BAG3 Negatively Regulates EBOV And Marv VLP Egress And Its Mechanisms Exploration

Ebola virms(EBOV)and Marburg virus(MARV)are members of the Filoviridae family which cause outbreaks of severe viral hemorrhagic fever syndrome in human and nonhuman primates.Filovirus is one of the most important zoonotic pathogens with high mortality rates.There are no approved vaccines,nor antiviral drugs available to prevent or treat filovirus infections,currently.The recent emergence of EBOV both in West African region and in other countries(e.g.United States)arouses the widespread attention of this virulent pathogen.The continuing and growing public health threat of filovirus underscores the critical need for a better understanding of the biology and pathogenesis of this emerging pathogen.Viral lifecycle starts from host cell entry,followed by virus replication,progeny virions assembly and egress,then spread to other cells.As the late stage of filovirus lifecycle,efficient budding of virion is the prerequisite to viral transmission and pathogenicity.Our research on EBOV and MARV seeks to elucidate the mechanisms by which these viruses assemble and bud from cells,and how host protein recruitment and virus-host interactions contribute to these processes.Filovirus major matrix protein VP40 plays a central role in virion assembly and egress,such that independent expression of VP40 in mammalian cells leads to the production and egress of virus-like particles(VLPs)which are morphologically indistinguishable from authentic virions and accurately mimic the budding process of live infectious virus.Thus,the use of EBOV and MARV VP40 VLP budding assay is a reliable experimental approach helps to provide us abundant and valuable insights into filovirus buddingLate budding domain(L-domain)is the core functional domain of VP40 protein and has been shown to be crucial for efficient VLP and virus budding.The four(or more)amino acid sequences,such as PPxY,P(T/S)AP,YPx(n)L,termed motifs,are the key components of late budding function.Viral L-domain is responsible for recruitment of specific host proteins required for completing virus-cell separation or "pinching-off" of virus particles.Both of EBOV and MARV VP40 proteins contain the PPxY motif,which is one of the typical WW domain ligands.WW domain is a module that mediates protein-protein interaction and widely distributed in mammalian proteins.Hence,VP40 can hijack the WW domain bearing host proteins to facilitate virus egress through PPxY-WW domain interaction.For example,Nedd4 and ITCH are WW domain containing HECT E3 ubiquitin ligases,which are recruited by the PPxY motif to mediate the ubiquitination of VP40 protein,and thus facilitate the budding of filovirus VLP or virion.To identify additional WW domain bearing host proteins that interact with VP40 through PPxY motif,we utilized an EBOV PPxY-containing peptide to"screen" the "proline rich" reading array which is comprised of 115 WW domains from mammalian or yeast proteins.Using this unbiased approach,we identified several host WW domains bound robustly to the EBOV PPxY peptide.BCL2 associated athanogene 3(BAG3)is one of these specific VP40 PPxY host interactors.GST-Pulldown assay and co-immunoprecipitation experiments conducted subsequently confirm that BAG3 interacts with EBOV and MARV VP40 both in vitro and in live cells.Furthermore,the co-immunoprecipitation results of VP40 PPxY mutants and BAG3 mutants indicate that VP40 interacts with BAG3 in a PPxY motif-WW domain dependent manner.The EBOV PPxY peptide only bounds to a select number of WW domains in our "proline rich" reading array,indicating that PPxY-WW domain interaction is highly specific and selected.Thus,it is tempting to hypothesize that this physical interaction might represent a biological function.Given that VP40 protein is essential for filovirus assembly and egress,we wonder if the expression of BAG3 influences the VP40 mediated VLP egress.Herein,we employed a well-established VLP budding assay to assess the biological consequence of BAG3—VP40 interaction.Intriguingly,we found that BAG3 significantly inhibits VLP egress in a dose-dependent manner,moreover,suppression of cellular endogenous BAG3 expression level enhances VLP egress,implying that BAG3 is contrary to all the previously identified WW domain bearing host interactors with VP40.The recruitment of BAG3 by VP40 cannot facilitate virus budding,instead,leads to inhibition of VLP production.Also,the anti-budding ability of BAG3 relies on the PPxY-WW domain interaction.Furthermore,our study found that BAG3 impedes the release of recombinant VSV-M40 virus,which contains the L-domain motifs of EBOV VP40 in its matrix protein.These results prove that the inhibitory effect of BAG3 on budding is not limited to VLP but extends to infectious virus as well.BAG3 is co-chaperone protein that regulates multiple major cellular biological processes and belongs to BAG protein family.As a cell stress-induced and pro-survival protein,BAG3 possesses anti-apoptotic and pro-autophagy activities.Moreover,BAG3 associated with chaperone mediated seclective autophagy by recognizing and sequestering misfolded or foreign proteins in to cellular aggresomes for autophagic-lysosomal pathway degradation,and thus helps to maintain cellular protein homeostasis.To illuminate the mechanism by which BAG3 inhibits VP40 mediated VLP egress,we utilized the confocal microscopy to visualize the intracellular localization of VP40 and BAG3.The representative live cells images indicate that the typical localization and accumulation of VP40 along the cell periphery and the formation of plasma membrane protrusions is remarkably reduced in the presence of BAG3.Consistently,our cytosol and plasma membrane protein fractionation and double-staining indirect immunofluorescence assay demonstrates that BAG3 expression relocates VP40 in the cytoplasm and sequestrates VP40 away from the budding sites at the plasma membrane,and hence impedes the VP40 meidated VLP assembly and budding.More importantly,BAG3 sequesters a portion of VP40 protein into perinuclear aggresomes,which contain the well-characterized autophagosome marker protein LC3-II.Therefore,we suppose that the pro-autophagy activity and selective autophagy associated function of BAG3 involve in its inhibitory effect on VLP egress.Besides,BAG3 competitively blocks the interactions of VP40 and other WW domain bearing host proteins that facilitate virus egress,such as Nedd4 and ITCH,and thus this ability may contribute to the negative role of BAG3 in virus budding,too.In conclusion,we have successfully identified a novel and functional EBOV and MARV VP40 host interactor,BAG3,which inhibits the egress of both EBOV and MARV VP40 VLPs,as well as live recombinant virus.As the first WW domain bearing host interactor of VP40 identified that uniquely plays a negative role in the budding process,BAG3 significantly alters the intracellular localization of VP40 and reduce the accumulation of VP40 at the plasma membrane.In addition,BAG3 sequesters a fraction of VP40 into aggresomes by selective autophagy associated function and competes for binding to VP40 with other host WW domain interactors that positively regulate virus budding.Overall,these abilities of BAG3 compose the mechanism to block the VP40 mediated VLP egress.Hereby,we propose that BAG3 represents a novel and specific host defense strategy to counteract the function of VP40 in promoting efficient egress and transmission of filovirus.