FIG. 1.

Mutations in the hydrophobic region (H₁₆₆PMSLHGM₁₇₃) in HIV-1 Nef C-loop-abrogated Nef-mediated CD4 downregulation. (A) L₁₆₄L₁₆₅ downstream sequence H₁₆₆PMSLHGM₁₇₃ is generally conserved among 259 Nef sequences. (B) FACS analysis shows CD4 downregulation by the Nef mutants indicated in each panel. Nef plasmids (10 (g) and pEGFP (2 (g) were cotransfected into the CD4 T cells by electroporation. The cells were surface stained with anti-CD4 PE for 2D FACS. (C) Effects of L₁₇₀ mutations on Nef-mediated CD4 downregulation. L₁₇₀ was mutated to Ala, Met, Lys, Glu, Pro, Gln, and Asn, respectively. The percentage of CD4 downregulation is calculated as described in Materials and Methods. The results are an average of three independent experiments. (D) Effects of M₁₆₈ mutations on Nef-mediated CD4 downregulation. M₁₆₈ was mutated to Ala, Leu, Lys, Glu, Pro, and Gln, respectively. (E) Comparison of the CD4 downregulation by Nef mutant M₁₆₈/A, L₁₇₀/A, and M₁₆₈L₁₇₀/AA. Nef plasmids of 2.5, 5.0, 10, or 15 (g and pEGFP of 2.5 (g were cotransfected into CD4 cells and analyzed as described in B. (F) Cellular expressions of the Nef mutants indicated at the top of each lane and numbered as shown in B. Whole cell lysates of 10⁶ transfected cells were resolved on SDS–PAGE and western blotted with anti-Nef antibody.