Exogenously expressed BAG3 was detected in eVP40-WT precipitates (Fig 3A, lane 3), but was not detected in preimmune IgG, nor eVP40-PT/PY precipitates (Fig 3A, lanes 1 and 4, respectively). as a specific VP40 PPxY interactor. Here, we demonstrate that this 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 usually 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 recognized that negatively regulates budding of VP40 VLPs and infectious computer 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. Author Summary The unprecedented magnitude and scope of the catastrophic 2014C2015 EBOV outbreak in West Africa, and its continued global emergence underscores the Resveratrol urgent need to better understand the biology and pathogenesis of this zoonotic pathogen. We have identified BAG3 as a novel and functional host VP40 interactor that Resveratrol negatively regulates VP40 VLP and computer virus egress in a PPxY/WW-domain dependent manner. As a cell survival protein and key regulator of chaperone-mediated autophagy (CMA), BAG3 sequesters EBOV and MARV VP40 away from the site of budding PIK3C2B at the plasma membrane, and thus may represent a novel host defense strategy to combat filovirus VP40-mediated egress and spread. Introduction 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 [1]. 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. The filovirus matrix protein VP40 is the most abundant protein in the virion and is essential for virus assembly and egress. Indeed, expression of VP40 alone is sufficient to form virus-like particles (VLPs), which are morphologically indistinguishable from infectious virions and are released from mammalian cells in a manner that recapitulates the release of authentic computer virus [2C6]. Although not required for EBOV replication [7], Late (L) domains (which contain PTAP and/or PPxY amino acid sequence motifs) are conserved within EBOV and MARV VP40 and promote efficient egress of VLPs and computer virus by recruiting host proteins that facilitate virus-cell separation [3,4,6,8C11]. For example, EBOV and MARV VP40 L-domains hijack specific host proteins associated with the ESCRT pathway, including Tsg101, Alix, and Nedd4 [3,6,8C13]. Viral proteins bearing PPxY motif each interact with a unique repertoire of WW-domain bearing host proteins with diverse functions [14C22]. For example, the PPxY L-domain within eVP40, mVP40, and other viral matrix proteins interacts specifically with WW-domains of: 1) host Nedd4; a HECT family E3 ubiquitin ligase that is linked with the cellular ESCRT machinery, 2) host ITCH; a HECT family E3 ubiquitin ligase involved in immune regulation and inflammatory signaling, and 3) host IQGAP1; a multifunctional scaffolding protein involved in regulating cell motility, actin polymerization, and filopodia formation [2,23C38]. In general, these previously characterized viral PPxY/WW-domain interactions promote efficient computer virus production. Here, we sought to identify additional WW-domain bearing proteins that interact with the eVP40 PPxY motif by screening a GST array of 115 host proteins containing one or more WW-domains [39] with an EBOV PPxY-containing peptide. By using this Resveratrol unbiased approach, we recognized WW-domain containing protein BAG3 as a novel eVP40 interactor. BAG3 is usually a stress-induced molecular co-chaperone involved in regulating cellular protein homeostasis by CMA. Since in general, viral PPxY-containing proteins tend to bind WW-domains with good specificity and selectivity [40], our identification of BAG3 suggests that.