Fig. 6.

The Nef-HFE interaction increases iron accumulation and HIV growth in monocytes/macrophages. (A) THP-1 cells were infected with lentiviral vectors encoding GFP or Nef-GFP and then permeabilized and analyzed for ferritin protein levels. Ex vivo macrophages homozygous for wild-type HFE or C282Y HFE were infected for 4 days with 50 ng/ml p24 HIV or Nef-deleted HIV and stained for p18/p55 gag and ferritin. The mean fluorescence intensity of 30,000-cell aliquots is given. Nef-GFP-expressing THP-1 cells contained double the ferritin of uninfected cells; GFP-infected cells have only a small increase in ferritin. HIV infection increases ferritin only in wild-type-expressing macrophages, and this effect is reduced in the absence of Nef. (B and C) THP-1 (B) and U937 (C), a monocytic cell line that does not express HFE, were transfected by nucleoporation with either Nef-GFP or GFP and incubated with 40 μg/ml [⁵⁹Fe]transferrin for 14 h, and iron accumulation was measured. Nef-GFP increased intracellular iron in THP-1 but not in U937 cells. The mean accumulation was calculated, and the asterisk indicates significance, compared with GFP control (P < 0.001 by Student's t test). (D) Ex vivo macrophages homozygous for either wild-type or C282Y HFE were infected with wild-type HIV or Nef-deleted HIV and, after 2 days, incubated with 33 μg/ml [⁵⁹Fe]transferrin for an additional 10 days. The cellular ⁵⁹Fe was then determined. HIV increased macrophage iron accumulation in a Nef- and wild-type-HFE-dependent manner. Experiments shown in A-D are representative of several giving similar results. (E) Ex vivo macrophages homozygous for wild-type HFE (four donors) or C282Y HFE (three donors) were incubated with 50 ng/ml p24 of M-tropic HIV, with or without nef, for 4 days, stained for p18/p55 gag expression, and analyzed by FACS to calculate the percentage of p18/p55-positive cells in the cultures. The data show that Nef induces a 2- to 3-fold increase in gag-expressing cells in wild-type HFE-expressing macrophages, but this effect is reduced in C282Y-expressing macrophages and is not induced by Nef.