Date Published: October 18, 2018
Publisher: Public Library of Science
Author(s): Thomas E. Eckland, Ronald A. Shikiya, Jason C. Bartz, Surachai Supattapone.
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.
Prion diseases are a group of transmissible neurodegenerative diseases that affect animals, including humans. Animal prion diseases include scrapie in sheep and goats, transmissible mink encephalopathy (TME) in ranch-raised mink, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy [1–10]. The human prion diseases can be acquired, inherited, or can occur sporadically and include Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease, fatal familial insomnia, and kuru [11–15]. Prion diseases have long asymptomatic incubation periods ranging from months to decades followed by a short symptomatic phase characterized by progressive cognitive and/or motor deficits [16,17]. During the asymptomatic phase, prions can be detected in the central nervous system and extraneural locations . Currently, effective treatment for prion diseases is not available, and they are inevitably fatal.
Mixtures of DY and 139H or DY and ME7H have differentiable PrPScWestern blot migration profiles. To determine the strain-specific migration of PrPSc from mixtures of strains, Western blot analysis was performed on mixtures of DY and 139H or DY and ME7H brain homogenates at ratios of 10:1, 1:1 and 1:10 (Fig 1). Samples that contained an excess of one strain had unglycosylated PrPSc polypeptide migration of the excess strain at either 21 or 19 kDa (Fig 1). Samples that contained an equal ratio of DY and 139H or DY and ME7H resulted in the unglycosylated PrPSc polypeptide migrating at 21 kDa and 19kDa (Fig 1A) that PrPSc migration analysis confirms as a band migrating from 21 to 19 kDa (Fig 1B and 1C). Overall, a mixture of DY and 139H or DY and ME7H resulted in a dual unglycosylated PrPSc polypeptide pattern that was resolved by Western blot if the ratio of the two strains was within 10-fold of each other.
Previous strain interference studies have examined the capacity of a long-incubation period strain that have low prion conversion efficiencies to interfere with a short-incubation strains that have relatively higher prion conversion efficiencies [46,51–55]. To investigate if two long-incubation period low conversion efficiency prion strains can interfere with each other we co-infected hamsters with DY and 139H or ME7H. These three strains have similar relatively low prion conversion efficiencies (Fig 4). Additionally, 139H and ME7H were chosen because 139H has a shorter incubation period compared to DY and the incubation period of ME7H is much longer. In addition, all three of these strains have a longer incubation period compared with short-incubation period hamster strains such as HY. We found that co-infection of hamsters with DY in combination with either 139H or ME7H resulted in animals developing clinical signs of disease with an incubation period comparable to animals inoculated with the shorter incubation period strain alone (Table 1), suggesting that strain interference was not occurring. Additionally, in the animals co-infected with DY and ME7H, we found evidence that the PrPSc in these animals contained a mixture of DY and ME7H PrPSc (Fig 3), providing evidence of independent strain amplification. Based on these in vivo experiments, however, we cannot exclude the possibility that DY and ME7H are not infecting the same population of neurons that is needed for strain interference to occur . For example, i.c. inoculation of hamsters with 139H scrapie prior to superinfection with Sc237 scrapie results in animals developing clinical signs, pathology and an incubation period similar to animals inoculated with Sc237 alone . This data suggests that 139H is unable to interfere with Sc237 prions. Recent work using the sciatic nerve route of inoculation (i.sc.) indicates that 139H can block HY, a strain that is similar to Sc237, from causing disease . HY and 139H initially replicate in ventral motor neurons (VMNs), following i.sc. inoculation, suggesting that the previous report of 139H and Sc237 failing to interfere with each other was not an inherent property of the strains, but rather was due to a failure to target both strains to the same location of prion conversion. We were unable to perform strain interference super-infection studies with these combinations of strains using i.sc. inoculation due to constraints on the relationship of incubation period of disease, the relative onset of PrPSc formation in VMNs and the lifespan of the host. To overcome this obstacle, we used PMCA to further examine strain interference between these long incubation period, low conversion efficiency prion strains since the relative onset of prion conversion of the strains governs which strain will emerge, independent if the strains were co-infected or super-infected .