Cells Expressing Anchorless Prion Protein Are Resistant to Scrapie Infection

Cell lines and viral transductions.Anchored and anchorless mouse PrP containing the epitope to mouse monoclonal antibody 3F4 (Mo3F4 GPI⁺ PrP-sen and Mo3F4 GPI⁻ PrP-sen, respectively) have been previously described (22). All clones were expressed in the retroviral vector pSFF, and viral transductions were performed with supernatant harvested from transfected retroviral packaging cells as previously described (18, 20). Mouse fibroblast cells (Ψ2) expressing Mo3F4 GPI⁻ PrP-sen were produced by viral transduction as previously described (21). Virally transduced cells were cloned to isolate a cell clone with high expression levels of Mo3F4 GPI⁻ PrP-sen (Ψ2F3). Fibroblast cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (Invitrogen), 100 U of penicillin (Invitrogen), and 100 μg of streptomycin (Invitrogen).

PrP knockout cells (CF10) were isolated as previously described (13). Knockout cells expressing Mo3F4 GPI⁺ PrP-sen, Mo3F4 GPI⁻ PrP-sen, or wild-type mouse PrP-sen were produced by viral transduction. The CF10 cells expressing Mo3F4 GPI⁻ PrP-sen were cloned to find cells that expressed higher levels of GPI⁻ PrP-sen. The CF10 cells and derivative cell lines were grown in OptiMem (Invitrogen) supplemented with 10% fetal bovine serum, 100 U of penicillin, and 100 μg of streptomycin.

Infection of cells with 22L scrapie.When passaged into wild-type mice, 22L from a mouse expressing GPI⁻ PrP-sen (10) is indistinguishable from wild-type 22L (Brent Race, personal communication). Based upon the detection of PrP-res at early passages, it also infects cells more efficiently in vitro than 22L scrapie from a wild-type mouse (data not shown). Thus, 22L scrapie from transgenic mice expressing GPI⁻ PrP-sen was used for all experiments with CF10 cells. The Ψ2F3 fibroblast cells were infected with 22L scrapie from a wild-type mouse.

Cells were plated at a density of 0.5 × 10⁵ per well into 24-well plates and allowed to attach overnight. The medium was then removed from the cells, and 200 μl of a 1:10 dilution (in OptiMem) of a 10% (wt/vol) 22L scrapie-infected brain homogenate was added to the cells for 4 h of incubation at 37°C, after which 400 μl of OptiMem containing 10% fetal bovine serum, 100 U of penicillin, and 100 μg of streptomycin was added to the cells. The cells were incubated for an additional 92 h at 37°C.

For detection of PrP-res in the cell lysates, medium was removed and cells at 96 h postinfection were lysed in 250 μl of lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA). Lysates were centrifuged at 10,600 × g for 5 min at 4°C. Supernatants were saved and stored at −20°C.

PK treatment of lysates and supernatants.All samples to be analyzed for PrP-res were incubated with proteinase K (PK; Roche) at a final concentration of 20 μg/ml for 1 h at 37°C. The reaction was stopped by addition of Pefablock (Roche) at a final concentration of 1 mM immediately after incubation with PK. Ice-cold methanol (four volumes) was added to each sample to precipitate the proteins, and samples were incubated at −20°C overnight. Samples were then centrifuged at 20,800 × g for 30 min at 4°C to pellet the precipitated proteins. The supernatant was removed, and the pellets were allowed to air dry. Protein pellets were sonicated in sample buffer (2.5% sodium dodecyl sulfate, 2 mM EDTA, 2% β-mercaptoethanol, 5% glycerol, 0.02% bromphenol blue, 63 mM Tris-HCl) with a cup-horn sonicator (Misonex).

