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. 2010 Nov 10;85(2):932–945. doi: 10.1128/JVI.01668-10

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Copyright © 2011, American Society for Microbiology

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FIG. 3. — Amino acid residues of hu-tetherin TM region required for Vpu interaction and counteraction. (A and B) BiFC assay of hu-tetherin TM mutants on Vpu interaction. Schematic diagram showing the amino acid alterations made in the C-terminal TM region of hu-tetherin. Identity is indicated by dashes. The KGN-Vpu-expressing cells were transfected with a series of KGC-hu-tetherin TM mutants and analyzed by flow cytometry as described in Materials and Methods. The protein expression of KGC-fused hu-tetherin and its mutants in cell lysates were detected using the indicated antibody. Relative MFI values are defined as the MFI of pKGN-Vpu-transfected or its mutant-transfected cells minus the MFI of untransfected cells, and results represent the means from three independent experiments plus standard deviations. (C) Tetherin activity and Vpu sensitivity of hu-tetherin TM mutants. HEK 293 cells were cotransfected with WT HIV-1 infectious DNA (pNL4-3) or its vpu-deleted DNA (pNL4-3/Udel) (1 μg) with or without (control) pKGC-hu-tetherin and its derivative expression plasmids (100 ng). The expression of HIV-1 (Pr55^Gag) in the cell lysates was detected using anti-p24 antibody. Results represent the means from more than three independent experiments. Statistical significance (Student's t test) is represented as P < 0.05 (**) and P < 0.01 (***) (A and B) or as numerical values (C). (D) Surface expression of tetherin and its mutants. HEK 293 cells were cotransfected with WT HIV-1 infectious DNA (pNL4-3) or its vpu-deleted DNA (pNL4-3/Udel) (1 μg) with or without (control) pKGC-hu-tetherin and its mutant expression plasmids (100 ng). The cell surface expression of tetherin was detected using anti-tetherin antibody. Statistical significance (Student's t test) is represented as P < 0.05 (*). (E) Amino acid alignment of seven primates and the mo-tetherin TM domain. Identity is indicated by dashes, and sequence gaps are indicated by an asterisk. Gray boxes indicate amino acid sequences corresponding to the mutants for which the BiFC signals were significantly decreased.

FIG. 3. — Amino acid residues of hu-tetherin TM region required for Vpu interaction and counteraction. (A and B) BiFC assay of hu-tetherin TM mutants on Vpu interaction. Schematic diagram showing the amino acid alterations made in the C-terminal TM region of hu-tetherin. Identity is indicated by dashes. The KGN-Vpu-expressing cells were transfected with a series of KGC-hu-tetherin TM mutants and analyzed by flow cytometry as described in Materials and Methods. The protein expression of KGC-fused hu-tetherin and its mutants in cell lysates were detected using the indicated antibody. Relative MFI values are defined as the MFI of pKGN-Vpu-transfected or its mutant-transfected cells minus the MFI of untransfected cells, and results represent the means from three independent experiments plus standard deviations. (C) Tetherin activity and Vpu sensitivity of hu-tetherin TM mutants. HEK 293 cells were cotransfected with WT HIV-1 infectious DNA (pNL4-3) or its vpu-deleted DNA (pNL4-3/Udel) (1 μg) with or without (control) pKGC-hu-tetherin and its derivative expression plasmids (100 ng). The expression of HIV-1 (Pr55^Gag) in the cell lysates was detected using anti-p24 antibody. Results represent the means from more than three independent experiments. Statistical significance (Student's t test) is represented as P < 0.05 (**) and P < 0.01 (***) (A and B) or as numerical values (C). (D) Surface expression of tetherin and its mutants. HEK 293 cells were cotransfected with WT HIV-1 infectious DNA (pNL4-3) or its vpu-deleted DNA (pNL4-3/Udel) (1 μg) with or without (control) pKGC-hu-tetherin and its mutant expression plasmids (100 ng). The cell surface expression of tetherin was detected using anti-tetherin antibody. Statistical significance (Student's t test) is represented as P < 0.05 (*). (E) Amino acid alignment of seven primates and the mo-tetherin TM domain. Identity is indicated by dashes, and sequence gaps are indicated by an asterisk. Gray boxes indicate amino acid sequences corresponding to the mutants for which the BiFC signals were significantly decreased.