51 research outputs found
Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity
<div><p>During prion infection, the normal, protease-sensitive conformation of prion protein (PrP<sup>C</sup>) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP<sup>Sc</sup>). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP<sup>Sc</sup> in prion-infected brain homogenates as an initiating seed to convert PrP<sup>C</sup> and trigger the self-propagation of PrP<sup>Sc</sup> over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP<sup>Sc</sup>. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP<sup>Sc</sup> seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP<sup>Sc</sup>. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.</p></div
Varying RNA and POPG concentration does not lead to rPrP-res that can induce PrP<sup>Sc</sup> formation in SN56 cells.
<p>(A) rPrP PMCA reactions seeded with rPrP-res and serially propagated in the presence of 30 µg/ml of RNA and 3 different concentrations of POPG lipid (POPG lanes) or in the presence of 4.4 µg/ml of POPG and 3 different concentrations of mouse liver RNA (RNA lanes) were digested with PK and assayed in duplicate by immunoblot. (B) SN56 cells inoculated with cell culture medium alone (Mock) or rPrP-res made in the presence of 30 µg/ml RNA plus 2.2 or 6.7 µg/ml of POPG lipid. (C) SN56 cells inoculated with cell culture medium alone (Mock) or rPrP-res made in the presence of 4.4 µg/ml POPG plus 15 or 45 µg/ml of RNA. (D) SN56 cells inoculated with rPrP-res (Passage 6). The concentration in µg/ml of the RNA and POPG used is indicated in the parentheses. Lanes labeled 1× indicate PMCA products generated using the original POPG and RNA concentrations of 4.4 µg/ml and 30 µg/ml, respectively. (E) SN56 cells inoculated with rPrP-res (Passage 10). Labeling is as in panel D. The first lane is a 1∶100 dilution of undigested PrP from SN56 cells. Based on a standard dilution curve of PrP<sup>Sc</sup> derived from SN56 cells persistently infected with 22L (first 3–4 lanes in panels B-D), the limit of detection of the immunoblot for PrP is equal to ∼1–3% of the total cell equivalents loaded. For all panels, blots were developed using the anti-PrP mouse monoclonal antibody 6D11. Molecular mass markers (kDa) are indicated on the right of each panel. Irrelevant lanes have been cropped and some lanes have been rearranged for clarity, but each immunoblot panel derives from a single film exposure.</p
Two different preparations of mouse rPrP do not spontaneously form rPrP-res capable of inducing the formation of mammalian PrP<sup>Sc</sup> in SN56 cells.
<p>(A) The products of eleven rounds of unseeded rPrP2 PMCA were PK digested and assayed by immunoblot with the C-terminal anti-PrP rabbit polyclonal antibody R20. The emergence of a13 kDa rPrP-res2 band (closed arrowhead) can be seen in Round 11. Unsonicated rPrP2 substrate mixture in the absence (PK-) or presence (PK+) of PK is shown in lanes 1 and 2. The PK- control sample was diluted 1∶100 prior to loading. Purified mouse rPrP from residues 90–230 is shown in the last lane as a molecular mass reference. Arrow: rPrP. (B) PMCA reaction products and unsonicated rPrP2 substrate (No Sonic) were digested with eight-fold dilutions of PK and assayed by immunoblot using the C-terminal anti-PrP rabbit polyclonal antibody R20. The PK- control sample was diluted 1∶100 prior to loading (lane 1), while the digested samples were loaded undiluted. Undigested rPrP peptide 23–230 (Lane 1, arrow) and rPrP peptide 90–230 (Lane 11) are used as references for molecular mass. In both panels A and B, the non-PrP bands from ∼18–66 kDa are derived from the digestion products of BSA and PK. Open arrowhead: 16 kDa rPrP-res; Closed arrowhead: 13 kDa rPrP-res. (C-E) SN56 cells were inoculated with cell culture medium alone (Mock), brain homogenate from mice with clinical 22L scrapie (22L), 120 ng of rPrP-res, or 7 ng of rPrP-res2. Cell lysates were PK-digested and analyzed by immunoblot with the anti-PrP mouse monoclonal antibody 6D11 at Passes 0–3 (C), 0–2 (D) and passage 10 (E). All rPrP-res and rPrP-res2 samples were loaded undiluted. In panels D and E, a dilution series of a 22L scrapie-infected cell lysate was used to demonstrate the limit of detection of PrP<sup>Sc</sup> (Lanes 1–3). For all gels, molecular mass markers (kDa) are indicated on the right. Irrelevant lanes have been cropped but each immunoblot panel derives from a single film exposure.</p
C57Bl/10 mice inoculated with rPrP-res do not accumulate detectable PrP<sup>Sc</sup> up to 626 days post inoculation.