Western blot analysis.Cells were lysed in lysis buffer and centrifuged at 10,600 × g for 5 min at 4°C to remove cell debris. For analysis of total PrP, 10% of the lysate was mixed with an equal volume of 2× sample buffer. For PrP-res analysis, samples were treated with PK as described above. All lysates were boiled for 5 min before loading. Protein lysates were subjected to gel electrophoresis on a 16% Tris-glycine Novex minigel (Invitrogen), transferred to polyvinylidene difluoride Immobilon-P membrane (Millipore), and probed with either a 1:3,000 dilution of mouse monoclonal antibody 3F4 or a 1:10,000 dilution of rabbit polyclonal antibody R30 (15, 25). Proteins were visualized with the enhanced chemiluminescence Western blotting detection reagent (GE Healthcare).

Detection of scrapie infectivity in passaged cells.Following exposure to scrapie-infected brain homogenate, cells were continuously cultured for a minimum of 12 passes to dilute out the original inoculum. Cells to be inoculated into Tga20 mice (12) were plated in 75-cm² flasks and allowed to grow to near confluence. Cells were harvested by treatment with phosphate-buffered saline-EDTA and pelleted by centrifugation at 524 × g for 5 min at 4°C. Supernatant was removed, and cell pellets were gently resuspended in OptiMem and 2% fetal bovine serum (FBS). Cells were stored at −80°C. Before inoculation into Tga20 mice, cells were thawed on ice and subjected to three freeze/thaw cycles. The solution was passed first through a 20-gauge needle and then through a 24-gauge needle and then heated at 70°C for 10 min. The solution was diluted 1:1 in phosphate-buffered balanced salt solution containing 2% FBS for injection, and 50 μl of inoculum was injected intracerebrally per mouse.

Cell supernatants were prepared by plating cells in 25-cm² flasks. When the cells were approximately 70% confluent, medium was removed and 2 ml of OptiMem containing 2% FBS was added to each flask. Cells were incubated overnight at 37°C. Supernatants were then harvested, centrifuged at 524 × g for 5 min at 4°C to remove cells, and stored at −80°C. Supernatants were thawed on ice, and 50 μl was injected intracerebrally per mouse. All animal experiments were approved by the Rocky Mountain Laboratory Animal Care and Use Committee. The Rocky Mountain Laboratories are fully accredited by the American Association for Laboratory Animal Care.

Metabolic labeling and immunoprecipitation of PrP-sen.Cells were plated into 25-cm² flasks so that they would be approximately 60 to 70% confluent the following day. Medium was removed from each flask, and the cells were washed three times with phosphate-buffered balanced salt solution. Methionine- and cysteine-free RPMI medium containing 1% dialyzed FBS and l-glutamine (2 mM) was added, and the cells were incubated at 37°C for 1 h. trans-[³⁵S]methionine/cysteine (150 μCi; Perkin-Elmer) was added, and the cells were incubated for 2 h at 37°C. Supernatants were removed from each flask and centrifuged to remove cells. A detergent solution (5% NP-40, 0.1 M EDTA, 0.2 M Tris, pH 7.4) containing a mini EDTA-free protease inhibitor tablet (Roche) was added at a 1:10 dilution to each supernatant. Cells were lysed in 1 ml of lysis buffer, and lysates were centrifuged at 10,600 × g for 5 min at 4°C. Ice-cold methanol (four volumes) was added to each lysate to precipitate proteins, and samples were stored at −20°C overnight. Samples were centrifuged to pellet proteins at 20,800 × g for 30 min at 4°C. Supernatants were aspirated, and pellets were sonicated into 1 ml of detergent lipid protein complex buffer (4.2 mg of l-α-phosphatidylcholine per ml, 123 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% N-lauroylsarcosine). Immunoprecipitation of supernatants and lysates was performed as follows. Five microliters of mouse polyclonal antibody R30 was added to immunoprecipitate PrP-sen, and samples were incubated at 4°C overnight. Protein A-Sepharose (PAS) was added, and samples were incubated at 4°C for 1 h with end-over-end rocking. Samples were centrifuged to pellet the PAS beads. The PAS beads were washed three times in wash buffer (50 mM Tris, 500 mM NaCl, 1% N-lauroylsarcosine, pH 7.0) and one time in distilled H₂O. Final pellets were resuspended in sample buffer without β-mercaptoethanol and boiled for 5 min before being subjected to gel electrophoresis. Radiolabeled proteins were visualized by autoradiography.

