Identification of RUVBL1 and RUVBL2 as Novel Cellular Interactors of the Ebola Virus Nucleoprotein

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.

Keywords: AAA+ proteins; Ebola; NP; R2TP; RUVBL1; RUVBL2.

Figure 1

Identification of candidate Ebola virus (EBOV) nucleoprotein (NP)-interacting host proteins. (A) Silver stain of HA-NP immunoprecipitations. (B) STRING analysis of NP interactor candidates. (C) Candidate proteins identified by mass spectrometry analysis.

Figure 2

Validation of protein-protein interactions between EBOV NP and RUVBL1 and RUVBL2. (A) RNase treatment was confirmed using agarose gel electrophoresis on samples that were left untreated or treated with RNase (designated by − RNase or +RNase). (B) Confirmation of HA-NP and FLAG-RUVBL1 interaction in the absence or presence of RNase. Whole cell lysates (WCL) samples were run in parallel with immunoprecipitations (IP) samples to confirm appropriate protein expression. (C) Reciprocal validation of protein-protein interaction between HA-NP and FLAG-RUVBL1 by FLAG IP and −/+ RNase treatments. (D) Confirmation of NP-V5 and HA-RUVBL2 interaction by V5 IP and −/+ RNase treatments. (E) Reciprocal validation of protein-protein interaction between NP-V5 and HA-RUVBL2 by HA IP and −/+ RNase treatments. Monoclonal antibodies against HA, FLAG, and V5 were used to detect target proteins.

Figure 3

Endogenous RUVBL1 and RUVBL2 colocalize with HA-NP. HeLa cells were transfected with vector control, HA-NP, or HA-VP35. Twenty-four h later, the cells were fixed and processed for immunofluorescence detection of endogenous RUVBL1 or RUVBL2 in the presence of vector control, HA-NP, or HA-VP35. Representative images of (A) endogenous RUVBL1 localization pattern with control vector (top panels), HA-NP (middle panels), or HA-VP35 (bottom panels) and (B) endogenous RUVBL2 localization pattern with control vector (top panels), HA-NP (middle panels), or HA-VP35 (bottom panels) are shown. HA-NP or HA-VP35 (green), RUVBL1/2 (red), and Hoechst 33342 nuclear stain (blue) were visualized by confocal microscopy. Scale bars = 20 µM.

Figure 4

RUVBL1/2 do not effect EBOV minigenome activity. (A) Schematic diagram of the EBOV minigenome system. The EBOV minigenome system consists of six plasmids: Four support plasmids encode replication complex components NP, L, VP35, and VP30. The EBOV minigenome plasmid encodes a firefly luciferase reporter gene flanked by the leader and trailer sequences of EBOV, and the plasmid that encodes Renilla luciferase is used for normalization. (B) Minigenome activity upon the knockdown of either RUVBL1, RUVBL2, or in combination. Below are protein levels confirmed by immunoblot. HeLa cells were transfected with 80 nM scrambled siRNA, 30 nM siRNA targeting RUVBL1, or 50 nM siRNA targeting RUVBL2. Twenty-four h after siRNA addition, the minigenome components were transfected. Forty-eight h later, minigenome reporter activity was measured. (C) Overexpression of FLAG-RUVBL1 and HA-RUVBL2 in the EBOV minigenome. HeLa cells were left untransfected, or transfected with vector control (VC), or increasing amounts of FLAG-RUVBL1 (125, 250, and 500 ng) or HA-RUVBL2 (125, 250, and 500 ng). Twenty-four h after exogenous transfection, the minigenome components were transfected. Forty-eight h later, minigenome reporter activity was measured. Data represent mean ± SEM from one representative experiment (n = 3) of at least three experiments (* p < 0.05).

Figure 5

The R2TP complex components RPAP3 and PIH1D1 colocalize with HA-NP. HeLa cells were transfected with vector control or HA-NP. Twenty-four hours later, the cells were fixed and processed for the immunofluorescence detection of endogenous PIH1D1 or RPAP3 in the presence of vector control or HA-NP. Representative images of endogenous localization pattern of (A) RPAP3 with vector control and (B) PIH1D1 with vector control. RPAP3 or vector control (green), PIH1D1 or vector control (red), and Hoechst 33342 nuclear stain (blue) were visualized by confocal microscopy. Scale bars = 20 µM. (C) Representative images of endogenous localization pattern of PIH1D1 and RPAP3 in the presence of HA-NP. HA-NP (green), RPAP3 (red), PIH1D1 (magenta), and Hoechst 33342 nuclear stain (blue) were visualized by SIM. Scale bars = 20 µM. (D) Minigenome activity upon the knockdown of RPAP. Below are protein levels confirmed by immunoblot. HeLa cells were transfected with 30 nM scrambled siRNA or 30 nM siRNA targeting RPAP3. Twenty-four hours after siRNA addition, the minigenome components were transfected. Forty-eight hours later, minigenome reporter activity was measured. Data represent mean ± SEM from one representative experiment (n = 3) of at least three experiments.