Date Published: January 11, 2017
Publisher: Public Library of Science
Author(s): Jingjing Liang, Cari A. Sagum, Mark T. Bedford, Sachdev S. Sidhu, Marius Sudol, Ziying Han, Ronald N. Harty, Christopher F. Basler.
Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.
Ebola (EBOV) and Marburg (MARV) viruses are virulent pathogens that cause severe hemorrhagic disease in humans and non-human primates. There are currently no FDA approved vaccines or antiviral drugs to prevent or treat infections by these Category A NIAID priority pathogens . The recent catastrophic outbreak of EBOV in West Africa underscores the urgent need to better understand the biology and pathogenesis of this global public health threat, and to decipher the molecular mechanisms by which EBOV interacts with the host to cause disease.
As the major filovirus matrix protein, VP40 plays a central role in directing virion assembly and egress from infected cells. Three minimal functional domains of VP40 are required for efficient VLP egress, including a membrane (M) binding region, a self-interaction (I) domain, and one or more late (L) domain motifs. The L-domain motifs hijack or recruit specific host cell proteins that facilitate or promote efficient virus-cell separation [3,4,6,48–53]. Here, we have identified host WW-domain containing protein BAG3 as a novel interactor with the PPxY L-domain motif of both eVP40 and mVP40. Moreover, we confirmed the physical and functional interaction between the WW-domain of BAG3 and the viral PPxY L-domain motif by using L-domain and WW-domain mutants in GST-pulldowns, co-immunoprecipitation, siRNA analysis, and/or VLP/virus budding assays. Intriguingly, unlike previously identified host WW-domain proteins that interact with VP40, including Nedd4 [2,28,32,35] and ITCH , BAG3 is the first host WW-domain interactor to negatively regulate egress of eVP40 and mVP40 VLPs, as well as infectious virus containing the eVP40 PPxY L-domain motif.