FIG 2 .

Strain C.BR92025 Nef shows a defect in MHC-I downregulation and protein expression. (A) Jurkat E6.1 cells were infected with lentiviral vectors expressing Nef from laboratory strain NL4.3, lacking Nef expression (dNef), consensus subtype C Nef, or Nef from reference strain B.JRFL or C.BR92025. Cell surface MHC-I levels were measured by flow cytometry at 48 h postinfection with a panspecific anti-MHC-I primary antibody (W6/32) and an Alexa Fluor 647-conjugated secondary antibody. Downregulation efficiency relative to that of NL4.3 was calculated by using MFI (****, P < 0.05; n = 4). (B) Uninfected (UI) Jurkat E6.1 cells or cells infected with the lentiviral vectors described above expressing nonfusion Nef proteins were collected and lysed at 48 h postinfection. Lysates were analyzed for Nef, EGFP, and actin protein levels by Western blotting. (C) Jurkat E6.1 cells were infected with lentiviral vectors encoding EGFP fused to the Nef proteins of reference strains B.JRFL and C.BR92025. A representative histogram from three independent experiments is shown.