Retrovirus infection strongly enhances scrapie infectivity release in cell culture
2006; Springer Nature; Volume: 25; Issue: 12 Linguagem: Inglês
10.1038/sj.emboj.7601162
ISSN1460-2075
AutoresPascal Leblanc, Sandrine Alais, Isabel Porto-Carreiro, Sylvain Lehmann, Jacques Grassi, Graça Raposo, Jean Luc Darlix,
Tópico(s)Viral Infections and Immunology Research
ResumoArticle25 May 2006free access Retrovirus infection strongly enhances scrapie infectivity release in cell culture Pascal Leblanc Corresponding Author Pascal Leblanc LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France IFR 128 Biosciences, Lyon-Gerland, France Search for more papers by this author Sandrine Alais Sandrine Alais LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France IFR 128 Biosciences, Lyon-Gerland, France Search for more papers by this author Isabel Porto-Carreiro Isabel Porto-Carreiro Institut Curie, CNRS-UMR144 Structure et Compartiments Membranaires, Paris Cedex, France Search for more papers by this author Sylvain Lehmann Sylvain Lehmann Institut de Génétique Humaine (IGH), CNRS, UPR 1142 Montpellier Cedex, France Search for more papers by this author Jacques Grassi Jacques Grassi CEA, Service de pharmacologie et d'immunologie, CEA/Saclay, Gif sur Yvette, France Search for more papers by this author Graça Raposo Graça Raposo Institut Curie, CNRS-UMR144 Structure et Compartiments Membranaires, Paris Cedex, France Search for more papers by this author Jean Luc Darlix Jean Luc Darlix LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France IFR 128 Biosciences, Lyon-Gerland, France Search for more papers by this author Pascal Leblanc Corresponding Author Pascal Leblanc LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France IFR 128 Biosciences, Lyon-Gerland, France Search for more papers by this author Sandrine Alais Sandrine Alais LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France IFR 128 Biosciences, Lyon-Gerland, France Search for more papers by this author Isabel Porto-Carreiro Isabel Porto-Carreiro Institut Curie, CNRS-UMR144 Structure et Compartiments Membranaires, Paris Cedex, France Search for more papers by this author Sylvain Lehmann Sylvain Lehmann Institut de Génétique Humaine (IGH), CNRS, UPR 1142 Montpellier Cedex, France Search for more papers by this author Jacques Grassi Jacques Grassi CEA, Service de pharmacologie et d'immunologie, CEA/Saclay, Gif sur Yvette, France Search for more papers by this author Graça Raposo Graça Raposo Institut Curie, CNRS-UMR144 Structure et Compartiments Membranaires, Paris Cedex, France Search for more papers by this author Jean Luc Darlix Jean Luc Darlix LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France IFR 128 Biosciences, Lyon-Gerland, France Search for more papers by this author Author Information Pascal Leblanc 1,2, Sandrine Alais1,2, Isabel Porto-Carreiro3, Sylvain Lehmann4, Jacques Grassi5, Graça Raposo3 and Jean Luc Darlix1,2 1LaboRétro unité de virologie humaine INSERM U758, Ecole Normale Supérieure de Lyon, Lyon Cedex, France 2IFR 128 Biosciences, Lyon-Gerland, France 3Institut Curie, CNRS-UMR144 Structure et Compartiments Membranaires, Paris Cedex, France 4Institut de Génétique Humaine (IGH), CNRS, UPR 1142 Montpellier Cedex, France 5CEA, Service de pharmacologie et d'immunologie, CEA/Saclay, Gif sur Yvette, France *Corresponding author. LaboRétro unité de virologie humaine, INSERM U758, Ecole Normale Supérieure de Lyon, 46 allée d'ltalie, 69364 Lyon Cedex 07, France. Tel.: +33 472728625; Fax: +33 472728080; E-mail: [email protected] The EMBO Journal (2006)25:2674-2685https://doi.org/10.1038/sj.emboj.7601162 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Prion diseases are neurodegenerative disorders associated in most cases with the accumulation in the central nervous system of PrPSc (conformationally altered isoform of cellular prion protein (PrPC); Sc for scrapie), a partially protease-resistant isoform of the PrPC. PrPSc is thought to be the causative agent of transmissible spongiform encephalopathies. The mechanisms involved in the intercellular transfer of PrPSc are still enigmatic. Recently, small cellular vesicles of endosomal origin called exosomes have been proposed to contribute