<p>Brain and spleen samples from C57Bl/10 mice inoculated with rPrP-res, 22L mouse scrapie, vehicle alone (Neg), or a mixture of unsonicated rPrP, POPG, and RNA in inoculation buffer (No sonic) were homogenized, PK digested, and assayed by immunoblot using the anti-PrP mouse monoclonal antibody 6D11. (A) Brain and spleen, 200 dpi. (B) Brain, 365 dpi. (C) Spleen, 365 dpi. (D) Spleen, 365 dpi, secondary antibody only. Samples assayed are identical to those in panel C. The first lane shows the reactivity of the secondary antibody with purified mouse IgG heavy and light chains. (E) Brain, 626 dpi. (F) Spleen, 626 dpi. For all panels, tissue samples were loaded undiluted (UD) unless otherwise noted. A standard dilution curve of 22L mouse brain homogenate containing PrP<sup>Sc</sup> was loaded on each gel as indicated and used to estimate the limit of detection of PrP as detailed in the Materials and Methods. A, B, and C represent individual mice assayed at each timepoint. IC = intracerebral; IP = intraperitoneal; PO = per ora. Molecular mass markers (kDa) are indicated on the right and the asterisk indicates the location of a cross-reactive proteinase K band.</p
Lack of spongiform change and PrP<sup>Sc</sup> in C57Bl/10 mice inoculated with rPrP-res.
<p>Sagittal sections from C57Bl/10 mice inoculated with 22L scrapie, rPrP-res, or the unsonicated rPrP mixture (No sonic) stained with D13 anti-PrP antibody. A representative region of the thalamus is shown. Mice inoculated with 22L showed clear spongiform change and PrP<sup>Sc</sup> deposition (brown stain). No spongiform change or PrP<sup>Sc</sup> was detected in any region of the brain from mice inoculated with rPrP-res. Scale bar = 100 µm.</p
rPrP-res is detergent insoluble.
<p>Unsonicated (No sonic) or sonicated (rPrP-res) PMCA substrate mixtures were subjected to ultracentrifugation for one hour at 100,000×<i>g</i> and the resulting supernatants (SN) and pellets were assayed by immunoblot with the anti-PrP mouse monoclonal antibody 6D11 either with (+) or without (−) PK digestion. Samples were loaded undiluted (UD) or diluted 1∶10 and 1∶100 as indicated. Molecular mass markers (kDa) are shown on the right. rPrP is indicated by the arrow and the 16 kDa protease-resistant band by an open arrowhead. The band at ∼66 kDa is bovine serum albumin which was added to the samples during methanol precipitation. The asterisk indicates the location of a cross-reactive proteinase K band. Lane numbers are shown at the bottom of the gel. Total = no centrifugation.</p
No evidence of clinical or subclinical infection following inoculation of rPrP-res into mice with different genetic backgrounds.
a<p>Mice inoculated with a 1∶10 dilution of a 22L mouse scrapie 10% brain homogenate. These mice were not inoculated in the same experiment as the rPrP-res inoculated mice.</p>b<p>Brains and spleens of two mice tested negative for PrP<sup>Sc</sup> at 200, 365, and 626 dpi.</p>c<p>Brains and spleens of two mice tested negative for PrP<sup>Sc</sup> at 365 dpi.</p>d<p>Brains and spleens of three mice tested negative for PrP<sup>Sc</sup> at 367 dpi.</p>e<p>Numbers in parentheses represent the number of scrapie positive mice over the total number of mice inoculated.</p>f<p>days post-inoculation (DPI). Numbers represent either average incubation time <u>+</u> SD or days post-inoculation when the experiment was terminated.</p
Generation of rPrP-res with a protease-resistant C-terminal core in PMCA reactions containing RNA and lipid.
