Acute Formation of Protease-resistant Prion Protein Does Not Always Lead to Persistent Scrapie Infection in Vitro
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m402576200
ISSN1083-351X
AutoresIna Vorberg, A Raines, Suzette A. Priola,
Tópico(s)Prion Diseases and Protein Misfolding
ResumoTransmissible spongiform encephalopathies are accompanied by the accumulation of a pathologic isoform of a host-encoded protein, termed prion protein (PrP). Despite the widespread distribution of the cellular isoform of PrP (protease-sensitive PrP; PrP-sen), the disease-associated isoform (protease-resistant PrP; PrP-res) appears to be primarily restricted to cells of the nervous and lymphoreticular systems. In order to study why scrapie infection appears to be restricted to certain cells, we followed acute and persistent PrP-res formation upon exposure of cells to different scrapie agents. We found that, independent of the cell type and scrapie strain, initial PrP-res formation occurred rapidly in cells. However, sustained generation of PrP-res and persistent infection did not necessarily follow acute PrP-res formation. Persistent PrP-res formation and scrapie infection was restricted to one cell line inoculated with the mouse scrapie strain 22L. In contrast to cells that did not become scrapie-infected, the level of PrP-res in the 22L-infected cells rapidly increased in the absence of a concomitant increase in the number of PrP-res-producing cells. Furthermore, the protein banding pattern of PrP-res in these cells changed over time as the cells became chronically infected. Thus, our results suggest that the events leading to the initial formation of PrP-res may differ from those required for sustained PrP-res formation and infection. This may, at least in part, explain the observation that not all PrP-sen-expressing cells appear to support transmissible spongiform encephalopathy agent replication. Transmissible spongiform encephalopathies are accompanied by the accumulation of a pathologic isoform of a host-encoded protein, termed prion protein (PrP). Despite the widespread distribution of the cellular isoform of PrP (protease-sensitive PrP; PrP-sen), the disease-associated isoform (protease-resistant PrP; PrP-res) appears to be primarily restricted to cells of the nervous and lymphoreticular systems. In order to study why scrapie infection appears to be restricted to certain cells, we followed acute and persistent PrP-res formation upon exposure of cells to different scrapie agents. We found that, independent of the cell type and scrapie strain, initial PrP-res formation occurred rapidly in cells. However, sustained generation of PrP-res and persistent infection did not necessarily follow acute PrP-res formation. Persistent PrP-res formation and scrapie infection was restricted to one cell line inoculated with the mouse scrapie strain 22L. In contrast to cells that did not become scrapie-infected, the level of PrP-res in the 22L-infected cells rapidly increased in the absence of a concomitant increase in the number of PrP-res-producing cells. Furthermore, the protein banding pattern of PrP-res in these cells changed over time as the cells became chronically infected. Thus, our results suggest that the events leading to the initial formation of PrP-res may differ from those required for sustained PrP-res formation and infection. This may, at least in part, explain the observation that not all PrP-sen-expressing cells appear to support transmissible spongiform encephalopathy agent replication. Transmissible spongiform encephalopathies (TSEs) 1The abbreviations used are: TSE, transmissible spongiform encephalopathy; BSE, bovine spongiform encephalopathy; PrP, prion protein; PrP-sen, protease-sensitive PrP; PrP-res, protease-resistant PrP; IC, intracranially; PK, Proteinase K; DMEM, Dulbecco's modified Eagle's medium. are progressive neurodegenerative diseases that include Creutzfeldt-Jakob disease, Gerstmann-Straüssler-Scheinker syndrome, and Kuru in humans as well as scrapie in sheep and goats, chronic wasting disease in mule deer and elk, transmissible mink encephalopathy, and bovine spongiform encephalopathy (BSE). A prerequisite for TSE infection is the expression of the protease-sensitive host prion protein (PrP-sen) (1Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1813) Google Scholar). PrP-sen is a highly conserved mammalian sialoglycoprotein of unknown function that is anchored to the cell membrane by a phosphatidylinositol moiety (2Haraguchi T. Fisher S. Olofsson S. Endo T. Groth D. Tarentino A. Borchelt D.R. Teplow D. Hood L. Burlingame A. Lycke E. Kobata A. Prusiner S.B. Arch. Biochem. Biophys. 