to the spread of prions in cell culture models. Retroviruses such as murine leukemia virus (MuLV) or human immunodeficiency virus type 1 (HIV-1) have been shown to assemble and bud into detergent-resistant microdomains and into intracellular compartments such as late endosomes/multivesicular bodies. Here we report that moloney murine leukemia virus (MoMuLV) infection strongly enhances the release of scrapie infectivity in the supernatant of coinfected cells. Under these conditions, we found that PrPC, PrPSc and scrapie infectivity are recruited by both MuLV virions and exosomes. We propose that retroviruses can be important cofactors involved in the spread of the pathological prion agent. Introduction The cellular prion protein (PrPC) is a GPI-anchored protein expressed in almost all tissues and predominantly in the central nervous system. PrPC is found in detergent-resistant microdomains (DRMs)/rafts and cycles between the cell surface and endosomal compartments (Vey et al, 1996; Naslavsky et al, 1997; Peters et al, 2003; Sunyach et al, 2003). The gene encoding PrPC, Prn-p, was shown to be essential for the susceptibility to transmissible spongiform encephalopathie (TSEs) in mice (Bueler et al, 1993). TSEs are neurodegenerative diseases characterized by the accumulation in the brain of a conformationally altered isoform of PrPC designated PrPSc (Sc for Scrapie; Prusiner, 1998). PrPSc differs from its normal isoform by its high content in β-sheet structure, its insolubility in mild detergents and its partial resistance to proteinase K (PK) treatment. The mechanism and the pathway by which the infectious prion spreads from cell to cell are still enigmatic. Indeed, it has been reported that conditioned medium of scrapie-infected GT1 cells is capable of transmitting infectivity to other cells, while in the case of scrapie-infected SMB cells, a direct cell-to-cell contact is required for transmission (Schatzl et al, 1997; Kanu et al, 2002). Recently, we found that both PrPC and PrPSc were released into the extracellular medium of scrapie-infected Rov and Mov cells in association with exosomes (Fevrier et al, 2004). Retroviruses and exosomes display many similarities with respect to lipid composition, cellular protein content and the site of assembly and release (Pelchen-Matthews et al, 2004). Retrovirus assembly is a multiple step process orchestrated by the viral Gag polyprotein, the genomic RNA and the cellular membrane in which the viral Envelope glycoproteins are anchored (Cimarelli and Darlix, 2002). In addition, several cellular proteins involved in membrane trafficking are required for particle trafficking and budding (Morita and Sundquist, 2004). Recent data indicated that retrovirus assembly is more complex than previously thought since murine leukemia virus (MuLV) and human immunodeficiency virus type 1 (HIV-1) assembly can take place in DRMs and, like exosomes, in intracellular compartments that display the hallmarks of late endosomes (Raposo et al, 2002; Basyuk et al, 2003; Pelchen-Matthews et al, 2003; Sherer et al, 2003). In agreement with this, several reports indicated that the site of virus assembly influences the protein and the lipid composition of the retroviral envelope. Indeed, host proteins from endosomal compartments or DRMs such as GPI-anchored proteins are recruited into nascent viral particles (Ott, 1997; Campbell et al, 2001; Raposo et al, 2002; Pelchen-Matthews et al, 2003) where they retain their biological function and are able to influence virus replication and the cell physiology (Ott, 1997; Campbell et al, 2001; Cantin et al, 2005). Since prion proteins and assembling retroviral particles seem to colocalize to the same intracellular compartments, this favors the notion that retroviruses could recruit prion proteins during assembly and budding. Such viruses may thus contribute to the release and the spread of prion proteins. Here we report that MoMuLV infection and viral particle production strongly enhance the release of PrPC, PrPSc and prion infectivity by coinfected cells. In addition, both PrPC and PrPSc are found associated with MoMuLV particles and exosomes. We propose that retroviruses could be cofactors involved in the spreading of the