Angiomotin Counteracts the Negative Regulatory Effect of Host WWOX on Viral PPxY-Mediated Egress

Abstract

Filoviridae family members Ebola (EBOV) and Marburg (MARV) viruses and Arenaviridae family member Lassa virus (LASV) are emerging pathogens that can cause hemorrhagic fever and high rates of mortality in humans. A better understanding of the interplay between these viruses and the host will inform about the biology of these pathogens, and may lead to the identification of new targets for therapeutic development. Notably, expression of the filovirus VP40 and LASV Z matrix proteins alone drives assembly and egress of virus-like particles (VLPs). The conserved PPxY Late (L) domain motifs in the filovirus VP40 and LASV Z proteins play a key role in the budding process by mediating interactions with select host WW-domain containing proteins that then regulate virus egress and spread. To identify the full complement of host WW-domain interactors, we utilized WT and PPxY mutant peptides from EBOV and MARV VP40 and LASV Z proteins to screen an array of GST-WW-domain fusion proteins. We identified WW domain-containing oxidoreductase (WWOX) as a novel PPxY-dependent interactor, and we went on to show that full-length WWOX physically interacts with eVP40, mVP40 and LASV Z to negatively regulate egress of VLPs and of a live VSV/Ebola recombinant virus (M40). Interestingly, WWOX is a versatile host protein that regulates multiple signaling pathways and cellular processes via modular interactions between its WW-domains and PPxY motifs of select interacting partners, including host angiomotin (AMOT). Notably, we demonstrated recently that expression of endogenous AMOT not only positively regulates egress of VLPs, but also promotes egress and spread of live EBOV and MARV. Toward the mechanism of action, we show that the competitive and modular interplay among WWOX-AMOT-VP40/Z regulates VLP and M40 virus egress. Thus, WWOX is the newest member of an emerging group of host WW-domain interactors (e.g. BAG3; YAP/TAZ) that negatively regulate viral egress. These findings further highlight the complex interplay of virus-host PPxY/WW-domain interactions and their potential impact on the biology of both the virus and the host during infection. Author Summary Filoviruses (Ebola [EBOV] and Marburg [MARV]) and arenavirus (Lassa virus; LASV) are zoonotic, emerging pathogens that cause outbreaks of severe hemorrhagic fever in humans. A fundamental understanding of the virus-host interface is critical for understanding the biology of these viruses and for developing future strategies for therapeutic intervention. Here, we identified host WW-domain containing protein WWOX as a novel interactor with VP40 and Z, and showed that WWOX inhibited budding of VP40/Z virus-like particles (VLPs) and live virus in a PPxY/WW-domain dependent manner. Our findings are important to the field as they expand the repertoire of host interactors found to regulate PPxY-mediated budding of RNA viruses, and further highlight the competitive interplay and modular virus-host interactions that impact both the virus lifecycle and the host cell.

WWOX-Mediated Degradation of AMOTp130 Negatively Affects Egress of Filovirus VP40 VLPs

“…In addition, AMOT stability and turnover is tightly regulated via PPxY/WW-domain interactions with Nedd4 E3 ubiquitin ligase family members (34)(35)(36)39). While Amot was previously shown to regulate assembly and egress of non-PPxY-containing viruses (49,50), we recently revealed a role for endogenous AMOT in positively regulating egress of PPxY-containing EBOV and MARV (12,13,31).…”

“…Tsg101, Nedd4, Itch, WWP1, and Smurf2) (11,16,17,42,44), the VP40 PPxY L-domain also engages with host proteins that negatively regulate VP40-mediated egress (e.g. BAG3, YAP/TAZ, and WWOX) (12,13,15). Thus, the modular and competitive nature of the PPxY-WW domain interplay likely impacts both host and virus functions (18).…”

“…The copyright holder for this preprint this version posted November 20, 2021. ; https://doi.org/10.1101/2021.11. 19.469355 doi: bioRxiv preprint process (11)(12)(13)(14)(15)(16)(17). These host proteins contain one or more modular WW-domains that interacted with the conserved PPxY Late (L) domain motif at the N-termini of both EBOV VP40 (eVP40) and MARV VP40 (mVP40).…”

“…For example, the VP40 PPxY motif interacts with several members of the HECT family of E3 ubiquitin ligases, such as Nedd4, Itch, WWP1, and Smurf2 which facilitate mono-ubiquitination of VP40 and subsequent engagement and re-localization of the host ESCRT machinery to the site of virus budding at the plasma membrane to enhance virus-cell separation and release of VLPs or infectious virus (8,11,16,17). In contrast to these positive regulators of virus budding, we have identified more recently a growing list of novel WW-domain interactors that negatively regulate egress, such as host proteins BAG3, YAP/TAZ, and WWOX (12,13,15). Notably, these negative regulators of budding are multifunctional proteins that regulate diverse pathways/processes in the cell including, apoptosis/autophagy, transcription, cytoskeletal dynamics, cell migration/morphology, and tight junction formation (19)(20)(21)(22)(23)(24).…”

“…CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It VLPs and live EBOV and MARV by counteracting the negative effects of YAP and WWOX (12,13,31).…”