Fulfillment of the parvovirus life cycle in infected hPBMCs upon treatment with type-I IFN and IFNR2 neutralizing antibodies (Neut. Abs).

Abstract

<p>(<b>A</b>, <b>B</b> and <b>C</b>) 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 (<b>A</b>, <b>B</b> and <b>C</b>) were harvested 24 hrs later by scraping in PBS and centrifuged in order to perform Western blot (<b>A</b>), RT-PCR (<b>B</b>), or Southern blot (<b>C</b>) experiments. (<b>A</b>) Cell pellets were re-suspended in complete Ripa buffer and Western blotting was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g006" target="_blank">Figure 6</a>. Actin was used as an internal loading control. Each presented blot is representative of 4 experiments which gave similar results. (<b>B</b>) Total RNA extraction and consecutive synthesis of cDNA were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g001" target="_blank">Figure 1</a>. 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. (<b>C</b>) Total DNA was extracted from cells and Southern blotting performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055086#pone-0055086-g002" target="_blank">Figure 2</a>. (dRF, dimmer replicative form; mRF, monomeric replicative form; ssDNA, single-stranded genome). The blot shown is representative of 4 experiments which gave similar results.</p

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