pathological prion agent. Results MoMuLV recruits murine PrPC Retroviruses such as MoMuLV use DRMs and endosomal compartments as sites for viral particle assembly and budding (Pickl et al, 2001; Basyuk et al, 2003; Sherer et al, 2003). This prompted us to analyze the colocalization of MoMuLV Gag and Env with murine prion protein. We first analyzed the presence of PrPC in isolated DRMs (see Supplementary Materials and methods) from NIH3T3 cells infected with MoMuLV (NIH3T3i). Results indicate that part of Gag and Env cofractionates with PrPc and GM1 in DRMs (Figure 1A, lanes 3–5). To determine if viral Gag and Env, and PrPc cofractionate in intracellular compartments, NIH3T3i cell extracts were fractionated through optiprep gradient as described previously (Kolesnikova et al, 2004). This method allows the separation of membrane proteins associated with the plasma membrane and intracellular compartments. Results presented in Figure 1B show that Gag and Env cofractionate with PrPC at the plasma membrane (lanes 1 and 2) and in intracellular compartments of endosomal/lysosomal origin as indicated by the presence of Lamp1 (lanes 8–11) and Rab11 (lanes 9, 10 and 17, 18). Immunogold labeling experiments (IEM) with anti-Lamp1 antibodies confirm the presence of virions in Lamp1-positive intracellular organelles where Lamp1 is associated with virions (data not shown). The cofractionation of PrPC with MoMuLV Gag and Env prompted us to further investigate the recruitment of PrPC by viral particles. To analyze the possible recruitment of PrPC by MoMuLV, virions were first concentrated and purified through a 6–18% optiprep gradient. Fractions were analyzed by Western blotting using anti-CAp30 and anti-PrP antibodies. Figure 2A shows that PrPC and MoMuLV CAp30 cofractionate (fractions 13–16) together with the GM1 raft marker and with reverse transcriptase (RT) (data not shown). To determine if PrPC was physically associated with virions, immunoprecipitation experiments were realized using magnetic beads conjugated with anti-PrP or the HIV-1 anti-CAp24 as a negative control (Supplementary Materials and methods). Results show that MoMuLV virions were specifically immunoprecipitated by the anti-PrP antibodies (Figure 2B). To confirm the association of PrPC with MoMuLV, IEM were realized (see Supplementary Materials and methods). To this end, ultrathin cryosections of NIH3T3i cells were immunogold labeled with anti-PrP and anti-CAp30 antibodies. Figure 2(c1–c4) reveals that large amounts of MoMuLV virions are present in intracellular compartments morphologically similar to multivesicular bodies (MVBs) and that viral particles consistently display PrP labeling at their surface (see arrows). These data show that PrPC is recruited by MoMuLV virions. To determine if incorporation of PrP can also take place with other retroviruses, we investigated the recruitment of PrP by a lentivirus such as HIV-1 (Leblanc et al, 2004). Our data revealed that human PrPC is also recruited by HIV-1 (Supplementary Figure 1). Figure 1.PrPC cofractionates with MoMuLV Gag and Env. (A) DRMs isolation from NIH3T3i cells. Fractions collected from the top of the gradient were analyzed by dot immunoblotting using anti-CAp30, anti-Envgp70 and anti-PrP antibodies. The GM1 raft marker was detected using the BCTx. (B) Fractionation of NIH3T3i cell extract by optiprep gradient centrifugation. Fractions were analyzed by immunoblotting using anti-CAp30, anti-Env and anti-PrP antibodies, and profiles were compared with the distribution of cellular marker proteins of the late endosomes/lysosome (Lamp1) compartments and recycling endosomes/small vesicles (Rab11). Download figure Download PowerPoint Figure 2.Recruitment of PrPC by MoMuLV. (A) Viral supernatants were recovered and virions were fractionated through a 6–18% optiprep gradient. Fractions were analyzed by Western blotting using anti-CAp30 and anti-PrP antibodies or biotinylated cholera toxin (BCTx) for the GM1 marker. As controls, the mock NIH3T3, the NIH3T3i-infected cell extracts and the 20% sucrose cushion-pelleted virions are shown on the right lanes. (B). Immunocapture of MoMuLV virions using anti-PrP antibodies. MoMuLV virions immunoprecipitated with magnetic beads