Abstract
The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
Highlights
Ebola virus (EBOV) is a zoonotic and emerging pathogen that continues to cause severe outbreaks of hemorrhagic fever in animals and humans
Images of EBOV VP40 (eVP40) virus-like particle (VLP) formation and egress obtained using total internal reflection fluorescence (TIRF) microscopy of transfected shCtrl or shAmot cells show that in the absence of Amot, the actin cytoskeleton appears less organized and is misaligned with eVP40 VLPs budding at the plasma membrane, whereas actin filaments observed in shCtrl cells appear to align with, and form a foundation for, budding eVP40 VLPs
To determine whether endogenous expression of Amot affected budding of eVP40 VLPs, shCtrl or shAmot cells were transfected with eVP40, and both cell extracts and
Summary
Results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot’s role in actin organization and dynamics contributes to promoting eVP40-mediated egress These findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. Images of eVP40 VLP formation and egress obtained using TIRF microscopy of transfected shCtrl or shAmot cells show that in the absence of Amot, the actin cytoskeleton appears less organized and is misaligned with eVP40 VLPs budding at the plasma membrane, whereas actin filaments observed in shCtrl cells appear to align with, and form a foundation for, budding eVP40 VLPs. In sum, our findings identify EBOV as a PPxY-containing virus whose egress and spread in vitro is regulated by host Amot-p130 via a mechanism involving competitive PPxY-mediated interactions with YAP1 and actin cytoskeleton dynamics at the plasma membrane
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