The Disulfide Bonds within BST-2 Enhance Tensile Strength during Viral Tethering

Abstract

Human BST-2/tetherin is a host factor that inhibits the release of enveloped viruses, including HIV-1, HIV-2, and SIV, from the cell surface by tethering viruses to the host cell membrane. BST-2 has an α-helical ectodomain that forms disulfide-linked dimers between two monomers forming a coiled coil. The ectodomain contains three cysteine residues that can participate in disulfide bond formation and are critical for viral tethering. The role of the disulfides in viral tethering is unknown but proposed to be for maintaining the dimer. We explored the role of the disulfides in the structure of BST-2 using experimental, biophysical methods. To understand the role of the disulfides in viral tethering, we used a new approach in viral tethering, steered molecular dynamics. We find that the disulfides coordinate the unfolding of the BST-2 monomers, which adds tensile strength to the coiled coil. Structural differences between oxidized and reduced BST-2 are apparent during unfolding, showing the monomers slide past each other in the absence of the disulfides. We found no evidence to support dissociation of the dimer upon reduction of the disulfide bonds. Moreover, the structure of BST-2 in the absence of the disulfides is similar to that of the oxidized form of BST-2, supporting previous X-ray crystallography and cellular work that showed the disulfides are not required for expression of BST-2. These data provide new insights into viral tethering by using novel techniques in the analysis of BST-2 to give amino acid level insight into functions of BST-2.

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

“…Therefore, we simulated the transition from the membrane bound to the host cell-viral particle bridging forms. In a previous study, we showed that a steered molecular dynamics simulation pulling the termini of the BST-2 ectodomain to mimic viral tethering correlated well with biochemical experiments 13 . Therefore, we turned to steered molecular dynamics to simulate the conformational changes BST-2 during the transition to the bridging form using our model of the membrane bound, full-length BST-2.…”

“…For comparison, the only other BST-2 homolog with a structure is the mouse homolog so we aligned the crystals structures of the human and murine homologs (data not shown) 8,6 . We did not observe any gaps in the murine coil and note that melting temperature for the ectodomain of the murine BST-2 is higher than the value for the human homolog 8,13 . These data suggest that the mouse homolog has a more rigid dimer interface which might alter the structure of the ectodomain during the transition from membrane to bridging position.…”

“…The antiviral protein BST-2 physically tethers budding viruses to the host cell membrane preventing the particle from spreading and infecting other cells 25 . While the cellular details of this process have been long studied and described, the mechanistic details regarding viral tethering are only now coming to light 11,13,16 . Using steered molecular dynamics, we simulated the transition of human and murine BST-2 from the host cell bound form to the host-virus bridging form (Figures 2, 3, and 4).…”

“…Several cellular and biochemical studies have described aspects of the mechanism of viral tethering by BST-2 [10][11][12][13][14] . 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 . Thus, the disulfides show a critical role in viral tethering by increasing the strength of the tether.…”