conjugated to anti-PrP antibody or with an anti-HIV-1-CAp24 and beads alone as negative controls. After extensive washing, RT activity was detected on immunocaptured virions. Results are representative of three independent experiments and show that anti-PrP antibodies immunoprecipitate MoMuLV virions. Error bars correspond to means±s.d. (C) Double immunogold labeling of ultrathin cryosections of NIH3T3i cells using anti-PrP (PAG15) and anti-CAp30 (PAG10) antibodies (c1–c4). Scale bar 100 nm. Download figure Download PowerPoint Since PrP remained raft-associated even when the GPI anchor is deleted (PrP-ΔGPI) (Walmsley et al, 2003), we wondered if the murine and human PrP-ΔGPI mutant could be recruited into MoMuLV and HIV-1 viral particles. Results indicate that murine and human PrP-ΔGPI are recruited by MoMuLV and HIV-1 viral particles respectively (Supplementary Figure 2). MoMuLV enhances PrPC and PrPSc proteins release To determine if PrPSc can be recruited by MoMuLV particles, we used NIH3T3 cells infected by the 22L scrapie prion strain (Vorberg et al, 2004b). For this purpose, NIH3T3-N and NIH3T3-22L cells, corresponding to normal and scrapie-infected cells, respectively, were infected with MoMuLV to generate the NIH3T3-N-MoMuLV and NIH3T3-22L-MoMuLV cell lines. Production of MoMuLV virions was assessed by Western blotting using anti-CAp30 and anti-Envgp70 antibodies (Figure 3A, lanes 2 and 4) and by RT activity (data not shown). The presence of PrPSc was determined by PK treatment followed by Western blotting (Figure 3B, lane 7). No modification of PrP PK resistance was observed in the presence of MoMuLV expression in NIH3T3-22L cells. As expected, PrPSc was not detected in NIH3T3-N-MoMuLV cells (lane 5). As reported in Figure 1A, Gag and Env cofractionated with PrPC but also with PrPSc in DRMs (Figure 3C, lanes 3–5) and soluble fractions (lanes 9–11). These results indicate that PrPSc cofractionates with assembling viral particles opening the possibility that PrPSc could be incorporated into MoMuLV virions during the assembly and/or budding process. Figure 3.Infection of NIH3T3-N and NIH3T3-22L cells by MoMuLV. (A) MoMuLV expression was verified by Western blotting using anti-Envgp70 (top panel), anti-CAp30 (medium panel) and anti-PrP (bottom panel) antibodies. Lane 1: NIH3T3-N; lane 2: NIH3T3-N-MoMuLV; lane 3: NIH3T3-22L; and lane 4: NIH3T3-22L-MoMuLV. (B) No modification of PrP PK resistance is induced by MoMuLV infection. Lanes 1–2: NIH3T3-N; lanes 3–4: NIH3T3-22L; lanes 5–6: NIH3T3-N-MoMuLV; lanes 7–8: NIH3T3-22L-MoMuLV; and lanes 1, 3, 5 and 7: PK treated. (C) MoMuLV Gag and Env cofractionate with PrPC and PrPSc in DRMs from NIH3T3-22L-MoMuLV cells. DRMs from NIH3T3-22L-MoMuLV and NIH3T3-N-MoMuLV cells were isolated by equilibrium centrifugation gradient. Fractions were analyzed by dot immunoblotting using the anti-Envgp70, anti-CAp30, anti-PrP antibodies and the BCTx for the GM1 raft marker. For PrPSc detection, each fraction from NIH3T3-N-MoMuLV (negative control) and NIH3T3-22L-MoMuLV (positive for PrPSc) was treated by PK. PK-resistant products were analyzed as above by dot immunoblotting. +PK and −PK indicate the presence or the absence of PK treatment. Download figure Download PowerPoint To investigate if MoMuLV infection can enhance the extracellular release of prion proteins, the supernatant of NIH3T3-22L cells infected by MoMuLV was harvested and submitted to sequential centrifugations with increasing centrifugation forces (Figure 4A, lanes 5–8). As controls, equal amounts of supernatant of NIH3T3-22L cells (lanes 1–4) and complete DMEM culture medium (lanes 9–12) were used. Western blotting analyses were realized using MoMuLV-specific anti-Envgp70 and anti-CAp30 antibodies, and the result confirmed that only NIH3T3-22L-MoMuLV cells release viral particles in the supernatant with most of them found in the 100 000 g pellet (i.e. 100K pellet, see Envgp70 and CAp30/Pr65Gag signals in Figure 4A, lane 8). No viral protein was recovered in the 100K pellet from the control cell supernatants (lanes 4 and 12). Analysis with the anti-PrP revealed a very faint PrP signal in the 100K pellet recovered from the NIH3T3-22L supernatant (lane 4). On the other hand, we