Preferential insertion of tetherin C-termini into virion envelopes.

<p>(<b>A</b>) Schematic representation of the polarities that would be adopted by the Flag N5Fac protein if HA- or FLAG-tagged proteolytic fragments are observed to partition with liberated virions. (<b>B</b>) Western blot analyses of 293T cells and liberated virions, obtained by the protease treatment of Flag N5Fac cells at various time points following infection. The samples were probed using anti-HA, anti-FLAG and anti-CA antibodies. (<b>C</b>) Western blot analyses of 293T cells expressing serial dilutions of the CA-HA-Flag protein and the PNGase-F-treated liberated virion lysates. The samples were probed with anti-CA, anti-HA and anti-FLAG antibodies. (<b>D</b>) The CA, HA and FLAG band intensities for the control protein were determined using a LiCOR Odyssey scanner and were plotted against the dilution (left panels). Regression analysis was performed over the linear range of signal intensities (right panels), and the unknown amounts of CA, HA and FLAG epitopes in the PNGase-F-treated, Factor Xa-liberated virion lysates were deduced by interpolation from the standard curves. (<b>E</b>) Western blot analyses of 293T cells stably expressing the Flag N5Fac tetherin protein and virions liberated upon Factor Xa or subtilisin A treatment. The samples were probed using an anti-CA antibody.