Positioning of Cysteine Residues within the N-terminal Portion of the BST-2/Tetherin Ectodomain Is Important for Functional Dimerization of BST-2

Welbourn, Sarah; Kao, Sandra; Pont, Kelly E. Du; Andrew, Amy J.; Berndsen, Christopher E.; Strebel, Klaus

doi:10.1074/jbc.m114.617639

Cited by 12 publications

(44 citation statements)

References 35 publications

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“…S2A in the supplemental material). Transfection with pEGFP-C3A-tetherin, a previously characterized tetherin mutant that does not form the disulfide bonds between monomers that contribute to dimer stability (26,53), failed to prevent HIV-1 release (see Fig. S2A).…”

Section: Clem and Cryo-et Of Hiv-1 Vlps Tethered To Human Cellsmentioning

confidence: 99%

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

Strauss

Hammonds

et al. 2016

J Virol

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Tetherin (BST2, CD317, or HM1.24) is a host cellular restriction factor that prevents the release of enveloped viruses by mechanically linking virions to the plasma membrane. The precise arrangement of tetherin molecules at the plasma membrane site of HIV-1 assembly, budding, and restriction is not well understood. To gain insight into the biophysical mechanism underlying tetherin-mediated restriction of HIV-1, we utilized cryo-electron tomography (cryo-ET) to directly visualize HIV-1 virus-like particles (VLPs) and virions tethered to human cells in three dimensions (3D). Rod-like densities that we refer to as tethers were seen connecting HIV-1 virions to each other and to the plasma membrane. Native immunogold labeling showed tetherin molecules located on HIV-1 VLPs and virions in positions similar to those of the densities observed by cryo-ET. The location of the tethers with respect to the ordered immature Gag lattice or mature conical core was random. However, tethers were not uniformly distributed on the viral membrane but rather formed clusters at sites of contact with the cell or other virions. Chains of tethered HIV-1 virions often were arranged in a linear fashion, primarily as single chains and, to a lesser degree, as branched chains. Distance measurements support the extended tetherin model, in which the coiled-coil ectodomains are oriented perpendicular with respect to the viral and plasma membranes. IMPORTANCETetherin is a cellular factor that restricts HIV-1 release by directly cross-linking the virus to the host cell plasma membrane. We used cryo-electron tomography to visualize HIV-1 tethered to human cells in 3D. We determined that tetherin-restricted HIV-1 virions were physically connected to each other or to the plasma membrane by filamentous tethers that resembled rods ϳ15 nm in length, which is consistent with the extended tetherin model. In addition, we found the position of the tethers to be arbitrary relative to the ordered immature Gag lattice or the mature conical cores. However, when present as multiple copies, the tethers clustered at the interface between virions. Tethered HIV-1 virions were arranged in a linear fashion, with the majority as single chains. This study advances our understanding of tetherin-mediated HIV-1 restriction by defining the spatial arrangement and orientation of tetherin molecules at sites of HIV-1 restriction.

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Section: Clem and Cryo-et Of Hiv-1 Vlps Tethered To Human Cellsmentioning

confidence: 99%

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

Strauss

Hammonds

et al. 2016

J Virol

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“…A canonical coiled-coil from vimentin or a non-coiled coil substitution showed reduced or absent function in viral tethering assays 15 . Further work using cysteine substitutions within the coiled-coil region (96 to 150) including substitutions at Gly109 and Ala130, which would strengthen the interaction between monomers, showed reduced or no viral tethering activity and reduced levels of dimerization 9 . In contrast, Cys substitutions in the N-terminal portion of the ectodomain (residues 50 to 95), which in our simulations was moved as a single unbroken region, showed mixed results with some substitutions performing viral tethering similar to the wild-type and all were able to dimerize 9 .…”

Section: Discussionmentioning

confidence: 99%

“…Structurally, BST-2 has four main features: a cytoplasmic region, an N-terminal transmembrane helix, an extracellular coiled coil domain, and a C-terminal membrane anchor which is either a GPI anchor or a protein based anchor depending on the species [4][5][6][7][8] . BST-2 is a constitutive dimer and the extracellular coiled coil intertwines the two monomers connected by at least one disulfide 9,10 . Finally, the protein is known to be glycosylated on at least one of two asparagine residues within the ectodomain which is important for trafficking the protein to the cell membrane 10 .…”

Section: Introductionmentioning

confidence: 99%

“…Andrew and coworkers showed that the size of the ectodomain was not strictly defined and that one of the three disulfides must be intact for effective viral tethering 10,15 . Welbourn and coworkers showed that the disulfides could be moved to several positions within the ectodomain if the structure of the coiled-coil was not disrupted 9 . These disulfides appear to stabilize the structure of the coiled-coil and make it more resistant to unwinding 9,13 .…”

Section: Introductionmentioning

confidence: 99%

“…Welbourn and coworkers showed that the disulfides could be moved to several positions within the ectodomain if the structure of the coiled-coil was not disrupted 9 . These disulfides appear to stabilize the structure of the coiled-coil and make it more resistant to unwinding 9,13 . Thus, the disulfides show a critical role in viral tethering by increasing the strength of the tether.…”

Section: Introductionmentioning

confidence: 99%

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Bending of the BST-2 coiled-coil during viral budding

Ozcan

Berndsen

2017

Preprint

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BST-2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV-1 and other viruses by inhibiting viral spreading. Structurally, BST-2 is a homodimeric coiled-coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C-terminal membrane anchor of BST-2 is inserted into the budding virus while the Nterminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST-2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST-2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated BST-2 and the cellvirus membrane bridging form. We observed that BST-2 did not transition as a rigid structure, but instead bent at sites with a reduced interface between the helices of the coiled-coil. The simulations for the human BST-2 were then compared with simulations on the mouse homolog, which has a more stable coiled-coil.We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled-coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.peer-reviewed)

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Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress

et al. 2019

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cAMP response element binding Protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane‐bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER‐stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumor suppression, and immune‐related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95 Å. The X‐ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre‐formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulfide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulfide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidizing conditions. Moreover, sequence alignment of bZIP sequences from chicken, human, and mouse reveals that only members of the CREB3 subfamily contain this cysteine residue, indicating that it could act as a redox‐sensor. Taken together, these results suggest that the activity of these transcription factors may be redox‐regulated and they may be activated in response to oxidative stress.

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Positioning of Cysteine Residues within the N-terminal Portion of the BST-2/Tetherin Ectodomain Is Important for Functional Dimerization of BST-2

Cited by 12 publications

References 35 publications

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

Three-Dimensional Structural Characterization of HIV-1 Tethered to Human Cells

Bending of the BST-2 coiled-coil during viral budding

Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress

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