observed a 20-fold increase in the PrP signal (compare lanes 4 and 8) in the 100K pellet from NIH3T3-22L-MoMuLV supernatant, indicating that MoMuLV infection causes a drastic enhancement of the prion protein release from the infected cells. Identical data were observed with the NIH3T3-N and NIH3T3-N-MoMuLV cell supernatants (data not shown). The observation that most of the PrP signal was associated with the 100K pellet indicates that PrP release in the supernatant is mediated through pelletable structures such as viral particles or, as recently reported, exosomes (Fevrier et al, 2004). To determine if exosomes release is enhanced upon MoMuLV infection, we searched for cellular proteins incorporated into exosomes but not found in MoMuLV virions. The translation elongation factor 1α (EF1α) appears to be a good candidate since analyses of purified exosomes by mass spectrometry revealed that EF1α is associated with exosomes (Thery et al, 2001; Fevrier et al, 2004) but absent from MoMuLV (Cimarelli and Luban, 1999). For this purpose, Western blotting analyses were carried out using antibodies directed against EF1α as an exosome marker. Results show that the production of MoMuLV is associated with a five-fold increase in the EF1α marker (Figure 4A, bottom panel, compare lanes 4 and 8), indicating that MoMuLV infection of NIH3T3-22L cells enhances exosome production. Figure 4.MoMuLV infection strongly enhances prion proteins release. (A) Supernatant from NIH3T3-22L (lanes 1–4), NIH3T3-22L-MoMuLV (lanes 5–8) and free complete medium as negative control (lanes 9–12) were submitted to differential centrifugation. Lanes 1, 5 and 9: 3000 g for 5 min; lanes 2, 6 and 10: 4500 g for 5 min; lanes 3, 7 and 11: 10 000 g for 30 min; and lanes 4, 8 and 12: 100 000 g for 1 h. The pellets were analyzed by Western blotting using the anti-Envgp70, anti-CAp30, anti-PrP and anti-EF1α antibodies. (B) To determine the presence of PrPSc in the 100K pellet from NIH3T3-22L-MoMuLV cells, the pellets from NIH3T3-N-MoMuLV (negative control, lane 1) and NIH3T3-22L-MoMuLV (lane 2) were treated with PK before immunoblotting with anti-PrP (lanes 3 and 4). Download figure Download PowerPoint To determine if PrPSc is released in the cell culture medium, the 100K pellet from NIH3T3-22L-MoMuLV supernatant was submitted to PK digestion before doing the Western blotting. As a negative control, we used the 100K pellet from the NIH3T3-N-MoMuLV supernatant. Results presented in Figure 4B revealed the presence of PK-resistant PrP in the 100K pellet of NIH3T3-22L-MoMuLV, thus corresponding to PrPSc (lane 4), whereas no signal was detected in the control pellet (lane 3). Fractionation of the 100K pellet on a 10–60% sucrose density gradient (Supplementary Materials and methods) revealed that PrP cofractionates with MoMuLV Gag and Env but also with the EF1α exosome marker at densities 1.1415 and 1.1612 g/cm3 in the RT peak (Supplementary Figure 3). Prion proteins are associated with MoMuLV virions and exosomes Because the anti-PrP antibodies do not specifically detect PrPSc, virions and exosomes preparations were treated with 3 M guanidine isothiocyanate to enhance PrPSc immunoreactivity (Taraboulos et al, 1990). Detection of PrPSc on virions and exosomes was carried out by IEM using the anti-PrP antibody followed by protein A gold (15 nm) labeling (Figure 5(a1–a3), see arrows for virions and arrowheads for exosomes). PrP labeling was increased by about two-fold in the presence of guanidine treatment (compare layers a3 and a8), suggesting that additional labelings were specific for PrPSc. Double immunogold labeling of CAp30/Pr65Gag (PAG 10 nm) and PrP (PAG 15 nm, layers a4–a6) confirmed the presence of PrPSc on virions. As expected, detection of EF1α revealed that it was exclusively detected on exosomes (layer a7, see white arrowheads for exosomes and white arrows for nonlabeled virions). No labeling was observed using the control (Cont.) antibody (layer a9). We concluded that MoMuLV infection enhances the extracellular release of prion proteins mediated by viral particles and exosomes. Figure 5.MoMuLV infection strongly enhances the release of prion infectivity. (A) PrPSc released is associated with MoMuLV virions and