Darwinian evolution of prions in cell culture

Abstract

Prions are infectious proteins consisting mainly of PrP(Sc), a beta sheet-rich conformer of the normal host protein PrP(C), and occur in different strains. Strain identity is thought to be encoded by PrP(Sc) conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating "mutants," and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, "cell-adapted" prions outcompeted their "brain-adapted" counterparts, and the opposite occurred when prions were returned from cells to brain. Similarly, the inhibitor swainsonine selected for a resistant substrain, whereas, in its absence, the susceptible substrain outgrew its resistant counterpart. Prions, albeit devoid of a nucleic acid genome, are thus subject to mutation and selective amplification.

Fig. 1

Prion characterization by the CPA. Cells (LD9, violet; CAD, brown; R33, green; PK1, light blue; PK1 + 12 μM swa, red) were exposed to the serially diluted samples indicated. The proportion of PrP^Sc-positive cells was plotted against log₁₀ dilution. (A) Brain[22L]-derived prions are swa resistant and R33 competent while those from 22L-infected PK1 cells (about 27 doublings after infection) are swa sensitive and R33 incompetent. (B) Transfer of prions from brain to PK1. PK1 cells were exposed to 10⁻⁴ brain[22L] for 1 day and propagated 9 days (P0); further propagation for 12 1:10 splits yielded P1 to P12. Conditioned media were analyzed on PK1 cells with (red) or without (blue) 2 μg/ml swa, and on R33 cells (green). Infection in presence of 10 μg/ml pentosan polysulfate, which abolishes prion replication, yielded no infectivity, documenting absence of inoculum. (C) Brain homogenates from terminal C57BL/6 mice inoculated with lysates of PK1[22L]_wp cells propagated in absence (a) or presence (b) of swa or with (c) 1% 22L-infected brain homogenate gave the same CPA responses. (d) The very different CPA response of brain RML is shown for comparison.

Fig. 2

Propagation of 22L-infected PK1 cells in swa results in swa-resistant prions. (A) Scheme. PK1[22L]_wp cells were either grown in swa (2 μg/ml) for up to 10 splits “S1-10”, without swa for up to 10 splits “C0-10”, or with swa for 5 splits followed by without swa for 5 splits “SC6-10”. (B) The level of PrP^Sc-positive cells propagated without swa (blue), as assessed by PK-ELISA. (C) Swa susceptibility of secreted prions propagated in the presence or absence of swa. 100× concentrated conditioned medium was assayed on PK1 cells in the presence (red) or absence (blue) of swa. (D) Swa-resistant prions are associated both with PrP^Sc carrying high-mannose glycans (S2-S5) and normally glycosylated PrP^Sc (SC7- SC8). PK1[22L]_wp cell lysates digested with PK and Endo H were subjected to immuno blotting with anti-PrP. After the first split in swa PrP bands shift to higher mobility reflecting inhibition of complex glycosylation, and Endo H digestion results in a large mobility increase due to removal of high-mannose glycans. After 2 splits without swa (SC7), normal glycosylation is restored but swa resistance is retained.

Fig. 3

Development of heterogeneity in cloned prion populations. Prions were cloned by end point dilution in cell culture and eight clones were tested for their ability to yield swa-resistant populations after propagation in swa. Values in parentheses indicate number of doublings. (A) PK1 cells were exposed to PK1[22L]_wp conditioned medium for 2 days, leading to infection of about 4% of the cells. Cells were distributed at 0.3, 1, 3, or 8 cells/well (a), along with about 1000 uninfected cells, grown to confluence (7 doublings) and split 1:10 five times (17 doublings) in 96-well plates. 23 of 602 wells scored positive by PK-ELISA. Eight clones were expanded for 7 doublings and all secreted swa-sensitive prions (b, altogether 31 doublings). After propagation in swa for five 1:20 splits, clones 8C4 and 3C6 yielded swa-resistant prions (c2), while the others were cured (c3). Propagation without swa yielded swa-sensitive prions (c1). Three of 6 clones that failed to yield swa-resistant populations after exposure to swa (8A8, 8B4, 8H6) were further passaged for 22 doublings without swa (c1) followed by 22 doublings with swa, whereupon one (8A8) yielded swa-resistant prions (d2) and two (8B4, 8H6) were cured (d3). Details in (17). (B) Swa-resistance was determined by assaying 100× concentrated conditioned medium on PK1 cells with or without swa.