(A) Circular dichrosim analyses of tetherin under native and reducing conditions (DTT). Disulfide bond reduction of tetherin(80–147) reduces the overall helical content, while tetherin(47–159) is less affected.
(B) Thermostability measurements of tetherin were performed at 222 nm under native and reducing conditions (DTT), revealing a dramatic change in Tm after disulfide bond reduction.

(a) Experimental scattering intensities obtained for tetherin(47–159) (upper curve) and tetherin(80–147) (lower curve) are shown as a function of resolution and after averaging and subtraction of solvent scattering. The scattering intensities calculated from representative models (presented in Figure 4B and C) with the lowest χ values are shown as red lines. The absolute values of the intensities of the upper curve are shifted by 2 logarithmic units.
(B) Ab initio models of tetherin(80–147) and (C) of tetherin(47–159) reveal elongated rodlike structures; the calculated bead model as well as the molecular envelopes with the docked coiled-coil structure are shown.

(A) Expression of tetherin_set1 and tetherin_set2 abolish the retention function of tetherin. Release of virions (left panel, lanes 3 and 4) is the same as in case of the vector control (lane 1), whereas wild-type tetherin prevents virion release (left panel, lane 2). The middle panel shows that intracellular Gag and its processing are not affected by the expression of mutant tetherin (lanes 3 and 4). The right panel shows the expression levels of mutant (lanes 3 and 4) and wild type tetherin (lane 2).
(B) Expression of tetherin_set3 (left panel) has no effect on tetherin function (lane 3) while tetherin_set4 abolishes the retention function of tetherin (lane 4) as indicated by the extracellular detection of CA. The middle panel shows that intracellular Gag and its processing are not affected by the expression of mutant tetherin (lanes 3 and 4). The right panel shows the expression levels of mutant and wild type tetherin. Note that the extensive posttranslational modification observed for wild type tetherin is absent in case of the set 4 mutant.
(C) Expression of the tetherin mutant Asn65Gln (N65Q)(left panel) has little effect on tetherin function (lane 3) compared to wild-type tetherin (lane 2). Intracellular Gag processing is not affected by the expression of the N65Q mutant (middle panel; lanes 2 and 3). The right panel shows that the expression pattern of N65Q (lane 3) is less complex than that of wild type tetherin (lane 2).
(D) Expression of tetherin(iHA) (left panel) has little effect on tetherin function (lane 2) when compared to wild-type tetherin (lane 3). The middle panel shows that intracellular Gag and its processing are not affected. The right panel shows the expression levels of tetherin(iHA) and wild-type tetherin.

(A) SEC analysis of tetherin(47–159) and tetherin(80–147).
(B) SDS-PAGE of tetherin(47–159) (lanes 1 and 3) and tetherin(80–147) under reducing (lanes 1 and 2) and non-reducing conditions (lanes 3 and 4).
(C) Reduced tetherin(47–159) and tetherin(80–147) still dimerize; tetherin(47–159) (lanes 1, 3, 4) and tetherin(80–147) (lanes 2, 5, 6) were treated with iodoacetamide and separated under non-reducing conditions (lanes 1 and 2), under reducing conditions, lanes 3 and 5 and after cross-linking with 5 mM EGS (lanes 4 and 6); dimers are indicate by *.

(A) Stereo image of the experimental electron density map obtained after SAD phasing and non-crystallographic symmetry averaging; the heptad d position occupied by Cys91 forming a disulfide bond is shown.
(B) Ribbon representation shows a 90 Å long parallel coiled-coil.
(C) Close-up of the heptad motifs and polar dimerization contacts.

Immunofluorescence of (A) MOCK transfected 293T cells showing DAPI staining.
(B) tetherin(iHA); the inset shoes a close-up of a section of the plasma membrane revealing patches of tetherin staining.
(C) tetherin(iHA-set1)
(D) tetherin(iHA-set2)
(E) tetherin(iHA-set3)
(F) tetherin(iHA-set4); all constructs reveal a similar plasma membrane staining pattern, indicating that the mutations do not affect their localization.