Functional mechanisms of the cellular prion protein (PrP(C)) associated anti-HIV-1 properties

Abstract

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.

Fig. 1

PrP^C and Shadoo, but not Doppel, affect HIV-1 expression and production. a Schematic representation of the PrP^C, Doppel and Shadoo GPI anchor proteins. b Viral supernatants from HIV-1/Control (CT)/PLAP (placental alkaline phosphatase)/human huPrP^C and /huPrP^CΔGPI or /murine moPrP^C,/moDoppel and/moShadoo co-transfected cells were harvested 48 h after transfection and the virus-associated reverse transcriptase (RT) activity measured. Data shown are representative of three independent experiments. Values are given as means ± SD. c Virus-producer cells were analyzed by Western blotting. Total protein (10 μg per lane) of HIV-1/CT (lane 1); HIV-1/PLAP (lane 2); HIV-1/WT-PrP^C (lane 3); HIV-1/PrP^CΔGPI (lane 4); HIV-1/moPrP^C (lane 5); HIV-1/moDoppel (lane 6); HIV-1/moShadoo (lane 7) was analyzed by SDS PAGE. Membranes were probed with antibodies directed against HIV-1 CAp24, PLAP, PrP^C, Flag tag for PrP^C and moDoppel, moShadoo. Cyclophilin A (CypA) and coomassie staining were used as loading controls

Fig. 2

Anti-viral properties of PrP^C map to the N-terminal region. a Schematic representation of PrP^C with amino acid positions and deleted domains. b Viral supernatants from 293T cells co-transfected with HIV-1 pNL43 and control vector (CT), full length WT PrP^C (WT), or PrP^C deletion mutants PrP-ΔGPI, Δ24-145, Δ24-28, Δ36-50, ΔOR, Δ95-110 or ΔHC were harvested 48 h after transfection and released virus associated RT activity was measured. Data shown are representative of six independent experiments. Values are given as means ± SD. c Virus producer cells were analyzed by Western blotting. Total protein (10 μg per lane) of HIV-1/CT (lane 1); HIV-1/PrP^C-WT (lane 2); HIV-1/PrP-ΔGPI (lane 3); HIV-1/PrP-ΔHC (lane 4); HIV-1/PrP-Δ24-28 (lane 5); HIV-1/PrP-Δ24-145 (lane 6) was analyzed by SDS PAGE. Membranes were probed with antibodies directed against HIV-1 CAp24 and PrP^C. Cyclophilin A (CypA) and Coomassie staining were used as loading controls. Note that the Pr55Gag protein level was strongly reduced in PrP^C-WT and ΔHC samples

Fig. 3

PrP^C and Tetherin affect HIV-1 in a different manner. a Viral supernatants from 293T cells coexpressing HIV-1 pNL43 (WT or ΔVpu mutant) and PrP^C-WT or Tetherin-HA or PrP^C-WT +Tetherin-HA were recovered 40 h after transfection, and virus-associated RT activity was monitored. Data shown are representative of three independent experiments. Values are given as means ± SD. Note the strong effect on HIV-1 virus production of PrP^C and Tetherin when they were coexpressed compared to PrP^C or Tetherin alone. b Virus producer cells were analyzed by Western blotting. Total protein (10 μg per lane) of HIV-1WT/CT (lane 1); HIV-1WT/WT-PrP^C (lane 2); HIV-1 WT/Tetherin-HA (lane 3); HIV-1 WT/WT-PrP^C + Tetherin-HA (lane 4) and the same but with the HIV-1Δvpu mutant (lanes 5–8) was analyzed by SDS-PAGE. After transfer, membranes were probed with antibodies directed against HIV-1 CAp24, PrP^C and HA epitope for Tetherin-HA fusion protein. Cyclophilin A (CypA) antibody and Coomassie staining were used as loading controls. Note that the Pr55Gag protein level was strongly reduced in PrP^C-FL and Tetherin coexpressing cells

