Abstract
Ebola virus (EBOV) is a filovirus that has become a global public health threat in recent years. EBOV is the causative agent of a severe, often fatal hemorrhagic fever. A productive viral infection relies on the successful recruitment of host factors for various stages of the viral life cycle. To date, several investigations have discovered specific host-pathogen interactions for various EBOV proteins. However, relatively little is known about the EBOV nucleoprotein (NP) with regard to host interactions. In the present study, we aimed to elucidate NP-host protein-protein interactions (PPIs). Affinity purification-mass spectrometry (AP-MS) was used to identify candidate NP cellular interactors. Candidate interactors RUVBL1 and RUVBL2, partner proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) superfamily, were confirmed to interact with NP in co-immunoprecipitation (co-IP) and immunofluorescence (IF) experiments. Functional studies using a minigenome system revealed that the siRNA-mediated knockdown of RUVBL1 but not RUVBL2 moderately decreased EBOV minigenome activity. Super resolution structured illumination microscopy (SIM) was used to identify an association between NP and components of the R2TP complex, which includes RUVBL1, RUVBL2, RPAP3, and PIH1D1, suggesting a potential role for the R2TP complex in capsid formation. Moreover, the siRNA-mediated knockdown of RPAP3 and subsequent downregulation of PIH1D1 was shown to have no effect on minigenome activity, further suggesting a role in capsid formation. Overall, we identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics.
Highlights
Ebola virus (EBOV) is in the family Filoviridae within the order Mononegavirales [1]
We identify RUVBL1 and RUVBL2 as novel interactors of EBOV NP and for the first time report EBOV NP recruitment of the R2TP complex, which may provide novel targets for broad-acting anti-EBOV therapeutics
The affinity-tagged NP protein was over-expressed in HEK 293T cells, and interacting host proteins were identified following affinity purification (AP); empty vector, transfected control cells were processed by identical AP procedures
Summary
Ebola virus (EBOV) is in the family Filoviridae within the order Mononegavirales [1]. The virus is the causative agent of severe, often fatal, hemorrhagic fever in humans, with case fatality rates as high as 90% [2]. Since its emergence in 1976 [3], sporadic localized outbreaks have typically characterized EBOV, but the unprecedented spread of the 2013–2016 West African epidemic and its high death toll highlights the global public health threat that it poses [4]. The recent 2017 and 2018 outbreaks in Democratic Republic of the Congo further emphasize the urgency for EBOV therapeutic interventions [5,6,7]. Novel therapeutic targets will be useful to the continued development of effective EBOV countermeasures
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