Fig. 7.
PrPC binds to HIV-1 genomic RNA and affects its translation. a Immunoprecipitation of PrPC in 293T HIV-1/PrPC coexpressing cells (lane 1). Coimmunoprecipitations were carried out using the specific monoclonal SAF32 anti-PrP (lane 3) or the mouse-IgG as a control antibody (lane 2). PrPC immunoprecipitation was performed by Western blotting using the anti-PrP antibody. Note the strong signal for the PrP immunoprecipitate (lane 3), whereas no signal was detected in the mouse IgG control (lane 2). b Coimmunoprecipitated RNAs associated with anti-PrP (lanes 5 and 6) or mouse IgG control antibodies (lanes 3 and 4) were extracted and DNAse treated. Purified RNAs were then used for cDNA synthesis in the presence or absence of reverse transcriptase (lanes 4, 6 and lanes 3, 5, respectively). PCR amplification was performed using specific HIV-1 primers located in the 5′UTR and in the gag gene (see Materials and Methods). Note the specific 657-bp HIV-1 PCR product only detected in the anti-PrP immunoprecipitates (lane 6). Positive RT-PCR samples were carried out with 293T HIV-1/PrPC cellular RNAs (lanes 1, 2). M: DNA molecular weight standards (in bp). c Schematic representation of the HIV-1 pNL43-renilla molecular clone. 293T cells were cotransfected with pNL43-luciferase molecular clone and expression constructs encoding the WT full length FL-PrPC or the inefficient mutant PrPΔ24-28. Measurement of HIV-1 Gag-luciferase activity and quantification of cytoplasmic HIV-1 luciferase-encoding RNAs by quantitative RT-PCR using GAPDH as an internal control was performed in the context of FL-PrPC or PrPΔ24-28. Total luciferase activity was measured 40 h post-transfection (left panel), and the amount of HIV-1 genomic RNA coding for cytoplasmic luciferase was quantified (middle panel). Translational efficiency (right panel) was calculated by normalizing the total HIV-1 Gag-luciferase activity by reference to the amount of cytoplasmic HIV-1 luciferase RNA. Note that FL-PrPC strongly affects HIV-1 translation, whereas no effect was observed with the PrPΔ24-28 mutant. d Schematic representation of the intronless luciferase coding vector used in this study (pcDNAGlobinRen) showing positions of the CMV promoter and BGH polyadenylation signal. Total luciferase activity was measured 40 h post-transfection (left panel) and the amount of cytoplasmic luciferase-encoding mRNAs was quantified (middle panel). Translational efficiency (right panel) was calculated by normalizing the total luciferase activity by reference to the amount of cytoplasmic luciferase mRNA. Note that PrPC has no effect