Elucidation of the Ebola Virus VP24 Cellular Interactome and Disruption of Virus Biology through Targeted Inhibition of Host-Cell Protein Function

Abstract

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

Elucidation of the Cellular Interactome of Ebola Virus Nucleoprotein and Identification of Therapeutic Targets

“…2 Co-immunoprecipitated samples were then analysed using label free mass spectrometry. To investigate whether the interaction of NP with selected cellular proteins was mediated by RNA binding an RNAse treatment (15 units of RNAse (QIAGEN; 19101) was performed after the coimmunoprecipitations the with GFP-Trap.…”

“…. 2 For western blot analysis the elution step was done in 100 l of 2xSDS-sample buffer (120mM Tris/Cl pH6.8; 20% glycerol; 4%SDS, 0.04% bromophenol blue; 10% -mercaptoethanol)…”

Temporal and spatial analysis of the 2014–2015 Ebola virus outbreak in West Africa

“…While there is amino acid variation in all of the genomes sampled, there were very few changes in viral protein 30 (VP30), viral protein 40 (VP40) and viral protein 24 (VP24), and these changes are only in less than ,2% of the genomes sampled. However, a single amino acid substitution in VP24 is associated with adaptation to a new host 4,8 , and this may be due to interactions with host-cell proteins 9,10 . While some of the variation may be attributed to a purely random molecular clock pattern, in GP, VP35, NP and L there are some amino acid variations that are present in over ,15% of the genomes sampled.…”

Determination of the interactome of non-structural protein12 from highly pathogenic porcine reproductive and respiratory syndrome virus with host cellular proteins using high throughput proteomics and identification of HSP70 as a cellular factor for virus replication

“…The interactome of PRRSV NSP12 was determined by high-affinity GFP pull-down coupled with label free mass spectrometry-based approach, which we have used previously to study virus/host protein interactions [16][17][18][19]. Our data identified112 cellular proteins probably interacted with NSP12, and these proteins were mainly nucleic acid binding proteins or chaperones and were grouped into several distinct functional clusters.…”

“…Third, 293T cells have already been successfully used in many studies to identify the cellular partners for different viral proteins [16][17][18][19]. Previous studies have reported that Marc-145 cells and PAMs behave quite differently in PRRSV infection [47].…”