Expression of Mo3F4 PrP-sen in CF10 cells.To study the role of the PrP-sen GPI anchor in scrapie infection, we expressed Mo3F4 GPI⁻ or Mo3F4 GPI⁺ PrP-sen in cells which contained no endogenous PrP-sen (CF10 cells). Western blot analysis was performed on cell lysates and supernatants harvested from each cell type to confirm the expression of the different forms of Mo3F4 PrP-sen. As expected, the CF10 cells alone showed no evidence of PrP-sen expression in either the cell lysate (Fig. 1A, lane 1) or the supernatant (Fig. 1A, lane 2). Lysates from cells expressing Mo3F4 GPI⁺ PrP-sen showed the three major glycosylated forms of GPI⁺ PrP-sen (Fig. 1A, lane 5), with very little detected in the supernatant (Fig. 1A, lane 6). Cells expressing Mo3F4 GPI⁻ PrP-sen showed mostly unglycosylated PrP-sen in the cell lysate (Fig. 1A, lane 3) and both un- and monoglycosylated forms in the supernatant (Fig. 1A, lane 4). There was no detectable decrease in the expression levels of either Mo3F4 GPI⁺ PrP-sen or Mo3F4 GPI⁻ PrP-sen after more than 50 passages, indicating that the proteins were stably expressed (data not shown).

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FIG. 1.

PrP-sen expression and acute PrP-res formation in CF10 cells. (A) Western blot analysis of PrP knockout (CF10) cells expressing either Mo3F4 GPI⁻ PrP-sen or Mo3F4 GPI⁺ PrP-sen. CF10 knockout cells do not express PrP-sen (lanes 1 and 2). CF10 cells expressing Mo3F4 GPI⁻ PrP sen express it in the cell lysate (L, lane 3) and secrete it into the cell supernatant (S, lane 4). Cells expressing Mo3F4 GPI⁺ PrP-sen express it in the cell lysate (L, lane 5), with very little GPI⁺ PrP-sen being detected in the supernatant (S, lane 6). The three glycoforms of Mo3F4 GPI⁺ PrP-sen are represented by the letters u (unglycosylated), m (monoglycosylated), and d (diglycosylated). (B) Western blot analysis of PrP-res from cells exposed to scrapie for 96 h. Cells expressing Mo3F4 GPI⁻ PrP-sen or Mo3F4 GPI⁺ PrP-sen were positive for newly made PrP-res (lanes 1 and 2, respectively). The Western blots were probed with mouse monoclonal antibody 3F4. The lower molecular weight of Mo3F4 GPI⁻ PrP is consistent with lack of the GPI anchor. The values on the left of each panel are molecular sizes in kilodaltons.

Acute formation of PrP-res in CF10 cells expressing Mo3F4 PrP-sen.To assess whether CF10 cells expressing Mo3F4 GPI⁻ or Mo3F4 GPI⁺ PrP-sen could produce PrP-res, the cells were exposed to a 22L scrapie-infected brain homogenate from a mouse expressing GPI⁻ PrP-sen. After 96 h, cell lysates were harvested and analyzed for newly converted PrP-res by Western blotting with the 3F4 antibody. The parental CF10 cells showed no detectable 3F4-positive PrP-res in the cell lysate (Fig. 2A and data not shown). Both the cells expressing Mo3F4 GPI⁻ PrP-sen and the cells expressing Mo3F4 GPI⁺ PrP-sen had newly converted PrP-res in the cell lysate (Fig. 1B, lanes 1 and 2, respectively). While Mo3F4 GPI⁺ PrP-res was normally detected after 96 h, detection of Mo3F4 GPI⁻ PrP-res was variable and occurred in ∼40% of the experiments performed (three of eight). Mo3F4 PrP-res was not detected in the supernatant of either cell type (data not shown). Thus, both Mo3F4 GPI⁻ and Mo3F4 GPI⁺ PrP-sen were converted to PrP-res acutely within the first 96 h of exposure to 22L scrapie.

