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. 2014 May;88(10):5845–5858. doi: 10.1128/JVI.03818-13

FIG 7.

FIG 7

HIV-2 envelope, SIVmac Nef, and KSHV K5 target tetherin (THN) isoforms with equal efficiency. (A and B) 293T cells were transfected with increasing amounts of rhesus (Rh) macaque WT, L-, or S-tetherin and SIVmac or SIVmacΔNef proviral plasmid. (A) Titers of infectious virus released from transfected cells were determined on TZM-bl HeLa reporter cells. Solid lines, SIVmac; dashed lines, SIVmacΔNef. RLU, relative light units. (B) Cell lysates and pelleted supernatant virions were subjected to SDS-PAGE and analyzed by Western blotting for Hsp90, rhesus tetherin, and p27 CA expression. The percentages of p27 CA in the supernatant, relative to virus release in the absence of tetherin, are indicated at the bottom. (C) 293T cells were transfected with increasing amounts of human WT, L-, or S-tetherin and HIV-2 ROD10 or the ROD10 GY-AA mutant proviral plasmid. Cell lysates and pelleted supernatant virions were subjected to SDS-PAGE and analyzed by Western blotting for Hsp90, tetherin, and p26 CA expression. The percentages of p26 CA in the supernatant, relative to virus release in the absence of tetherin, are indicated at the bottom. (D) 293 cells stably expressing L- or S-tetherin were transfected with HIV-2 ROD10 Env-IRES-GFP or HIV-2 ROD10 GY-AA mutant Env-IRES-GFP. At 48 h posttransfection, cells were analyzed by flow cytometry for surface GFP and tetherin expression. (E) HT1080 cells stably expressing tetherin isoforms were transduced with K5 or mutant K5 (K5NTR) defective for tetherin antagonism and selected. Cells were then analyzed by flow cytometry for surface tetherin expression. Dotted gray lines, tetherin-negative HT1080 cells; gray lines, nontransduced tetherin-expressing HT1080 cells; black lines, tetherin-expressing cells transduced with WT K5 (top) or a mutant form (K5NTR; bottom). The graph indicates the surface tetherin expression on transduced cell lines, shown as percentages of the fluorescence of the corresponding nontransduced (Untransd.) tetherin-expressing cell line. (F) Lysates of transduced and nontransduced cells were subjected to SDS-PAGE and analyzed by Western blotting for Hsp90 and tetherin expression. Tetherin expression in lysates as a percentage of that in nontransduced cells is shown at the bottom.