Coinfecting prion strains compete for a limiting cellular resource

Abstract

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP(Sc), in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP(Sc) and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP(Sc) does not convert all of the available PrP(C) to PrP(Sc) in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP(C) and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP(Sc) in animals where DY TME had completely blocked HY TME from causing disease.

FIG. 1.

The DY TME agent does not significantly inhibit retrograde transport or cause death of VMNs. Lumbar spinal cord sections from hamsters that were injected in the sciatic nerve with dextran conjugated to Alexa Fluor 568 at 120 days after sciatic nerve inoculation with either an uninfected brain homogenate (A) or DY TME agent (B). Insets in panels A and B depict dextran-positive ventral motor neurons. (C) The number of VMNs containing dextran in the lumbar spinal cord from the DY TME agent infected did not differ significantly (P > 0.05) compared to the negative control animals. The number of VMNs in Nissl-stained sections of lumbar spinal cord collected from hamsters at 120 days after sciatic nerve inoculation with the DY TME agent contralateral (D) or ipsilateral (E) to the side of inoculation did not significantly (P > 0.05) differ (F). The yellow regions in the schematic insets depict the location of the spinal cord that were imaged. Scale bar, 100 μm.

FIG. 2.

PrP^Sc deposition is present in the absence of spongiform degeneration, astrocytosis, microgliosis, and synaptic loss in the lumbar spinal cord at 134 days after DY TME agent inoculation. Hamsters were inoculated in the sciatic nerve with either the DY TME agent or uninfected (Mock) brain homogenate and lumbar spinal cord was collected at 134 days postinfection or at clinical disease. Lumbar spinal cord sections were stained either with hematoxylin and eosin (H&E) or immunohistochemistry was performed using antibodies directed against PrP^Sc, glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba-1) or synaptophysin. The yellow regions in the schematic insets depict the location of the brain that were imaged for each column. Arrows indicate PrP^Sc deposits in the panel E and G insets. Scale bar, 50 μm.

FIG. 3.

Strain-specific characteristics of HY and DY PrP^Sc are recapitulated by PMCA. (A) PMCA reactions were seeded with uninfected (UN), DY TME-infected (DY), or HY TME-infected brain homogenates (HY) and one round of PMCA resulted in detection of PK digestion-resistant PrP^Sc in the DY TME- and HY TME-seeded reactions but not in the negative control uninfected seeded reactions. (B) HY PrP^Sc accumulated to a higher abundance compared to DY PrP^Sc, suggesting that HY TME amplifies more efficiently than DY PrP^Sc. (C) Western blot analysis of PK-digested brain homogenates from HY TME (HY), DY TME (DY), or uninfected hamster (UN) before (lanes 1 to 3, 7, and 8) or after PMCA (lanes 4 to 6, 9, and 10) treated without (1-6) or with (7-10) PNGase indicates that the strain-specific migration of HY TME and DY TME PrP^Sc is maintained after PMCA. The migration of the 19- and 21-kDa unglycosylated PrP^Sc polypeptide is indicated on the left of the panel. (D) Survival of Syrian hamsters after i.c. inoculation of HY TME agent (n = 5, ⧫), DY TME agent (n = 5, ⋄), or the tenth serial PMCA round of HY TME (n = 5, ▪)-, DY TME (n = 4, □)-, or mock (n = 5, •)-seeded reactions.

FIG. 4.

Protein misfolding cyclic amplification strain interference. Known amounts of HY TME-infected and DY TME-infected brain homogenate were combined and added to PMCA reactions, and 10 rounds of serial PMCA were performed. (A) After four rounds of serial PMCA, Western blot analysis determined that PrP^Sc migration was consistent with HY PrP^Sc when the initial ratio of DY TME to HY TME brain homogenate ranged from 1:1 to 1:1,000, respectively (lanes 3 to 6) and was consistent with DY PrP^Sc when the ratio of DY TME to HY TME brain homogenate was greater than 1:10,000 (lanes 7 to 11). (B) When the ratio of DY to HY TME brain homogenate was 1:10,000, the migration of PrP^Sc was consistent with DY PrP^Sc during the first eight rounds of serial PMCA (lanes 3 to 8) but then changed to a migration pattern consistent with HY PrP^Sc during rounds 9 and 10 (lanes 11 to 12).

FIG. 5.

Persistence of HY TME in animals coinfected with the DY TME and HY TME agents. Four rounds of serial PMCA was performed on brain homogenate from uninfected (UN) hamsters, hamsters infected with either the HY TME agent (HY) or the DY TME agent (DY), or hamsters infected under conditions where the DY TME agent completely blocks the HY TME agent from causing disease. Western blot analysis of PK-digested sample from the uninfected negative control PMCA indicated that it did not amplify PrP^Sc (lane 3). The HY TME and DY TME positive control PMCA reactions amplified PrP^Sc that maintained the 21- and 19-kDa strain-specific PrP^Sc migration pattern (lanes 4 and 5, respectively). In the group of superinfected hamsters, one round of PMCA results in a 19-kDa PrP^Sc migration pattern consistent with DY PrP^Sc (compare lanes 5 and 6). After four rounds of serial PMCA, the PrP^Sc migration pattern is consistent with HY PrP^Sc (compare lanes 9 to 10). The migration of the 19- and 21-kDa unglycosylated PrP^Sc polypeptide is indicated on the left of the panel.