title: Conserved motifs within Ebola and Marburg virus VP40 proteins are important for stability, localization, and subsequent budding of virus-like particles.
abstract: The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the (96)LPLGVA(101) sequence of eVP40 and the corresponding (84)LPLGIM(89) sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the (96)LPLGVA(101) motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the (96)LPLGVA(101) motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-DeltaLPLGVA failed to rescue the budding defective eVP40-DeltaLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-DeltaLPLGIM successfully rescued budding of mVP40-DeltaLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.
authors.affiliation: Laboratory 412, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Liu
authors.forename: Luis
authors.initials: L
authors.lastname: Cocka
authors.forename: Atsushi
authors.initials: A
authors.lastname: Okumura
authors.forename: Yong-An
authors.initials: YA
authors.lastname: Zhang
authors.forename: J Oriol
authors.initials: JO
authors.lastname: Sunyer
authors.forename: Ronald N
authors.initials: RN
authors.lastname: Harty
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D029043:Ebolavirus / Q000235:genetics*; D006801:Humans; D042541:Intracellular Space / Q000821:virology; D029024:Marburgvirus / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009698:Nucleoproteins / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014758:Viral Core Proteins / Q000235:genetics*; D014771:Virion / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D019065:Virus Assembly / Q000235:genetics; D057074:Virus Release
chemical_list: D009698:Nucleoproteins; D014758:Viral Core Proteins; C084819:nucleoprotein VP40, Ebola virus