Combining epitope tag-based immunoprecipitation with a mass spectrometry-based proteomics approach, Takahashi et al. (2013) investigated interactome co-factors important in replication of the Ebola virus (EBOV). They focused on the role played by key viral protein L (EBOL), an RNA-dependent RNA polymerase necessary for EBOV replication, and on its interaction with host cellular proteins following infection.1
The researchers used cell cultures transfected with FLAG-tagged EBOL constructs as their primary experimental source. First, they determined the activity of the FLAG-tagged EBOL constructs using a mini-genome assay and discovered that only N-terminal tagging (FLAG-EBOL) retained polymerase activity similar to wild type. In all following experiments, only this construct was used.
Takahashi and colleagues established viable cultures of human embryonic kidney (HEK293), baby hamster kidney (BHK) and Madin-Darby canine kidney (MDCK) cell lines, all capable of sustaining viable EBOV infection in vitro. They successfully transfected all cell lines, using cell lysates and culture media for subsequent experiments.
Immunoprecipitation of the FLAG-tagged EBOL constructs pulled down the associated host cell proteins. The researchers separated the protein clusters with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), visualizing the bands with silver staining. Following in-gel trypsin digestion, Takahashi et al. analyzed the peptides via liquid chromatography–tandem mass spectrometry (LC-MS/MS).
LC-MS/MS analysis identified 65 candidate proteins that co-precipitated as the EBOL interactome. Of these, the researchers chose nuclear protein DNA topoisomerase 1 (TOP1) for further investigation because previous studies had implicated this enzyme in RNA viral replication. TOP1 first unwinds the nucleic acid helical structure to allow transcription and replication by cleaving phosphodiester bridges, and it then repairs the cut ends. To begin, Takahashi and co-authors used Western blotting to confirm that TOP1 co-precipitated with EBOL. They also revealed cellular location with immunofluorescence—demonstrating that, following treatment with Venus-EBOL, TOP1 co-localized in the cytoplasm as well as in the nucleus.
To study the effect of TOP1 on viral replication, the team infected cells with an EBOV mutant in the presence or absence of small interfering TOP1 RNA (siTOP1). They found that treatment with siTOP1 downregulated TOP1 and reduced viral replication. Treatment did not, however, affect in vitro infection using two other viruses, vesicular stomatitis virus (VSV) or influenza virus, suggesting the response was EBOV-specific. Furthermore, using a mini-genome assay to investigate RNA-dependent polymerase activity, the scientists found that knockdown of TOP1 reduced EBOV enzyme activity but not that of the influenza virus.
In order to understand and further confirm the interaction between EBOL and the host cell protein, Takahashi et al. next examined whether TOP1’s DNA and RNA phosphodiester bridge-cleaving action was involved in promoting viral replication. Using mutant TOP1 constructs, the researchers found that following siTOP1 downregulation of cellular EBOL polymerase, transfection with a TOP1 mutant containing the phosphodiester bridge-cleaving activity restored activity of the viral enzyme. For further confirmation of the EBOL co-factor in EBOV replication, the researchers saw that treating infected HEK293 cells with irinotecan (CPT-11) and topotecan, inhibitors of TOP1, also reduced specific polymerase activity.
The authors propose that information arising from the characterization of the EBOV interactome in host cells will aid in drug discovery for the treatment of this deadly disease.
1. Takahashi, K., et al. (2013) “DNA Topoisomerase 1 Facilitates the Transcription and Replication of the Ebola Virus Genome,” Journal of Virology, 87 (pp. 8862–9), doi: 10.1128/JVI.03544-12.
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