title: 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.
abstract: The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrP(C)) into the pathogenic isoform (PrP(Sc)). Diverse mammalian species possess the cofactors required for in vitro replication of PrP(Sc) 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 PrP(Sc) seeds and Bac-PrP(Sc) 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 PrP(Sc) 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 PrP(Sc) of some prion strains.
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Kato
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Okada
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan ; Research Area of Pathology and Pathophysiology, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Miyako
authors.initials: M
authors.lastname: Yoshioka
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Iwamaru
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Shirou
authors.initials: S
authors.lastname: Mohri
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
authors.affiliation: Influenza • Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Murayama
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D015153:Blotting, Western; D019286:Endopeptidase K / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006358:Hot Temperature; D016133:Polymerase Chain Reaction; D018621:PrPC Proteins / Q000378:metabolism*; D018620:PrPSc Proteins / Q000378:metabolism*; D011328:Prions / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D017261:Glycosylphosphatidylinositols; D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions; D011994:Recombinant Proteins; D019286:Endopeptidase K