exosomes. The 100K pellets from NIH3T3-22L-MoMuLV cells were analyzed by IEM for PrP (PAG15), CAp30/Pr65Gag (PAG10) or EF1α (PAG10) after treatment by 3 M guanidium isothiocyanate (5 min). Note the presence of PrP labeling on typical dense viral particle structures (see arrows layers a1–a3) and on light spherical structures corresponding to exosomes (see black arrowheads layers a1 and a3). Single and double IEM for CAp30/Pr65Gag, PrP and EF1α, respectively, on permeabilized virions/exosomes preparation. Layers a4–a6: double IEM for CAp30/Pr65Gag and PrP on MoMuLV virions. Layer a7: EF1α labeling on exosomes (white arrowhead) and not on virions (see white arrows). Layer a9: control (Cont.) irrelevant antibody. Scale bar is 100 nm. (B) Coculture assay. The NIH3T3-N cells (target cells) were grown on the bottom surface of a six-well plate and the NIH3T3-N, NIH3T3-N-MoMuLV, NIH3T3-22L and NIH3T3-22L-MoMuLV on the surface of an insert of 0.4 μm pore size. Coculture was carried out over 4 days and target cells were passaged over 4 weeks (seven passages). Transmission of MoMuLV from the insert to the well was controlled by RT assay. Transmission of PrPSc from the insert cells to the well cells was analyzed by cell blotting in the presence or absence of PK treatment. Immunoblotting was carried out using anti-PrP. Download figure Download PowerPoint MoMuLV infection enhances the release of prion infectivity To investigate if MoMuLV infection enhances the release of prion infectivity, coculture experiments were realized (Figure 5B). For this purpose, we cocultured normal NIH3T3-N cells with NIH3T3-N (+/− MoMuLV) and NIH3T3-22L (+/− MoMuLV) as separate cell populations. The first one (NIH3T3-N) was cultured on the bottom surface of a six-well plate (these cells will be named hereafter ‘target cells’), while the other cells (NIH3T3-N, NIH3T3-N-MoMuLV, NIH3T3-22L or NIH3T3-22L-MoMuLV) were on four independent inserts containing membranes with 0.4 μm diameter pores, permitting transfer of exosomes and viral particles. Cocultures were realized over 4 days and the target cells were submitted to seven passages (4 weeks). As a control for exchanges between the inserts and the wells, RT activity was monitored on the target cell supernatants 15 days after passage of the cells. As expected, RT activity was detected in wells 2 and 4 (Figure 5B), confirming the passage of virions from the insert to the well and the infection of NIH3T3-N cells. At the end, the target cells were harvested and the presence of PrPSc was determined using the cell blot assay (Figure 5B; Bosque and Prusiner, 2000). Results revealed that in the absence of PK treatment, all cells express similar levels of PrP. On the contrary, after PK treatment, we observed that only the NIH3T3-N cells cocultured with NIH3T3-22L-MoMuLV cells express PK resistant PrP indicating that the scrapie infectivity was efficiently transmitted under the present experimental conditions. After 16 passages, we failed to detect PK resistant PrP in NIH3T3-N cells cocultured with NIH3T3-22L cells. This observation correlates with the very low level of PrP detected in the NIH3T3-22L supernatant (Figure 4A, lane 4) and confirms that prion infectivity spreading in NIH3T3 cells is not efficient (Vorberg et al, 2004a). No prion infectivity transmission was observed when cocultures were realized using inserts containing membranes with 20 nm diameter pores confirming the association of prion infectivity with structures larger than 20 nm (data not shown). Anti-MoMuLV Envelope antibodies immunoprecipitate prion infectivity Since PrPSc is recruited by MoMuLV particles, we analyzed if antibodies directed against the MoMuLV Envelope glycoproteins can immunoprecipitate prion infectivity. For this purpose, magnetic beads were coated with antibodies directed against Envgp70 or the Simian immunodeficiency virus Vpx protein used as an irrelevant antibody. Immunoprecipitations were realized on NIH3T3-22L or NIH3T3-22L-MoMuLV supernatants. RT detection carried out on each immunoprecipitate revealed that condition 4 was positive for RT activity (data not shown) indicating that, as expected, virions were immunoprecipitated. After extensive washings, beads were layered on NIH3T3-N