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FIG. 2.

Persistent scrapie infection requires the PrP-sen GPI anchor. Western blot analysis of PrP-res from cells that were exposed to 22L scrapie. After 96 h, cells were harvested and then replated for continual passage. Pass 1 refers to the first passage after replating. Each passage was lysed, and lysates were treated with 20 μg/ml PK for 1 h at 37°C to remove PrP-sen. The membrane was probed with mouse monoclonal antibody 3F4. Only CF10 cells expressing Mo3F4 GPI⁺ PrP-sen produced PrP-res at all of the passes tested after exposure to 22L scrapie. (A) CF10 cells. (B) CF10 cells expressing Mo3F4 GPI⁺ PrP-sen. (C) CF10 cells expressing Mo3F4 GPI⁻ PrP-sen. The values on the left of each panel are molecular sizes in kilodaltons.

Persistent scrapie infection in CF10 cells expressing Mo3F4 PrP-sen.To determine if CF10 cells expressing GPI⁻ or GPI⁺ Mo3F4 PrP-sen were able to support a persistent TSE infection, cells that were acutely exposed to 22L scrapie for 96 h were subsequently passaged. At each passage, cells were lysed and lysates were analyzed by Western blotting for the presence of newly formed PrP-res. As expected, the CF10 cells were negative for PrP-res at all passes (Fig. 2A). The cells expressing Mo3F4 GPI⁺ PrP-sen produced PrP-res at all passes, suggesting that these cells were able to support a persistent TSE infection (Fig. 2B). Despite the early formation of anchorless PrP-res (Fig. 1B), once the CF10 cells expressing Mo3F4 GPI⁻ PrP-sen were passaged, no PrP-res was detected, suggesting that these cells were unable to sustain PrP-res formation over multiple passages (Fig. 2C). The inability of the cells expressing Mo3F4 GPI⁻ PrP-sen to support a persistent TSE infection, in contrast to cells expressing Mo3F4 GPI⁺ PrP-sen, emphasizes the importance of the GPI anchor for long-term PrP-res accumulation in vitro.

Detection of scrapie infectivity in passaged cells.The reduction in Mo3F4 GPI⁻ PrP-res in the passaged cells compared with the acutely exposed cells suggested that the GPI anchor was necessary for persistent TSE infection in vitro. However, previous work has shown that PrP-res is not always detectable in scrapie-infected cells by Western blotting (24). To test whether these cells were infected despite the lack of detectable GPI⁻ PrP-res, we inoculated the cells into Tga20 mice, which overexpress mouse PrP-sen and are highly susceptible to mouse scrapie (12). To ensure that the original inoculum was no longer present in the cell culture, all of the cell lines inoculated into the mice were passaged more than 12 times.

None of the uninfected cells caused disease in Tga20 mice (Table 1). As a positive control, brain homogenate from a mouse expressing GPI⁻ PrP-sen infected with 22L was inoculated into Tga20 mice. These mice died of scrapie, with an average incubation time of 87 ± 8 days (Table 1, line 1). Mice inoculated with the parental CF10 cells exposed to the same 22L homogenate have not shown clinical signs of scrapie infection (Table 1, line 2). In contrast, mice inoculated with 22L-infected CF10 cells expressing Mo3F4 GPI⁺ PrP-sen died of scrapie with an average incubation time of 148 ± 47 days (Table 1, line 4). Supernatant from these cells has not transmitted disease to mice (>278 days, Table 1, line 7), suggesting that any infectivity in the supernatant is present at much lower levels than in the cells. Mice inoculated with the 22L-exposed cells expressing Mo3F4 GPI⁻ PrP-sen have not shown clinical signs of scrapie infection for greater than 574 days (Table 1, line 3), nor has the supernatant from these cells caused disease for greater than 490 days (Table 1, line 6). These data strongly suggest that CF10 cells expressing Mo3F4 GPI⁻ PrP-sen were not persistently infected following exposure to 22L scrapie.

Coexpression of wild-type mouse PrP-sen in the cells expressing Mo3F4 GPI⁻ PrP-sen.The striking difference between the cells expressing Mo3F4 GPI⁻ or Mo3F4 GPI⁺ PrP-sen in their ability to support a persistent scrapie infection suggested that while the GPI anchor is not required for initial PrP-res formation in cells, it is critically important for the establishment of a long-term infection. To test whether expression of an anchored form of PrP-sen would alter the susceptibility of the cells expressing GPI⁻ PrP-sen to scrapie infection, we expressed wild-type mouse PrP-sen (MoPrP GPI⁺) in the cells expressing Mo3F4 GPI⁻ PrP-sen. If the anchor is necessary, the presence of a GPI-anchored form of PrP-sen should allow the formation of PrP-res and establishment of a persistent infection.

Cells expressing Mo3F4 GPI⁻ PrP-sen were stably transduced with wild-type mouse PrP-sen. To determine if the cells were expressing both types of mouse PrP-sen, they were labeled with [³⁵S]methionine/cysteine. PrP-sen was immunoprecipitated from the cell lysate and supernatant with the R30 antibody and analyzed by size migration of the ³⁵S-labeled proteins. No PrP-sen was immunoprecipitated from the CF10 cell lysate or supernatant (Fig. 3, lanes 1 and 2, respectively). Immunoprecipitated PrP-sen from cells expressing Mo3F4 GPI⁺ PrP-sen showed the characteristic glycosylation pattern expected for wild-type PrP-sen in the cell lysate (Fig. 3, lane 3), with little or no GPI⁺ PrP-sen detected in the cell supernatant (Fig. 3, lane 4). Cells expressing only Mo3F4 GPI⁻ PrP-sen showed unglycosylated GPI⁻ PrP-sen, as well as a less abundant band representing monoglycosylated GPI⁻ PrP-sen, in the cell lysate (Fig. 3, lane 5). Both of these forms of PrP-sen could also be detected in the cell supernatant, indicating that they were secreted (Fig. 3, lane 6). The lower-molecular-weight species in the supernatant are likely GPI⁻ PrP-sen degradation products. The cells expressing Mo3F4 GPI⁻ PrP-sen that were transduced with wild-type PrP-sen express both Mo3F4 GPI⁻ PrP-sen and MoPrP GPI⁺ PrP-sen in the cell lysate (Fig. 3, lane 7). However, only Mo3F4 GPI⁻ PrP-sen was detected in the cell supernatant (Fig. 3, lane 8). Thus, these cells express both the Mo3F4 GPI⁻ and MoPrP GPI⁺ PrP-sen proteins.

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FIG. 3.

Coexpression of MoPrP GPI⁺ PrP-sen in cells expressing Mo3F4 GPI⁻ PrP-sen. Cells were radiolabeled with [trans-³⁵S]methionine/cysteine, and PrP-sen was immunoprecipitated from cell lysates or supernatants with the R30 antibody. CF10 knockout cells showed no PrP-sen in either the lysate or the supernatant (lanes 1 and 2, respectively). Cells expressing Mo3F4 GPI⁺ PrP-sen show the characteristic glycoforms (u, unglycosylated; m, monoglycosylated; d, diglycosylated) of PrP-sen in the cell lysate (lane 3) but not in the cell supernatant (lane 4). Cells expressing Mo3F4 GPI⁻ PrP-sen show unglycosylated and monoglycosylated forms of Mo3F4 GPI⁻ PrP-sen in the cell lysate (lane 5). These two glycoforms are also observed in the cell supernatant (lane 6). The lower-molecular-weight products in the supernatant are likely degradation products of PrP-sen. Cells expressing Mo3F4 GPI⁻ PrP-sen that were transduced with MoPrP GPI⁺ PrP-sen express Mo3F4 GPI⁻ PrP-sen like the parental cells in both the lysate (lane 7) and the cell supernatant (lane 8) but also express GPI⁺ PrP-sen in the cell lysate (lane 7). L, cell lysate; S, cell supernatant.

Persistent scrapie infection of CF10 cells coexpressing the Mo3F4 GPI⁻ and MoPrP GPI⁺ PrP-sen proteins.To test if the CF10 cells expressing both the Mo3F4 GPI⁻ and MoPrP GPI⁺ forms of mouse PrP-sen were susceptible to persistent scrapie infection, we exposed them to 22L scrapie and analyzed them for the ability to produce PrP-res over multiple passes. In contrast to the cells expressing Mo3F4 GPI⁻ PrP-sen alone (Fig. 2C), the cells expressing both forms of PrP-sen became stably infected over time, as indicated by the presence of PrP-res at all passes (Fig. 4A). Moreover, Western blot analysis with the 3F4 antibody clearly demonstrated the presence of Mo3F4 GPI⁻ PrP-res at late passes, which was not seen in the cells expressing only Mo3F4 GPI⁻ PrP-sen (compare Fig. 4B to Fig. 2C). These results indicate that coexpression of GPI-anchored wild-type PrP-sen enabled the cells expressing anchorless PrP-sen to persistently produce both anchored and anchorless PrP-res and strongly suggest that the cells are persistently infected with 22L scrapie.

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FIG. 4.

Persistent infection of cells coexpressing Mo3F4 GPI⁻ and MoPrP GPI⁺ PrP-sen. Western blot analysis of PrP-res from PK-treated cell lysates of 22L-exposed cells with the R30 antibody (A) or the 3F4 antibody (B) is shown. Both MoPrP GPI⁺ and Mo3F4 GPI⁻ PrP-res were produced at late passes. Note that the reduced signal in passages 1, 2, 9, and 10 is reflective of fewer cells at the time of harvest. The values on the left of each panel are molecular sizes in kilodaltons.

To determine if endogenous levels of wild-type GPI⁺ PrP-sen would also enable cells expressing anchorless PrP-sen to be infected with scrapie, Ψ2 fibroblast cells, which express endogenous mouse PrP-sen, were virally transduced to express Mo3F4 GPI⁻ PrP-sen. Following exposure to 22L scrapie, Mo3F4 GPI⁻ PrP-res was detected in the cell lysate (Fig. 5, lane 1) but not in the cell supernatant (Fig. 5, lane 2). Analysis with the R30 antibody showed both GPI⁻ and GPI⁺ PrP-res in the cell lysate (Fig. 5, lane 3) but not in the supernatant (Fig. 5, lane 4). Inoculation of the fibroblast cells into Tga20 mice caused disease in 110 ± 9 days, confirming that the cells were persistently infected with scrapie (Table 1, line 5). Taken together, these results demonstrate that expression of an anchored form of PrP-sen allows the persistent formation of anchorless PrP-res, even when the anchored form of PrP-sen is expressed at endogenous levels.

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FIG. 5.

Fibroblast cells expressing Mo3F4 GPI⁻ PrP-sen can be persistently infected with 22L scrapie. Mouse fibroblast cells expressing both endogenous mouse PrP-sen and Mo3F4 GPI⁻ PrP-sen were exposed to 22L scrapie and passaged. Cell lysates and supernatants were analyzed for PrP-res with the 3F4 antibody (A) to detect Mo3F4 GPI⁻ PrP-res (lanes 1 and 2) and the R30 antibody (B) to detect total PrP-res (lanes 3 and 4). Both GPI⁻ and GPI⁺ PrP-res were present in the cell lysate but not in the supernatant. L, cell lysate; S, cell supernatant. The band below 37 kDa is a nonspecific band that cross-reacts with the ECL anti-mouse secondary antibody. The values on the left of panel A and the right of panel B are molecular sizes in kilodaltons.