Independent amplification of co-infected long incubation period low conversion efficiency prion strains

Abstract

Prion diseases are caused by a misfolded isoform of the prion protein, PrPSc. Prion strains are hypothesized to be encoded by strain-specific conformations of PrPSc and prions can interfere with each other when a long-incubation period strain (i.e. blocking strain) inhibits the conversion of a short-incubation period strain (i.e. non-blocking). Prion strain interference influences prion strain dynamics and the emergence of a strain from a mixture; however, it is unknown if two long-incubation period strains can interfere with each other. Here, we show that co-infection of animals with combinations of long-incubation period strains failed to identify evidence of strain interference. To exclude the possibility that this inability of strains to interfere in vivo was due to a failure to infect common populations of neurons we used protein misfolding cyclic amplification strain interference (PMCAsi). Consistent with the animal bioassay studies, PMCAsi indicated that both co-infecting strains were amplifying independently, suggesting that the lack of strain interference is not due to a failure to target the same cells but is an inherent property of the strains involved. Importantly PMCA reactions seeded with long incubation-period strains contained relatively higher levels of remaining PrPC compared to reactions seeded with a short-incubation period strain. Mechanistically, we hypothesize the abundance of PrPC is not limiting in vivo or in vitro resulting in prion strains with relatively low prion conversion efficiency to amplify independently. Overall, this observation changes the paradigm of the interactions of prion strains and has implications for interspecies transmission and emergence of prion strains from a mixture.

Fig 1. Western blot migration analysis of PrP^Sc from 139H, DY and ME7H-infected animals.

Western blot (A) and migration analysis (B, C) of PrP^Sc from 139H, DY or ME7H-infected PK digested brain homogenates at ratios of 1:10, 1:1, or 10:1. In lanes 1, 2, 8, and 9 the unglycosylated PrP^Sc polypeptide migrates at 21 kDa and in lanes 4, 5, and 6 the unglycosylated PrP^Sc polypeptide migrates at 19 kDa. In lane 3 and 7 the migration of the unglycosylated PrP^Sc polypeptide migrates from 19 kDa and 21 kDa. This experiment was repeated a minimum of three times with similar results.

Fig 2. Effect of prion infection on weight gain over the incubation period of disease.

Hamsters were inoculated with either uninfected brain homogenate (blue triangles), DY-infected brain homogenate (orange diamonds), 139H-infected brain homogenate (gray circles) or an equal mixture of 139H and DY-infected brain homogenate (yellow squares) and their weights were recorded weekly. Solid grey triangle and open yellow triangle indicates when the weight of 139H-infected or 139H/DY co-infected hamsters, respectively, significantly (p<0.05) differed from mock-infected animals.

Fig 3. Western blot migration of 139H and DY interference or ME7H and DY interference animal bioassay.

Western blot (A) and migration analysis (B, C) of PK digested brain material from hamsters inoculated with either uninfected (UN) brain homogenate, DY-infected brain homogenate, 139H-infected brain homogenate, ME7H-infected brain homogenate or an equal mixture of either DY and 139H or DY and ME7H. In lanes 1 and 7 the unglycosylated PrP^Sc polypeptide of the DY positive controls migrates at 19 kDa. In lanes 2, 3, and 6 the unglycosylated PrP^Sc polypeptide of the 139H and ME7H-infected positive controls migrates at 21 kDa. In lane 8 the migration of the unglycosylated PrP^Sc polypeptide from the animals co-infected with DY and ME7H migrates from 19 to 21 kDa.

Fig 4. DY, 139H, and ME7H have similar PMCA conversion coefficients.

Western blot (A) detection of PrP^Sc from PK digested PMCA reactions seeded with serial dilutions of either DY, 139H or ME7H-infected brain homogenate after one round of PMCA. The PMCA-CC of DY, 139H, or ME7H seeded reactions (B) did not significantly (p>0.05) differ. This experiment was repeated a minimum of three times with similar results.

Fig 5. Low conversion efficiency prion strains do not interfere in PMCA.

Western blot (panels A, B, E, F) and PrP^Sc migration analysis (panels C, D, G, H) of PMCAsi reactions seeded with either DY and 139H (A through D) or DY and ME7H-infected brain homogenate (E through H). Shown is PMCAsi round 4 with the tested ratios of DY and 139H (panels A and C) or DY and ME7H (panels E and G) or PMCAsi rounds 1 through 10 of and equal mixture of DY and 139H (panels B and D) or DY and ME7H (panels F and H). This experiment was repeated a minimum of three times with similar results.

Fig 6. Preservation of strain specific PrP^Sc migration following PMCA.

Western blot analysis (A) and PrP^Sc migration analysis (B) of PK digested brain material from hamsters inoculated with either 10^th serial round PMCA reactions seeded with uninfected (UN) brain homogenate, DY, 139H or an equal mixture of DY and 139H-infected brain homogenate.

Fig 7. PrP^C abundance is not limiting for low conversion efficiency prion strains in PMCAsi.

Epitope accessibility immunoassay measurement of the fold change of PrP^C (blue squares) and PrP^Sc (red circles) after one round of PMCA or PMCAsi from reactions seeded with either uninfected (UN), HY-infected, DY-infected, 139H-infected, ME7H-infected, or co-infected with DY and 139H scrapie-infected, or DY and ME7H-infected brain homogenates. Error bars represent SEM. The experiment was repeated a minimum of three times with similar results.