Prion strain mutation determined by prion protein conformational compatibility and primary structure

Abstract

Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

Fig. 1

Primary transmission of deer and elk CWD prions to Tg(CerPrP)1536^+/− mice. (A) Blue and red circles, mice with CWD1 and CWD2 patterns of neuropathology, respectively, analyzed by IHC or histoblotting; open circles, mice not analyzed; gray circle, ambiguous strain pattern; squares, asymptomatic mice. Animal identification codes are shown on the x axis. Total CWD1 and CWD2, total number of mice with each strain pattern. Incubation time differences between groups were established using an unpaired, two-tailed t test (p < 0.0001). d, days. (B) Summary of strain profiles after primary passage of elk and deer. The mean (± SD) incubation times and numbers of all mice in each category are shown. The low titer CWD1 012-22012 elk isolate was excluded from the summary. (C) Neuronal vacuolation was quantified in nine brain regions: 1, medulla; 2, cerebellum; 3, midbrain; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, paraterminal body; 8, cerebral cortex at hippocampus; and 9, cerebral cortex at septum. Blue circles, eight mice with rapid incubation periods; red circles, seven mice with prolonged incubation periods; open circles, uninoculated, age-matched mice. Error bars represent the average number of vacuoles per field ± SEM. Differences were established using an unpaired, two-tailed t test; asterisks indicate the degree of significance.

Fig. 2

Serial transmission of deer and elk CWD prions to Tg(CerPrP)1536^+/− mice. (A) Symbols are the same as in Fig. 1A. Animal identification codes are shown on the x axis. Asterisks indicate the degree of significance. (B) Summary of strain profiles on secondary passage. The mean (± SD) incubation times and numbers of mice in each category are shown. (C) Six representative transmissions demonstrating instability of CWD1 and CWD2. For each transmission, the single circles represent diseased mice after the first passage of CWD isolates (p1); boxed symbols represent mice resulting from serial passage of those brains (p2).

Fig. 3

Representative distributions of CerPrP^Sc in the brains of diseased Tg(CerPrP)1536^+/− mice. Sections encompassing the hippocampus and cortex were analyzed by IHC using anti-PrP mAb 6H4 (A, B, E, and F) or by using antibodies against glial fibrillary acidic protein (C and G). (D and H) CerPrP^Sc distribution was evaluated by histoblotting of coronal sections of similar regions. (I) Asymmetrical distribution of cortical florid plaques and associated neuronal vacuolation in CWD2-infected mice. Scale bars in (A) and (E), 1 mm; in (B), 50 µm; in (C), (F), (G), and (I), 100 mm. Isolate passage number (p) and incubation times of mice are shown (d, days).

Fig. 4

Biochemical properties of CerPrP^Sc associated with CWD1 and CWD2. (A) Immunoblots probed with mAb 6H4. (B) Immunoblots of samples from mice infected with CWD1 or CWD2 probed with mAbs as indicated. Samples were treated as indicated with proteinase K (PK). The bands on immunoblots show the various glycoforms of proteaseresistant CerPrP^Sc and, in samples from phosphate-buffered saline–inoculated mice not treated with PK, CerPrP^C. Positions of protein molecular mass markers at 37, 25, and 20 kD (top to bottom) are shown. (C) Ratio of CerPrP^Sc glycoforms. Data points represent the mean (± SD) relative proportions from three or four animals of di-(purple squares), mono-(green squares), and unglycosylated (yellow squares) CerPrP^Sc. Aggregated values for all mice exhibiting CWD1 or CWD2 strain properties are also shown. n, number of analyzed mice of each strain type. (D) Percentage of CerPrP^Sc as a function of Gdn.HCl concentration in 17 mice infected with CWD1 (blue circles) and 20 mice affected with CWD2 (red circles). (Gdn.HCl)_1/2 values are shown for Tg(CerPrP)1536^+/− mice. Tg(CerPrP-E226)5037^+/− mice were infected with elk isolates that produced CWD1 (12389, blue squares) or CWD2 (04-0306, red squares). Fapp, fraction of apparent PK-resistant PrP^Sc = (maximum signal – individual signal)/(maximum signal – minimum signal); error bars indicate SD of mean values from three animals analyzed in each study group.