title: Positioning of cysteine residues within the N-terminal portion of the BST-2/tetherin ectodomain is important for functional dimerization of BST-2.
abstract: BST-2/tetherin is a cellular host factor capable of restricting the release of a variety of enveloped viruses, including HIV-1. Structurally, BST-2 consists of an N-terminal cytoplasmic domain, a transmembrane domain, an ectodomain, and a C-terminal membrane anchor. The BST-2 ectodomain encodes three cysteine residues in its N-terminal half, each of which can contribute to the formation of cysteine-linked dimers. We previously reported that any one of the three cysteine residues is sufficient to produce functional BST-2 dimers. Here we investigated the importance of cysteine positioning on the ectodomain for functional dimerization of BST-2. Starting with a cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced at various locations throughout the ectodomain. The resulting BST-2 variants were tested for expression, dimerization, surface presentation, and inhibition of HIV-1 virus release. We found significant flexibility in the positioning of cysteine residues, although the propensity to form cysteine-linked dimers generally decreased with increasing distance from the N terminus. Interestingly, all BST-2 variants, including the one lacking all three ectodomain cysteines, retained the ability to form non-covalent dimers, and all of the BST-2 variants were efficiently expressed at the cell surface. Importantly, not all BST-2 variants capable of forming cysteine-linked dimers were functional, suggesting that cysteine-linked dimerization of BST-2 is necessary but not sufficient for inhibiting virus release. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell.
authors.affiliation: From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460 and.
authors.forename: Sarah
authors.initials: S
authors.lastname: Welbourn
authors.affiliation: From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460 and.
authors.forename: Sandra
authors.initials: S
authors.lastname: Kao
authors.affiliation: Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Du Pont
authors.affiliation: From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460 and.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Andrew
authors.affiliation: Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Berndsen
authors.affiliation: From the Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0460 and kstrebel@nih.gov.
authors.forename: Klaus
authors.initials: K
authors.lastname: Strebel
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D053586:Virus Internalization; D000074010:Bone Marrow Stromal Antigen 2
chemical_list: D015703:Antigens, CD; D003545:Cysteine; D058851:GPI-Linked Proteins; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2
keywords: BST-2; Cysteine-mediated Cross-linking; Gene Expression; Microbiology; Protein Complex; Protein Structure; Tetherin; Virus Release