Fulfillment of the parvovirus life cycle in infected hPBMCs upon treatment with type-I IFN and IFNR2 neutralizing antibodies (Neut. Abs).
(A, B and C) hPBMCs collected from the blood of healthy donors were distributed into 6-well plates at 1×107 cells/5 ml culture medium/well. They were then treated or not with a mixture of neutralizing antibodies directed against hIFN-αs, hIFN-β, and hIFNR-2 (+Neut. Abs) at a concentration of 1 µg each/ml for 5 hrs and then mock-treated or infected with MVMp or H-1PV at 20 PFUs/cell in presence of the neutralizing antibodies. Cells (A, B and C) were harvested 24 hrs later by scraping in PBS and centrifuged in order to perform Western blot (A), RT-PCR (B), or Southern blot (C) experiments. (A) Cell pellets were re-suspended in complete Ripa buffer and Western blotting was performed as described in Figure 6. Actin was used as an internal loading control. Each presented blot is representative of 4 experiments which gave similar results. (B) Total RNA extraction and consecutive synthesis of cDNA were performed as described in Figure 1. Expression of the indicated transcripts was assessed using specific pairs of primers. Transcripts encoding the human 18S ribosomal protein were used as internal loading controls. Data shown are representative of 4 experiments which gave similar results. (C) Total DNA was extracted from cells and Southern blotting performed as described in Figure 2. (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded genome). The blot shown is representative of 4 experiments which gave similar results.