Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

被引:8
作者
Imamura, Morikazu [1 ]
Kato, Nobuko [1 ]
Okada, Hiroyuki [1 ]
Yoshioka, Miyako [1 ,2 ]
Iwamaru, Yoshifumi [1 ]
Shimizu, Yoshihisa [1 ]
Mohri, Shirou [1 ]
Yokoyama, Takashi [1 ]
Murayama, Yuichi [1 ]
机构
[1] Natl Inst Anim Hlth, Influenza Prion Dis Res Ctr, Tsukuba, Ibaraki 305, Japan
[2] Natl Inst Anim Hlth, Res Area Pathol & Pathophysiol, Tsukuba, Ibaraki 305, Japan
来源
PLOS ONE | 2013年 / 8卷 / 12期
关键词
IN-VITRO; PRESYMPTOMATIC DETECTION; CYCLIC AMPLIFICATION; SURFACE RETENTION; INFECTIOUS PRIONS; PRPSC; AGGREGATION; REPLICATION; PROPAGATION; ABSENCE;
D O I
10.1371/journal.pone.0082538
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrPC) into the pathogenic isoform (PrPSc). Diverse mammalian species possess the cofactors required for in vitro replication of PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrPSc seeds and Bac-PrPSc generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrPSc were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrPSc of some prion strains.
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页数:10
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