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Title: Independent amplification of co-infected long incubation period low conversion efficiency prion strains}
Article:
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.
Introduction:
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[18]. Currently, effective treatment for prion diseases is not available, and they are inevitably fatal. The prion agent is comprised mainly, if not entirely, of PrPSc which is an abnormal isoform of the host encoded prion protein, PrPC[19–23]. Prion propagation is thought to occur in a three-step process where PrPSc first binds to PrPC followed by a conformational conversion of PrPC to PrPSc. Next, fragmentation of the growing PrPSc polymer results in the generation of new PrPSc free ends for PrPC to bind. Repeated cycles of this process are thought to encompass prion formation in vivo and are recapitulated in vitro by protein misfolding cyclic amplification( PMCA)[24]. Prion strains are operationally defined as a phenotype of disease under a fixed set of agent and host parameters. Under experimental conditions where these parameters are precisely controlled, distinct phenotypes of disease correspond with prion strains[25–27][28–36]. Differences in the distribution and relative intensity of spongiform degeneration in select areas of the central nervous system( CNS) are, currently, the most well-accepted criteria to distinguish strains[37, 38]. Prion strains can differ in incubation period, clinical signs of disease, tissue tropism, and host range. Importantly, the strain specific phenotype is maintained upon serial passage and is, therefore, heritable. Prion strain diversity may be encoded by distinct conformations of PrPSc. Studies using the hyper( HY) and drowsy( DY) strains of hamster-adapted TME were the first to show that PrPSc could have strain-specific differences in the proteinase K( PK) cleavage site, with the unglycosylated PrPSc polypeptide migrating at 21 and 19 kDa respectively, relative PK resistance and detergent insolubility[39–41]. Consistent with these findings, similar strain-specific differences in PrPSc migration properties of human prion isolates were preserved upon transmission to transgenic mice expressing chimeric mouse-human PrPC[42]. Structural studies of PrPSc using Fourier transform infrared spectroscopy indicated that strain-specific differences in PrPSc secondary structure may underlie strain-specific properties of PrPSc such as PK cleavage site[43, 44]. The conformation dependent immunoassay( CDI) measures changes in immunoreactivity of PrPSc compared to immunoreactivity of PrPC under conditions of increasing denaturation. CDI has identified strain-specific differences in PrPSc conformation from several rodent prion strains[45]. The relationship between the strain-specific biochemical features of PrPSc and the outcome of disease are poorly understood. Prion strains, when present in the same host, can interfere with each other. This was first observed in mice where inoculation of the long incubation period blocking strain, 22C, prior to superinfection with the short incubation period strain, 22A, could extend the incubation period of 22A[46]. Short and long incubation period strains are categorized relative to the minimum and maximum incubation periods of known strains for a given host under experimental transmission parameters. As the interval between inoculation of 22C and 22A is increased, 22C can extend the incubation period or completely block 22A from causing disease[46]. This is consistent with subsequent studies indicating that the relative onset of conversion of the blocking and superinfecting strain influences strain emergence, and conversion of the blocking strain PrPSc corresponds with the occurrence of strain interference[47–49]. Additionally, the blocking and superinfecting strains must infect the same cells for strain interference to occur and potentially compete for PrPC[47, 50]. Prion strain interference is a common property of prions and numerous host strain combinations have been identified where prion strains interfere with each other or completely block one strain from causing disease[16, 46, 51–55]. Most of these studies, however, investigated combinations of prion strains that have large differences in incubation period and/or prion agent conversion efficiencies. It is unknown if two relatively long-incubation period, low efficiency converting prion strains can interfere with each other. In this study we investigated the ability of relatively low prion conversion efficiency strains to interfere with each other in vivo and in vitro and the contribution of PrPC in this process.
