed by emerging respiratory infections are a significant concern, particularly in children and infants. Traditional culture-based detection methods are time-consuming and typically require 1-3 days. Herein, we developed and evaluated a 23-plex common respiratory pathogen mass spectrometry assay that enables the simultaneous detection of 18 common respiratory pathogens in children. This assay combines matrix-assisted laser desorption/ionization time of flight mass spectrometry with multiplex reverse transcription-PCR and targets 11 bacterial and 7 viral pathogens (including 10 subtypes), and two internal controls. The detection limit of the common respiratory pathogen mass spectrometry assay was as low as 1 copy/µL, with no cross-reactivity with other organisms. We assessed the clinical performance of the common respiratory pathogen mass spectrometry assay using respiratory samples from 450 children. The total 450 clinical specimens underwent analysis via matrix-assisted laser desorption/ionization time of flight mass spectrometry, and the outcomes were juxtaposed with those derived from real-time reverse-transcriptase PCR conducted concurrently. The concordance between these methods was 96.0%, and the multiple infection identification rate was 7.1%. This innovative approach enables the simultaneous analysis of numerous outcomes from a solitary examination across 192 specimens within a timeframe of approximately 7 hours, with a dramatically reduced sample use and cost. In summary, the common respiratory pathogen mass spectrometry assay is a sensitive, accurate, and cost-effective method for detecting common respiratory pathogens in children and has the potential to revolutionize the diagnosis of respiratory tract infections. IMPORTANCE This study aimed to present and evaluate a novel co-detection method that enables the simultaneous identification of 11 bacterial and 7 viral pathogens in about 7 hours using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Our approach utilizes a combination of multiplex reverse transcription-PCR and matrix-assisted laser desorption/ionization time of flight mass spectrometry, which overcomes the limitations of conventional assays, which include a long assessment time, technical difficulty, and high costs. As a screening method for common respiratory pathogens in children, common respiratory pathogen mass spectrometry assay has the potential to revolutionize the diagnosis of respiratory tract infections by providing an accurate etiological diagnosis. The common respiratory pathogen mass spectrometry assay is expected to be a critical tool for the diagnosis of respiratory infections in children, offering a more efficient, cost-effective, and accurate approach for the detection of common respiratory pathogens.

Dataset Information

Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

Publications

Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification.

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