When a virus infects a cell, it commandeers host proteins to influence cell behavior that promotes viral interests, i.e., viral replication and release to infect more host cells. The Ebola virus (EBOV) is no different. In addition to targeting viral biology itself, researchers are therefore now looking at host protein interactions as potential therapeutic targets for this deadly disease.
Garcia-Dorival and colleagues (2014) examined the viral–host protein interactome in order to investigate infectivity and potential therapeutics.1 Using tagged EBOV protein constructs, they transfected suitable cell lines in vitro, then mapped the interactions through high-affinity purification followed by a label-free quantitative proteomics approach.
Of the eight proteins produced by EBOV, virion protein 24 (VP24) has been identified as a major factor in virulence. It is a secondary matrix protein associated with the virion envelope and plays a crucial role in dodging the host immune response. It is therefore highly probable that VP24 interacts with a number of host cellular proteins, and characterizing this interactome could lead to novel therapeutic antivirals.
First, the researchers developed EGFP-labeled VP24 fusion constructs, tagging the protein at both 5’ and 3’ termini (EGFP-VP24 and VP24-EGFP). They transfected these constructs into human embryonic kidney cells (HEK293T) that are characterized as capable of supporting EBOV infection in vitro. Garcia-Dorival and co-authors collected transfected cells from five replicates, immunoprecipitating the lysates using GFP-Trap agarose before digesting the samples with trypsin. They then examined the peptide digests using an LTQ Orbitrap Velos hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific). The researchers used MaxQuant (incorporating the Andromeda search engine) for label-free quantification, then analyzed the data with the String 9.05 program to determine protein–protein interactions and functionality.
Combining the data from five replicates, the researchers identified, via immunoprecipitation, 50 proteins implicated as interacting with VP24. The team confirmed mass spectrometry results using Western immunoblotting following sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). They also confirmed the proposed interactome constituents by using reverse immunoprecipitation, looking for VP24 immunoreactivity in the captured products.
Among the 50 candidate proteins, the team identified previously established co-factors such as importin subunit karyopherin α1 and α6, VDAC-1 and lamin B1, in addition to discovering the novel interactome protein ATP1A1 (Na/K transporting ATPase subunit α-1), among others.
Garcia-Dorival et al. further characterized the functional role of ATP1A1 in EBOV infection. Using ouabain, a cardiac glycoside and small molecule inhibitor of ATP1A1, the researchers measured the effect of treatment on cells infected with EBOV. Data combined from triplicate experiments showed that although ouabain had no effect on EGFP-VP24 levels in human fibroblast lung (MRC-5) cultures transfected with the construct, it did reduce levels of EBOV virus RNA released into the growth medium. In other words, treatment with ouabain appeared to reduce viral replication.
The authors acknowledge that their experimental details preclude direct conclusion that the anti-viral activity of ouabain on ATP1A1 affects its association with VP24. They do, however, comment on the drug’s known anti-viral activity in herpes simplex, for example, suggesting that their findings may be applicable in the treatment of EBOV infection.
As a conclusion, Garcia-Dorival et al. offer that proteomic characterization of viral protein interactomes represents a valid strategy for discovering new therapeutic targets. They suggest that interfering with host–virus interactions by targeting the host proteins themselves could also avoid development of drug resistance.
Reference
1. Garcia-Dorival, I., et al. (2014, August) “Elucidation of the Ebola virus VP24 cellular interactome and disruption of virus biology through targeted inhibition of host cell protein function,” Journal of Proteome Research, doi: 10.1021/pr500556d [e-pub ahead of print].
Post Author: Amanda Maxwell. Mixed media artist; blogger and social media communicator; clinical scientist and writer.
A digital space explorer, engaging readers by translating complex theories and subjects creatively into everyday language.
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