Discussion:
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[47]. 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[57]. 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[58]. 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[47]. Low prion conversion efficiency strains amplify independently when present together in PMCA. Strain interference can be recapitulated with PMCA similar to what is observed in vivo[50]. Since both prion strains are present in the same PMCA reaction, this in vitro system mimics when both strains are infecting the same cell in vivo. We found that in PMCAsi the strain that started in greater abundance maintained its strain properties through all 10 rounds of serial PMCAsi, suggesting the other strain was not able to interfere( Tables 2 and 3; Fig 5). Importantly, we found that when both strains started at an equal ratio, a mixture of the 19 and 21 kDa migrating unglycosylated PrPSc polypeptide was maintained for 10 rounds of PMCAsi( Tables 2 and 3; Fig 5). Based on the limitations of Western blot to resolve a mixture of PrPSc from both strains( Fig 1) and the strains used have similar PMCA-CC values( Fig 4) and a new prion strain is not generated( Table 4, Fig 6) we conclude that the strains are amplifying independently. This suggests that the failure of these strain combinations to interfere with each other in vivo is not due to a failure to target the same cell, but instead is an inherent property of the strain combinations. The concept that not all prion strain combinations result in interference suggests that strain diversity in naturally-infected animals may be greater than previously thought. Prion strain interference occurs when PrPC abundance is limiting. Prion formation is dependent on PrPC, and evidence suggests that prion strains compete for PrPC[50, 59]. By definition, therefore, if the amount of available PrPC for prion conversion is limiting, strain interference will occur. We hypothesized that PrPC abundance was not limiting with the long-incubation period, low prion conversion efficiency strains used in this study. To investigate this hypothesis, we used EAI to measure the PK resistant fraction of PrP and the fraction of PrP that binds to the monoclonal anti-PrP antibody 3F4 in the native state, which we interpret as PrPC( Fig 7). We cannot exclude the possibility that other forms of PrP( e. g. PK sensitive PrPSc) that bind to 3F4 in the native state, in addition to PrPC, contribute to the observed results, however, previous reports suggest this is unlikely[60, 61]. Using this assay, we determined the abundance of PrPC and PrPSc in the PMCA reactions before and after amplification, and we found that PrPC levels following one round of PMCA were higher in reactions seeded with either DY, 139H or ME7H compared to PMCA reactions seeded with the relatively higher prion conversion efficiency strain HY( Fig 7). This is consistent with the observation that DY, 139H and ME7H have lower amounts of PrPSc produced after one round of PMCA and lower PMCA-CC( i. e. lower prion conversion efficiencies) compared to HY( Figs 4 and 7). When two low prion conversion efficiency strains are present in PMCA, after one round of PMCA, the level of PrPC is significantly higher compared to reactions seeded with HY( Fig 7). Overall, we conclude that the PrPC requirements of DY, 139H and ME7H are sufficiently low as to not result in PrPC abundance becoming limiting under the conditions tested; therefore, strain interference does not occur. Not all forms of PrPC are equally suitable templates for prion conversion. Recent work suggests that the sialylation state of PrPC N-linked glycosylation can influence prion formation. Importantly, prion strains may convert a subset of PrPC, based on sialylation, more efficiently than other forms of PrPC[62–64]. Based on these findings, it is possible that the subset of PrPC that is convertible by both strains may have a greater influence on prion strain interference than the total PrPC abundance would suggest. In addition to post-translational modifications of PrPC, tissue specific ratios of the C1 and C2 cleavage fragments of PrPC may affect the abundance of convertible PrPC[65–67] and can affect strain emergence. Prion strain interference can affect the emergence of a strain from a mixture. Interspecies transmission can generate new prion strains that, following intraspecies transmission, result in the emergence of a dominant strain[16, 68–73]. Interference between prion strains that compete for limiting PrPC is thought to be an important parameter affecting this process[73]. Consistent with this observation, overexpression of PrPC can lead to the emergence of prion strains that are not identified when PrPC is expressed at physiological levels[74]. Additionally, extraneural prion inoculation can result in the emergence of novel strains, further suggesting that changes in the population of PrPC that is initially infected can result in profound differences in prion strain emergence[75]. This finding is consistent with previous work indicating that the initial population of cells infected by a mixture of strains has a large effect on prion strain emergence[47]. Overall, we hypothesize that the availability of strain-specific convertible PrPC can influence strain interference and alter the emergence of a strain from a mixture.
This is the first example of prion strains that replicate independently in vitro and in vivo . This observation changes the paradigm of the interactions of prion strains and provides further evidence that strains are a dynamic mixture of substrains . Strain interference is influenced by the abundance of PrPC that is convertible by the strains involved . These observations have implications for interspecies transmission and emergence of prion strains from a mixture .