FIG 3 .

Cells expressing strain C.BR92025 Nef have reduced CD4 downregulation. (A) CD4⁺ HeLa cells were transfected with plasmids encoding EGFP fusion proteins from laboratory strain NL4.3, consensus subtype C Nef, or Nef from reference strain B.JRFL or C.BR92025, as well as nonfused EGFP as a negative control. Cell surface CD4 levels were measured by flow cytometry with an APC-conjugated anti-CD4 antibody at 24 h posttransfection. Downregulation efficiency relative to that of NL4.3 was calculated by using MFI (*, P < 0.05; n = 3). (B) Lysates of transfected CD4⁺ HeLa cells were collected and lysed at 24 h postinfection. Lysates were analyzed for fusion protein expression by Western blotting with an anti-GFP antibody. (C) Jurkat E6.1 cells were infected with lentiviral vectors either expressing NL4.3 Vpu or not expressing NL4.3 Vpu (dVpu) in addition to expressing Nef of laboratory strain NL4.3, lacking Nef expression (dNef), consensus subtype C Nef, or Nef of reference strain B.JRFL or C.BR92025. Cell surface levels of CD4 were measured by flow cytometry with an APC-conjugated anti-CD4 antibody at 48 h postinfection. Downregulation efficiency relative to that of NL4.3 was calculated by using MFI (*, P < 0.05; n = 3).