Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1.

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins.DOI: http://dx.doi.org/10.7554/eLife.02362.001

Characterization of the BST2/Tetherin cytoplasmic tail and understanding BST2-mediated NF-κB activation

BST2/Tetherin is an interferon-induced anti-viral host protein that restricts the release of newly assembled virions of enveloped viruses at the plasma membrane (PM) of infected cells. It also signals to activate NF-κB in a manner that is dependent on the tyrosine 6 residue of its cytoplasmic tail domain (CD). The main goal of this dissertation is to understand the role of BST2 cytoplasmic tail in signaling and trafficking and how BST2 signaling is modulated in response to proteins of Ebola virus.I first asked whether the cytoplasmic domain (CD) of BST2 directly binds to the host μ1 protein of the AP1 trafficking complex. Vpu of HIV-1 also binds to this complex and traps BST2 within internal membranes leading to enhanced virion release. I showed, using yeast two-hybrid assays, that the BST2 CD interacts with μ1 via a YxYxxV motif within BST2, in which both Ys and the V are required. Next, I asked whether both tyrosine residues are required for the activation of NF-κB by BST2. I found that tyrosine 6, but not tyrosine 8, is required for signaling likely due to its phosphorylation by Syk kinase. These results indicate that the determinants of membrane sorting and signaling by BST2 CD are overlapping but genetically separable.To understand the relationship between virion-entrapment and activation of NF- κB by BST2, I studied the modulation of BST2-mediated NF-κB activation in response to the expression of the Ebola virus (EBOV) matrix protein (VP40) and glycoprotein (GP1,2). I asked whether the relief of virus-like particle (VLP) restriction by EBOV GP1,2 would reduce or inhibit BST2 signaling. VP40 was used as a virion model because it forms VLPs that resemble actual virions of EBOV, whose release is restricted by BST2. Under the conditions of this study, I found that GP1,2 and VP40 cooperated with BST2 to induce NF-κB activity. Using a mutant of BST2 lacking the C-terminal GPI anchor, which is defective for virion-entrapment and is not expressed at the PM, I found that BST2 signaling does not require trapping VLPs at the PM and that cooperative signaling with EBOV proteins occurs within internal membranes

Characterization of the BST2/Tetherin cytoplasmic tail and understanding BST2-mediated NF-κB activation

BST2/Tetherin is an interferon-induced anti-viral host protein that restricts the release of newly assembled virions of enveloped viruses at the plasma membrane (PM) of infected cells. It also signals to activate NF-κB in a manner that is dependent on the tyrosine 6 residue of its cytoplasmic tail domain (CD). The main goal of this dissertation is to understand the role of BST2 cytoplasmic tail in signaling and trafficking and how BST2 signaling is modulated in response to proteins of Ebola virus.I first asked whether the cytoplasmic domain (CD) of BST2 directly binds to the host μ1 protein of the AP1 trafficking complex. Vpu of HIV-1 also binds to this complex and traps BST2 within internal membranes leading to enhanced virion release. I showed, using yeast two-hybrid assays, that the BST2 CD interacts with μ1 via a YxYxxV motif within BST2, in which both Ys and the V are required. Next, I asked whether both tyrosine residues are required for the activation of NF-κB by BST2. I found that tyrosine 6, but not tyrosine 8, is required for signaling likely due to its phosphorylation by Syk kinase. These results indicate that the determinants of membrane sorting and signaling by BST2 CD are overlapping but genetically separable.To understand the relationship between virion-entrapment and activation of NF- κB by BST2, I studied the modulation of BST2-mediated NF-κB activation in response to the expression of the Ebola virus (EBOV) matrix protein (VP40) and glycoprotein (GP1,2). I asked whether the relief of virus-like particle (VLP) restriction by EBOV GP1,2 would reduce or inhibit BST2 signaling. VP40 was used as a virion model because it forms VLPs that resemble actual virions of EBOV, whose release is restricted by BST2. Under the conditions of this study, I found that GP1,2 and VP40 cooperated with BST2 to induce NF-κB activity. Using a mutant of BST2 lacking the C-terminal GPI anchor, which is defective for virion-entrapment and is not expressed at the PM, I found that BST2 signaling does not require trapping VLPs at the PM and that cooperative signaling with EBOV proteins occurs within internal membranes

Intersection of Polycystic Ovary Syndrome and the Gut Microbiome.

The etiology of polycystic ovary syndrome (PCOS) remains unclear, although studies indicate that both genetic and environmental factors contribute to the syndrome. In 2012, Tremellen and Pearce proposed the idea that dysbiosis of the intestinal (gut) microbiome is a causative factor of metabolic and reproductive manifestations of PCOS. In the past 5 years, studies in both humans and rodent models have demonstrated that changes in the taxonomic composition of gut bacteria are associated with PCOS. Studies have also clearly shown that these changes in gut microbiota are associated with PCOS as opposed to obesity, since these changes are observed in women with PCOS that are both of a normal weight or obese, as well as in adolescent girls with PCOS and obesity compared with body mass index- and age-matched females without the disorder. Additionally, studies in both women with PCOS and rodent models of PCOS demonstrated that hyperandrogenism is associated with gut microbial dysbiosis, indicating that androgens may modulate the gut microbial community in females. One study reported that the fecal microbiome transplantation of stool from women with PCOS or exposure to certain bacteria resulted in a PCOS-like phenotype in mice, while other studies showed that exposure to a healthy gut microbiome, pre/probiotics, or specific gut metabolites resulted in protection from developing PCOS-like traits in mice. Altogether, these results suggest that dysbiosis of the gut microbiome may be sufficient to develop PCOS-like symptoms and that modulation of the gut microbiome may be a potential therapeutic target for PCOS

Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1.

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins.DOI: http://dx.doi.org/10.7554/eLife.02362.001