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. Author manuscript; available in PMC: 2013 Sep 13.
Published in final edited form as: Cell Host Microbe. 2012 Sep 13;12(3):373–380. doi: 10.1016/j.chom.2012.07.008

Figure 2. Functional activity of Vpu and Nef proteins of a CPZ-adapted HIV-1 strain.

Figure 2

(A–D) Levels of (A, B) CD4 and (C, D) tetherin cell surface expression in the presence of Vpu or Nef relative to levels measured in cells transfected with the GFP control vector (100%). Values are averages (±SD) from three experiments. **, p<0.01; ***, p<0.001.

(E, F) Infectious virus yield from 293T cells cotransfected with the HIV-1 NL4-3 ΔVpuΔNef construct and vectors expressing the indicated (E) vpu or (F) nef alleles in combination with various quantities of plasmids expressing HU or CPZ tetherin. Panels E to G give average values derived from triplicate infections of TZM-bl indicator cells relative to those obtained in the absence of tetherin expression vector (100%). All results were verified by p24 ELISA.

(G) The JC16 clone uses both Nef and Vpu to antagonize tetherin. Virus release from 293T cells following transfection with 2.5 μg of a proviral JC16 wt construct or mutants lacking either nef (U+N−), vpu (U−N+) or both (U−N−) and varying amounts of plasmids expressing HU or CPZ tetherin.

(H) The JC16 Nef targets amino acid residues that are missing in the human tetherin orthologue. 293T cells were transfected with plasmids expressing HU tetherin or a variant in which the N-terminal deletion was restored (HU-INS). The middle panel shows the enhancement of infectious virus release relative to the eGFP only control derived from triplicate infections of TZM-bl indicator cells. The right panel shows the reduction of the surface expression levels of the respective tetherin variant in the presence of JC16 or GAB1 Nefs relative to those measured in cells transfected with the GFP only vector. The deletion in HU tetherin is highlighted by a yellow box. (See also Figure S1).