title: Palmitoylation Strengthens Cholesterol-dependent Multimerization and Fusion Activity of Human Cytomegalovirus Glycoprotein B (gB).
abstract: Herpesviruses are a large order of animal enveloped viruses displaying a virion fusion mechanism of unusual complexity. Their multipartite machinery has a conserved core made of the gH/gL ancillary complexes and the homo-trimeric fusion protein glycoprotein B (gB). Despite its essential role in starting the viral infection, gB interaction with membrane lipids is still poorly understood. Here, evidence is provided demonstrating that human cytomegalovirus (HCMV) gB depends on the S-palmitoylation of its endodomain for an efficient interaction with cholesterol-rich membrane patches. We found that, unique among herpesviral gB proteins, the HCMV fusion factor has a Cys residue in the C-terminal region that is palmitoylated and mediates methyl-β-cyclodextrin-sensitive self-association of purified gB. A cholesterol-dependent virus-like particle trap assay, based on co-expression of the HIV Gag protein, confirmed that this post-translational modification is functional in the context of cellular membranes. Mutation of the palmitoylated Cys residue to Ala or inhibition of protein palmitoylation decreased HCMV gB export via Gag particles. Moreover, purified gBC777A showed an increased kinetic sensitivity in a cholesterol depletion test, demonstrating that palmitoyl-gB limits outward cholesterol diffusion. Finally, gB palmitoylation was required for full fusogenic activity in human epithelial cells. Altogether, these results uncover the palmitoylation of HCMV gB and its role in gB multimerization and activity.
authors.affiliation: From the Animal Cell Technology Unit, iBET Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal, the Biocrystallography Unit, DIBIT Fondazione Centro San Raffaele, 20132 Milano, Italy, and patrone.marco@hsr.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Patrone
authors.affiliation: From the Animal Cell Technology Unit, iBET Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal, the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2784-505 Oeiras, Portugal.
authors.forename: Ana Sofia
authors.initials: AS
authors.lastname: Coroadinha
authors.affiliation: From the Animal Cell Technology Unit, iBET Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal, the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2784-505 Oeiras, Portugal.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Teixeira
authors.affiliation: From the Animal Cell Technology Unit, iBET Instituto de Biologia Experimental e Tecnológica, 2780-901 Oeiras, Portugal, the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2784-505 Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D003587:Cytomegalovirus / Q000502:physiology*; D006801:Humans; D007915:Lepidoptera; D008051:Lipid Bilayers / Q000378:metabolism; D054878:Lipoylation; D008561:Membrane Fusion; D021962:Membrane Microdomains; D008954:Models, Biological; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D008051:Lipid Bilayers; D011994:Recombinant Proteins; C546870:UL55 protein, human cytomegalovirus; D014759:Viral Envelope Proteins; D002784:Cholesterol; D003545:Cysteine
keywords: cholesterol; herpesvirus; human cytomegalovirus; lipid raft; membrane protein; protein palmitoylation; protein-lipid interactions; protein-protein interaction; virion fusion protein