target cells and let over 4 days in contact (Figure 6A). After 4 days, cells were extensively washed to eliminate magnetic beads. Surprisingly, we found that beads from condition 4 were still attached to the cell surface (Figure 6B). This ‘sticky’ phenotype observed in condition 4 was most probably due to the interaction of the cell surface MoMuLV receptor with free Envgp70 associated with virions bound to the antibody–beads complex. No beads were observed in the other conditions (compare conditions 1–3 with 4). Figure 6.MoMuLV anti-Env antibodies immunoprecipitate prion infectivity. (A) Experimental strategy. Magnetic beads coated with MoMuLV anti-Env (conditions 2 and 4) or with SIV anti-Vpx (as irrelevant antibody; conditions 1 and 3) were used for the immunoprecipitation experiments on NIH3T3-22L (conditions 1 and 2) and NIH3T3-22L-MoMuLV (conditions 3 and 4) supernatant. After extensive washing, RT activity was only detected on beads from condition 4 (open circle). Beads were put in contact with NIH3T3-N cells over 4 days. (B) Visualization of cell culture by direct light microscopy revealed that beads from condition 4 were still attached to the cell surface after extensive washing. (C) Cells were passaged over 6 weeks until the complete loss of beads from the culture. PK-resistant PrP was detected by cell blotting experiment in the presence or absence of PK treatment. As controls of PK treatment, NIH3T3-N and scrapie NIH3T3-22L cells were used (left panel). Download figure Download PowerPoint Target cells were then passaged over 6 weeks until the complete clearance of beads. Then, target cells were analyzed by cell blot assays to detect the presence of PK-resistant PrP (Figure 6C). Results indicate that PrPSc was only detected in condition 4. No PrPSc was observed in control experiments, indicating that neither beads nor Vpx were able to immunoprecipitate prion infectivity (Figure 6A–C, conditions 1 and 3). No PrPSc was detected in condition 2, indicating that prion infectivity was specifically immunoprecipitated by MoMuLV anti-Env in condition 4 (Figure 6C, compare conditions 2 and 4). Thus, these experiments show that MoMuLV anti-Env can immunoprecipitate prion infectivity. MoMuLV Gag as a key factor for PrP release The retroviral Gag precursor protein alone is necessary and sufficient to drive the formation and the release of virus-like particles (VLPs). To examine how MoMuLV infection enhances PrP release, we analyzed the respective contribution of GagPol and Env to this process (Figure 7A). For this purpose, NIH3T3-22L cells were transfected with plasmids encoding MoMuLV-GagPol (lane 2), MoMuLV−Env (lane 3), or GagPol+Env (lane 4, see Supplementary Materials and methods). As negative controls non-transfected NIH3T3-22L cells were used (lane 1). After 3 days, the expression of CAp30/Pr65Gag, Envgp70 and PrP was confirmed by Western blotting (Figure 7A, lanes 1–4). No modification of intracellular PrP expression was observed under these different conditions (bottom panel). The release of VLPs and virions was monitored by the detection of the RT activity in the cell supernatant (Figure 7B). Results indicated that RT activity was only detected in the supernatant of GagPol and GagPol+Env cotransfected cells. As expected, the level of RT activity was high in cotransfected cells due to the presence of replicative infectious virions (Figure 7B). To determine the role of GagPol and Env, or both in PrP release, the cell supernatant was recovered and centrifuged. The 100K pellets were analyzed by Western blotting using the anti-CAp30 and anti-PrP antibodies (Figure 7C). Results indicate that GagPol production enhanced the PrP release (lane 1), whereas Env expression had no effect (compare lane 2 with lanes 1 and 3). As expected, MoMuLV replication in NIH3T3-22L cells was associated with a strong PrP release (compare lanes 1 and 3), suggesting that the formation and release of VLPs are critical for the enhancement of PrP release. Figure 7.MoMuLV Gag as a key factor involved in PrP release. (A) Western blotting analysis of NIH3T3-22L cellular lysate (15 μg) from cells transfected with MoMuLV GagPol, Env and GagPol+En
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