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. 2017 Feb 28;91(6):e02177-16. doi: 10.1128/JVI.02177-16

FIG 4.

FIG 4

Effect of HIV-1 RBF206 Vpu on different tetherin isoforms and orthologs. (A) Amino acid alignment of the long and short isoforms of human tetherin. The region targeted by O-Nefs is indicated. A dual tyrosine endocytosis motif is shown in red. (B and C) Downmodulation of wild-type and Y6AY8A mutant (B) and the long and short isoforms of human tetherin (C). HEK293T cells were cotransfected with plasmids expressing the indicated tetherin and constructs expressing the indicated vpu alleles. Twenty-four hours later, cells were stained for tetherin and analyzed via flow cytometry. Shown are the levels of cell surface expression relative to those obtained in the presence of the vector expressing only eGFP (100%). (D) Expression of various tetherin isoforms and orthologs. HEK293T cells were cotransfected with plasmids expressing the indicated tetherin variants and examined by Western blotting 2 days later. All tetherin variants show variations in electrophoretic mobility due to differences in glycosylation. (E and F) Effects of various Vpus on infectious virus yield (E) and p24 release (F) in the presence of short tetherin. HEK293T cells were cotransfected with an HIV-1 NL4-3 Δvpu Δnef construct and vectors expressing the indicated Vpu proteins and the short isoform of human tetherin. Infectious virus yield was determined by infection of TZM-bl cells, and p24 release was determined via ELISA. The values obtained in the absence of tetherin represent 100%. (G) Downmodulation of the indicated tetherin orthologs by NL4-3 and RBF206 Vpus was determined as described for panels B and C. (H) Expression of the long and short forms of human tetherin in the presence of the indicated Vpu or Nef proteins. HEK293T cells were cotransfected with plasmids expressing the indicated tetherin variants and Vpu or Nef proteins and examined by Western blotting 2 days later. (I) Levels of L and S tetherin expression determined by Western blotting as shown in panel H. Values are mean percentages (±SEM, n = 3) obtained for the Vpu and Nef expression constructs relative to the vector control (100%). *, P < 0.05; **, P < 0.01; ***, P < 0.001.