The formation of cysteine-linked dimers of BST-2/tetherin is important for inhibition of HIV-1 virus release but not for sensitivity to Vpu.
BACKGROUND
The Human Immunodeficiency virus type 1 (HIV-1) Vpu protein enhances virus release from infected cells and induces proteasomal degradation of CD4. Recent work identified BST-2/CD317 as a host factor that inhibits HIV-1 virus release in a Vpu sensitive manner. A current working model proposes that BST-2 inhibits virus release by tethering viral particles to the cell surface thereby triggering their subsequent endocytosis.


RESULTS
Here we defined structural properties of BST-2 required for inhibition of virus release and for sensitivity to Vpu. We found that BST-2 is modified by N-linked glycosylation at two sites in the extracellular domain. However, N-linked glycosylation was not important for inhibition of HIV-1 virus release nor did it affect surface expression or sensitivity to Vpu. Rodent BST-2 was previously found to form cysteine-linked dimers. Analysis of single, double, or triple cysteine mutants revealed that any one of three cysteine residues present in the BST-2 extracellular domain was sufficient for BST-2 dimerization, for inhibition of virus release, and sensitivity to Vpu. In contrast, BST-2 lacking all three cysteines in its ectodomain was unable to inhibit release of wild type or Vpu-deficient HIV-1 virions. This defect was not caused by a gross defect in BST-2 trafficking as the mutant protein was expressed at the cell surface of transfected 293T cells and was down-modulated by Vpu similar to wild type BST-2.


CONCLUSIONS
While BST-2 glycosylation was functionally irrelevant, formation of cysteine-linked dimers appeared to be important for inhibition of virus release. However lack of dimerization did not prevent surface expression or Vpu sensitivity of BST-2, suggesting Vpu sensitivity and inhibition of virus release are separable properties of BST-2.
Andrew
Amy J
AJ
Laboratory of Molecular Microbiology, Viral Biochemistry Section, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892-0460, USA. andrewa@niaid.nih.gov
Miyagi
Eri
E
Kao
Sandra
S
Strebel
Klaus
K
D019943:Amino Acid Substitution / Q000235:genetics; D015703:Antigens, CD / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides; D058851:GPI-Linked Proteins; D006031:Glycosylation; D015497:HIV-1 / Q000276:immunology* / Q000502:physiology*; D054298:Human Immunodeficiency Virus Proteins / Q000502:physiology*; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D054334:Viral Regulatory and Accessory Proteins / Q000502:physiology*; D014779:Virus Replication; D000074010:Bone Marrow Stromal Antigen 2; D000086082:Viroporin Proteins
D015703:Antigens, CD; D003545:Cysteine; D004220:Disulfides; D058851:GPI-Linked Proteins; D054298:Human Immunodeficiency Virus Proteins; D008562:Membrane Glycoproteins; D054334:Viral Regulatory and Accessory Proteins; C094572:BST2 protein, human; D000074010:Bone Marrow Stromal Antigen 2; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins