Cells expressing anchorless prion protein are resistant to scrapie infection

Abstract

The hallmark of transmissible spongiform encephalopathies (TSEs or prion diseases) is the accumulation of an abnormally folded, partially protease-resistant form (PrP-res) of the normal protease-sensitive prion protein (PrP-sen). PrP-sen is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. In vitro, the anchor and the local membrane environment are important for the conversion of PrP-sen to PrP-res. In vivo, however, the anchor is not necessary because transgenic mice expressing anchorless PrP-sen accumulate PrP-res and replicate infectivity. To clarify the role of the GPI anchor in TSE infection, cells expressing GPI-anchored PrP-sen, anchorless PrP-sen, or both forms of PrP-sen were exposed to the mouse scrapie strain 22L. Cells expressing anchored PrP-sen produced PrP-res after exposure to 22L. Surprisingly, while cells expressing anchorless PrP-sen made anchorless PrP-res in the first 96 h postinfection, no PrP-res was detected at later passes. In contrast, when cells expressing both forms of PrP-sen were exposed to 22L, both anchored and anchorless PrP-res were detected over multiple passes. Consistent with the in vitro data, scrapie-infected cells expressing anchored PrP-sen transmitted disease to mice whereas cells expressing anchorless PrP-sen alone did not. These results demonstrate that the GPI anchor on PrP-sen is important for the persistent infection of cells in vitro. Our data suggest that cells expressing anchorless PrP-sen are not directly infected with scrapie. Thus, PrP-res formation in transgenic mice expressing anchorless PrP-sen may be occurring extracellularly.

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.

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.

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.

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.

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.