In Situ Photodegradation of Incorporated Polyanion Does Not Alter Prion Infectivity

PLoS Pathogens

Justin R. Piro¹  Surachai Supattapone¹  Brent T. Harris² 

[1] Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, United States of America;Department of Pathology, Dartmouth Medical School, Hanover, New Hampshire, United States of America

关键词: Animal prion diseases;    Oligonucleotides;    Hamsters;    Nucleic acids;    Light pulses;    Scrapie;    Cofactors (biochemistry);    Gel electrophoresis;   

DOI  :  10.1371/journal.ppat.1002001

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrPSc aggregates in vivo [1], [2]. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrPSc, we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrPC substrate, PC-oligo was incorporated into physical complexes with PrPSc molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrPSc molecules alone.

【 授权许可】

CC BY