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. 2017 Sep 27;91(20):e00812-17.
doi: 10.1128/JVI.00812-17. Print 2017 Oct 15.

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

Ziying Han  1 , Cari A Sagum  2 , Fumio Takizawa  1 , Gordon Ruthel  1 , Corbett T Berry  1 , Jing Kong  1 , J Oriol Sunyer  1 , Bruce D Freedman  1 , Mark T Bedford  2 , Sachdev S Sidhu  3 , Marius Sudol  4 , Ronald N Harty  5
Affiliations

Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

Ziying Han et al. J Virol. .

Abstract

Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress.IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.

Keywords: E3 ubiquitin ligase; Ebola virus; L-domain; PPXY; VLPs; VP40; WW domain; WWP1; budding.

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Figures

FIG 1
FIG 1
Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).
FIG 2
FIG 2
IP/Western analysis of WW-domain-dependent interactions between WWP1 and eVP40. (A) HEK293T cells were mock transfected or transfected with the indicated plasmids; extracts were first immunoprecipitated (IP) with either nonspecific (IgG) or polyclonal anti-eVP40 antiserum, and the indicated Flag-tagged WWP1 protein was detected in precipitated samples by Western blotting (WB) using anti-Flag antiserum (lanes 2 to 9). (B) Western blots of expression controls for eVP40, the indicated WWP1 protein, and actin. (C) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with HA-tagged WWP1-WT (lanes 2 and 4). Cell extracts were first immunoprecipitated with either anti-eVP40 antiserum (lanes 1 and 2) or anti-HA (WWP1) antiserum (lanes 3 and 4), and HA-tagged WWP1 was detected in precipitated samples by Western blotting. (D) Western blots of expression controls for WWP1 and actin in cell extracts used in the experiment shown in panel C.
FIG 3
FIG 3
Confocal microscopy to visualize colocalization of eVP40 and WWP1. HEK293T cells were cotransfected with eVP40 plus WWP1-WT (top) or eVP40 plus mutant WWP1-ΔWW1–4 (bottom). Deconvolved confocal images are shown for eVP40 alone (green), WWP1-WT or WWP1-ΔWW1–4 alone (red), and merged panels, with the boxed insets enlarged. The white arrowheads highlight colocalization (yellow) of eVP40 and WWP1, the long white arrow highlights budding VLPs, and the short white arrows highlight long, thin projections typical of inhibited VLP budding.
FIG 4
FIG 4
siRNA knockdown of endogenous WWP1 regulates eVP40 VLP egress. (A) Western blot of cell extracts and VLPs from HEK293T cells mock transfected (lane 1) or transfected with the indicated siRNAs and eVP40 (lanes 2 and 3). (B) Relative budding efficiency or mean relative VLP intensity (normalized to that of random siRNA) of eVP40 VLPs from cells treated with a WWP1-specific siRNA relative to that from a random siRNA control. Error bars represent the standard deviation of the mean from three independent experiments (n = 3). **, P = 0.002.
FIG 5
FIG 5
Enzymatically active WWP1 promotes enhanced eVP40 VLP egress in a WW-domain-dependent manner. (A) Western analysis of cell extracts and VLPs from HEK293T cells mock transfected (lane 1) or transfected with eVP40 plus the indicated plasmids (lanes 2 to 9). (B) Budding efficiency or mean relative VLP intensity (normalized to empty vector) and standard error are shown for n = 5 experiments. *, P < 0.05; **, P < 0.01.
FIG 6
FIG 6
WWP1 ubiquitinates eVP40 and reduces expression of higher-MW oligomers of eVP40. (A) Western analysis of cell extracts and VLPs from HEK293T cells transfected with eVP40 plus an empty vector (lane 1), WWP1-WT, or mutant WWP1-C890A. Cell extracts were harvested and analyzed under nondenaturing (nonD) conditions where indicated. Higher-molecular-mass forms of eVP40 were detected under nondenaturing conditions and migrated at approximately 80 and 240 kDa, compared to monomeric eVP40 detected at approximately 40 kDa. (B) Western analysis of cell extracts and VLPs from HEK293T cells transfected with eVP40 plus ubiquitin (Ub) alone (lane 1), WWP1-WT plus Ub (lane 2), or mutant WWP1-C890A plus Ub (lane 3). Cell extracts were harvested and analyzed under nondenaturing (nonD) conditions where indicated. The asterisks (*) represent potential ubiquitinated forms of eVP40 (lane 2).
FIG 7
FIG 7
Gel filtration analysis of eVP40 in the presence of WWP1-WT or mutant WWP1-C890A. HEK293T cells were transfected with eVP40 plus either Ub, Ub plus WWP1-WT, or Ub plus WWP1-C890A as indicated. Lysates were separated by size on a Superdex-200 10/30 high-resolution fast-performance liquid chromatography column. (A) The chromatogram for eVP40 elution is shown as absorbance (Abs) versus elution volume. Additionally, molecular weight standards were plotted along the dashed line as log values versus elution volume. (B) Western blot analysis for eVP40 in the indicated elution volumes. Input controls for eVP40, WWP1-WT, and WWP1-C890A are shown.
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