Figure 3. hHR23A is required for Vpr-mediated stimulation of HIV-1 replication in non-dividing MAGI-CCR5 cells and macrophages.

A.i. Cell proliferation of MAGI-CCR5 cells in different FBS concentration. MAGI-CCR5 cells were plated at 7,000 cells/well in DMEM containing 10% or 0.1% of FBS. Cellular proliferation was measured over a period of 5 days. Cells in 0.1% FBS were viable over the experimental period, as they remained adherent to plates. ii. Depletion of hHR23A significantly reduces viral replication in non-dividing MAGI cells in a Vpr-dependent manner. MAGI-CCR5 cells were transfected with hHR23A-targeting (+) or control (−) siRNA, plated in DMEM with 10% or 0.1% FBS, and infected with HIV-1_Ada Vpr(+) or Vpr(−) 24 hrs after hHR23A knockdown. Viral replication was evaluated 48 hrs after infection by staining; blue cells were counted as infected. Results are presented as percent of control, i.e., the number of blue cells in cultures transfected control siRNA and infected with Vpr-positive HIV-1 and show average ± SE of quadruplicate determinations. B. Vpr-dependent HIV-1 viral replication in macrophages is mediated through hHR23A. i. Monocyte-derived macrophages pretreated with hHR23A siRNA or control (Ctr) siRNA were infected with HIV-1_Ada (Vpr+) or (Vpr−) viruses. Cells were collected 72 hrs p.i. and viral replication was determined by measuring p24. Results are presented as inhibition of HIV-1 replication in cells treated with hHR23A siRNA relative to cells treated with control siRNA, and show mean ± SE of three independent experiments with cells from different donors, each performed in triplicate. Statistical analysis was performed using Student's t-test, and p value is shown. ii. Monocyte-derived macrophages were transfected with hHR23A siRNA or control siRNA. Cells were collected 72 hrs p.t. and subjected to Western blot analysis using anti-Rad23A and anti-β-actin antibodies.