Fig. 4

PrP^C is expressed in HIV-1 target cells. a Western blotting analysis of PrP^C expressing cells; 10 μg of total protein extracts of cell lines or peripheral blood mononuclear cells was analyzed by SDS-PAGE, and transferred membranes were probed with the monoclonal SAF32 anti-PrP. Lane 1 lymphoblastoid cell line CEM-GFP; lane 2 monocytes; lane 3 monocyte-derived dendritic cells (MDDC); lane 4 activated peripheral blood lymphocytes (PBLs); lane 5 monocyte-derived macrophages (MDM); lane 6 the glioblastoma cell line U251MG. b Distribution of PrP^C by confocal immunofluorescence in U251MG cells (panel 1); CEM lymphoblastoid cells (panel 2); primary monocytes (panel 3); monocyte-derived dendritic cells (MDDC, panel 4); PBLs, panel 5; monocyte-derived macrophages (MDM, panel 6). Note the presence of PrP^C signal (green) at the plasma membrane, in filopodia (see white arrows for U251MG cells), in tunneling nanotubes (see white arrowheads in CEM and MDDC) and at the immunological synapse (see double white arrowheads for activated PBL). Scale bars are 10 μm

Fig. 5

PrP^C colocalizes with HIV-1 Gag and Env in infected T lymphocytes. a CEM cells infected with HIV-1 were recovered 5 days after infection, fixed and analyzed by confocal immunofluoresence using anti-PrP (panel 2, green), anti-Envgp120 (panel 3, red) and anti-MAp17 (panel 4, blue) antibodies. Images are single sections through the middle of the cell with the corresponding transmission image (panel 1): areas of green/red/blue colocalization appear white (panel 5). Region of interest (ROI; white arrow) is depicted in the merge panel (panel 5). The plot profile of PrP^C (green)/Env (red) and Gag (blue) colocalization along the ROI was constructed and analyzed using Image J software. b HIV-1 infected CEM-GFP cells were put in contact with activated PBLs for 3 h at 37°C on SuperFrost^RPlus microscope slides in an humidified chamber and then fixed. Images are single sections through the middle of the cell with the corresponding Nomarski image (panel 1). Confocal immunofluorescence was carried out using anti-PrP (panel 3, green), anti-Envgp120 (panel 4, red) and anti-MAp17 (panel 5, blue) antibodies. Panel 2 corresponds to GFP fluorescence (grey staining) indicating the HIV-1 CEM-GFP-positive cell. Areas of PrP (green)/Env (red)/Gag (blue) colocalization appear white (see arrowheads in merge, panel 6). Note the strong signal of PrP^C at the virological synapse. Scale bars are 10 μm

Fig. 6

Knock-down of PrP^C affects HIV-1 replication in T cells. a T-lymphocytes with strong PrP^C 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 PrP^C expression (arrowhead) display low expression of HIV-1 Gag and Env, whereas cells with low PrP^C 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-PrP^C) or the luciferase gene (negative control; miRShRNAs-Lu). After puromycin selection, transduced cells [knock-down (KD) Lu and PrP^C 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 PrP^C expression in KD-Lu cells. Solid red lines PrP^C expression in KD-PrP^C cells. Dotted green and red lines negative control KD-Lu and KD-PrP^C cells. Note that silencing of PrP^C is not absolute. c Kinetics of HIV-1 infection in KD-Lu or KD-PrP^C CEM-GFP cells. KD-Lu and KD-PrP^C 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-PrP^C cells. Note that HIV-1 replication is enhanced in KD-PrP^C 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-PrP^C CEM-GFP cells. Double-transduced KD-Lu and KD-PrP^C 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-PrP^C cells. Note that HIV-1 replication is twofold enhanced in KD-PrP^C 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-PrP^C cells are more infectious compared to virions released by KD-Lu cells

Fig. 7

PrP^C binds to HIV-1 genomic RNA and affects its translation. a Immunoprecipitation of PrP^C in 293T HIV-1/PrP^C 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). PrP^C 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/PrP^C 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-PrP^C 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-PrP^C 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-PrP^C 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 PrP^C has no effect

Fig. 8

Cellular distribution of WT and mutant PrPs in 293T HIV-1 coexpressing cells. Cellular distribution of WT and mutant PrPs (WT, Δ24-28, ΔGPI, ΔHC) in 293T coexpressing cells by immunofluorescence using the SAF32 anti-PrP antibody. Left panel corresponds to the transmission. Medium panel is the PrP labeling for a HIV-1/PrP^C-WT, b HIV-1/PrP-Δ24-28 cells, c HIV-1/PrP-ΔGPI and d HIV-1/PrP-ΔHC cells. Right panel is a 2.6 magnification of the selected region (white box in the medium panel). Scale bar is 15 μm