Fig. 6.
Knock-down of PrPC affects HIV-1 replication in T cells. a T-lymphocytes with strong PrPC expression have reduced expression of HIV-1 Env and Gag. CEM-GFP cells (panel 1, transmission) infected with HIV-1 (panel 2, GFP expression grey) were recovered 5 days after infection, fixed and analyzed by confocal immunofluorescence using anti-PrP (panel 3, green), anti-Envgp120 (panel 4, red) and anti-MAp17 (panel 5, blue) antibodies. Images are single sections through the middle of the cell. Note that cells with high PrPC expression (arrowhead) display low expression of HIV-1 Gag and Env, whereas cells with low PrPC expression (arrow) have high HIV-1 Gag and Env expression. Scale bars are 10 μm. b CEM-GFP cells were transduced with lentiviral vectors expressing small hairpin RNAs (ShRNAs) surrounded by miR30 sequences directed against the prnp gene (miRShRNAs-PrPC) or the luciferase gene (negative control; miRShRNAs-Lu). After puromycin selection, transduced cells [knock-down (KD) Lu and PrPC CEM-GFP cells] were analyzed by Western blotting (15 μg of protein extracts) using anti-PrP or anti-cyclophilin A (CypA; loading and expression control) or by FACS analyses. Solid green lines PrPC expression in KD-Lu cells. Solid red lines PrPC expression in KD-PrPC cells. Dotted green and red lines negative control KD-Lu and KD-PrPC cells. Note that silencing of PrPC is not absolute. c Kinetics of HIV-1 infection in KD-Lu or KD-PrPC CEM-GFP cells. KD-Lu and KD-PrPC CEM-GFP cells were infected with WT HIV-1, cell culture supernatants were recovered each day for 9 days, and released reverse transcriptase (RT) activity was monitored. Dotted black lines RT activity released by HIV-1-infected KD-Lu cells. Solid black lines RT activity released by HIV-1-infected KD-PrPC cells. Note that HIV-1 replication is enhanced in KD-PrPC cells. d Second silencing strategy. CEM-GFP cells were first transduced as previously described above, selected and/or transduced a second time with the same vectors. Three days after transduction, cells were analyzed by Western blotting using anti-PrP antibodies. Loading control was monitored by Coomassie gel staining. S is simple-transduction and D is double transduction. Lanes 1, 2 correspond to KD-Lu cells and lanes 3, 4 to KD-PrP cells. e Kinetics of HIV-1 infection in double-transduced KD-Lu or KD-PrPC CEM-GFP cells. Double-transduced KD-Lu and KD-PrPC CEM-GFP cells were infected with WT HIV-1, at low MOI (80 ng CAp24/1 × 106 cells) or high MOI (400 ng CAp24/1 × 106 cells). Cells and cell culture supernatants were recovered each day for 6 days and released reverse transcriptase (RT) activity monitored. Dotted black lines RT activity released by HIV-1-infected KD-Lu cells. Solid black lines RT activity released by HIV-1-infected KD-PrPC cells. Note that HIV-1 replication is twofold enhanced in KD-PrPC cells at low MOI. f Virus infectivity. Virions released at days 4 and 5 (low MOI) were standardized for their RT activity and used for infection of HeLa P4 indicator cell line (CD4+ LTR-LacZ+). Infected cells were revealed by in situ staining for β-galactosidase activity (blue cells) 40 h post infection. Infection was quantified using a β-galactosidase-based colorimetric assay. Note that virions released by KD-PrPC cells are more infectious compared to virions released by KD-Lu cells