Down-modulation of human tetherin by chimeras between HIV-1 and SIVcpz/gor Vpus. (A) Expression of wild-type Vpu proteins and chimeras between the TMD of HIV-1 NL4-3 Vpu and the CP of SIVcpz or SIVgor Vpus. 293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Vpus and eGFP. Mock transfected cells were used as negative control. ß-actin and eGFP expression levels were analyzed to control for loading and transfection efficiency, respectively. For HeLa cells two different western blots are shown to allow comparison of Vpu signal intensities (middle) and to show that all Vpus can be detected after long exposure times (bottom). (B) FACS analysis of 293T cells co-transfected with a vector expressing tetherin and plasmids expressing eGFP alone or together with the indicated vpu allele. The range of eGFP expression levels used to calculate receptor down-modulation and the mean fluorescence intensities (MFI) are indicated. (C, D) Vpu-dependent reduction of tetherin surface expression in (C) 293T and (D) HeLa cells. Shown are the levels of receptor cell surface expression relative to those measured in cells transfected with the eGFP only control vector (100%). Data represent average values (±SEM) derived from three or more experiments. (E) Expression of tetherin in untransfected 293T or HeLa cells and in 293T cells transfected with a construct expressing human tetherin (293T teth). (F) Correlation between the levels of tetherin expression on 293T and HeLa cells expressing wild-type or chimeric Vpus proteins.