<p>(A) The products of twenty rounds of serial PMCA were PK digested and assayed by immunoblot using the anti-PrP mouse monoclonal antibody 6D11. The number of PMCA rounds is indicated at the top of the gel. Lanes 1–4 represent a standard curve of 1∶100 to 1∶2700 dilutions of the non-PK digested, unsonicated rPrP substrate mixture (No sonic). Based on the standard curve, 0.3–3% of the input rPrP was converted to rPrP-res. Lane numbers are shown at the bottom of the gel. For all panels, the arrow on the left indicates rPrP, the open arrowhead on the right indicates rPrP-res, and molecular mass markers in kDa are indicated on the right. (B) Two independent preparations of rPrP-res (A and B) were digested with increasing concentrations of PK and assayed by immunoblot using the 6D11 antibody. The non-PK digested, unsonicated rPrP substrate mixture (No sonic) was diluted 1∶100 prior to loading. All other samples were loaded without dilution. The asterisk to the right indicates the location of a cross-reactive proteinase K band. (C) rPrP-res was PK-digested and assayed by immunoblot with three anti-PrP antibodies: 8B4 (residues 37–44), POM1 (multiple residues between140–212, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071081#pone.0071081-Baral1" target="_blank">[38]</a>), and R20 (residues 218–231). Unless otherwise indicated, all samples were loaded without dilution. No sonic = non-PK digested, unsonicated rPrP substrate mixture. (D) rPrP-res was digested with varying concentrations of PK and assayed by immunoblot using the anti-PrP antibody R20. The rPrP PK-minus control sample (No sonic) was diluted 1∶100 prior to loading while all PK-digested samples were loaded undiluted. The closed arrowhead indicates the 13 kDa rPrP-res fragment.</p
The ultrastructure of rPrP-res is fibrillar and not granular.
<p>Transmission electron microscopy by negative stain techniques was used to examine rPrP-res (A, B), unsonicated PMCA substrate (C), mouse liver extract alone (D), and mouse liver extract treated with amylase and amyloglucosidase (E). (F) Fibrils of rPrP-res were immune-labelled with the anti-PrP antibody 6D11 and gold-conjugated anti-mouse IgG secondary antibody, prior to negative staining. The glycogen granule in the upper left of the panel is not immunogenic, while the nearby fibrils are specifically labelled. The inset is magnified 2×. (G) rPrP-res stained with secondary antibody only. Scale bars = 100 nm.</p
VZV mutants deleted for the IDE binding domain of gE are impaired for syncytia formation.
<p>(A) Melanoma cells were infected with VZV ROka or ROka68D32-71 and stained with DAPI (top panels) or mouse monoclonal antibody to gE followed by anti-mouse-Alexa-488 and visualized by immunofluorescence microscopy. Magnification 40X. (B) Melanoma cells were infected with VZV ROka-GFP or ROka68D32-71-GFP and visualized directly by fluorescence microscopy. Magnification 40X. (C) Quantification of the number of nuclei in syncytia per nucleus was performed using confocal microscopy (Leica SP2, Leica Microsystems, Exton, PA). Sequential Z-sections of DAPI stained infected cells were acquired for 3D reconstruction of representative cells with Imaris software (version 6.2, Bitplane AG, Zurich, Switzerland). The numbers of nuclei were automatically determined using spot function in Imaris software and manually corrected for errors by independent investigators in Biological Imaging, Research Technologies Branch, NIH. Vertical lines show standard deviations. n represents the number of syncytia analyzed in which nuclei were counted by a microscopist who was unaware of the expected outcome of the experiment.</p
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