1989; 274: 1-13Crossref PubMed Scopus (186) Google Scholar, 3Bendheim P.E. Potempska A. Kascsak R.J. Bolton D.C. J. Infect. Dis. 1988; 158: 1198-1208Crossref PubMed Scopus (36) Google Scholar). During TSE infection, PrP-sen is converted to its partially protease-resistant, pathologic isoform PrP-res. This abnormal form of PrP is closely associated with TSE infectivity and has been proposed as the protein-only agent responsible for TSE diseases (4Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4121) Google Scholar). PrP-sen is required for susceptibility to TSE disease (1Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1813) Google Scholar). At the cellular level, however, it is unlikely that PrP-sen expression is the sole prerequisite for TSE infection. PrP-sen is expressed in a wide variety of tissues (5Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1253) Google Scholar, 6Harris D.A. Lele P. Snider W.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4309-4313Crossref PubMed Scopus (74) Google Scholar, 7Pammer J. Suchy A. Rendl M. Tschachler E. Lancet. 1999; 354: 1702-1703Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), yet PrP-res formation appears to be restricted primarily to cells of the nervous and lymphoreticular systems (8Bolton D.C. McKinley M.P. Prusiner S.B. Science. 1982; 218: 1309-1311Crossref PubMed Scopus (1014) Google Scholar, 9Kitamoto T. Muramoto T. Mohri S. Doh-Ura K. Tateishi J. J. Virol. 1991; 65: 6292-6295Crossref PubMed Google Scholar). The reason for this discrepancy in PrP-sen expression and PrP-res formation is unknown. However, the fact that different scrapie strains induce PrP-res formation in different brain areas suggests that PrP-res formation is both cell type- and scrapie strain-dependent (10Hecker R. Taraboulos A. Scott M. Pan K.M. Yang S.L. Torchia M. Jendroska K. DeArmond S.J. Prusiner S.B. Genes Dev. 1992; 6: 1213-1228Crossref PubMed Scopus (193) Google Scholar, 11DeArmond S.J. Sanchez H. Yehiely F. Qiu Y. Ninchak-Casey A. Daggett V. Camerino A.P. Cayetano J. Rogers M. Groth D. Torchia M. Tremblay P. Scott M.R. Cohen F.E. Prusiner S.B. Neuron. 1997; 19: 1337-1348Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Dickinson A.G. Fraser H. Meikle V.M. Outram G.W. Nat. New Biol. 1972; 237: 244-245Crossref PubMed Scopus (96) Google Scholar, 13Bruce M.E. Baker H. Ridley R.M. Methods in Molecular Medicine: Prion Diseases. Humana Press, Totowa, NJ1996: 223-226Google Scholar). Evidence for scrapie strain-dependent PrP-res formation and infection also comes from infection studies with tissue culture cells, which demonstrate that the same cell line is susceptible to some scrapie strains but not to others (14Milhavet O. McMahon H.E. Rachidi W. Nishida N. Katamine S. Mange A. Arlotto M. Casanova D. Riondel J. Favier A. Lehmann S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13937-13942Crossref PubMed Scopus (190) Google Scholar, 15Birkett C.R. Hennion R.M. Bembridge D.A. Clarke M.C. Chree A. Bruce M.E. Bostock C.J. EMBO J. 2001; 20: 3351-3358Crossref PubMed Scopus (135) Google Scholar, 16Vorberg I. Raines A. Story B. Priola S.A. J. Infect. Dis. 2004; 189: 431-439Crossref PubMed Scopus (108) Google Scholar, 17Rubenstein R. Deng H. Race R.E. Ju W. Scalici C.L. Papini M.C. Kascsak R.J. Carp R.I. J. Gen. Virol. 1992; 73: 3027-3031Crossref PubMed Scopus (33) Google Scholar). Unfortunately, the cellular prerequisites needed for PrP-res formation and infection remain elusive. One possible explanation for the inability of TSE strains to persistently infect specific cell populations may be that these cells cannot support the formation of new PrP-res induced by these strains. However, attempts to study the very early events in cell culture leading to PrP-res formation have been difficult, since 1) only a few cell culture systems have been identified that appear to be susceptible to the scrapie agent, 2) newly formed PrP-res must be discriminated from PrP-res present in the inoculum, and 3) the amount of PrP-res produced in susceptible cell lines is often relatively low. In this study, we have used cell culture systems that allow us to follow PrP-res formation following exposure of the cells to scrapie-infected brain homogenates (18Korth C. Kaneko K. Prusiner S.B. J. Gen. Virol. 2000; 81: 2555-2563Crossref PubMed Scopus (52) Google Scholar, 19Vorberg I. Priola S.A. J. Biol. Chem. 2002; 277: 36775-36781Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We have found that PrP-res is detectable within 24 h of exposure to mouse scrapie agents. This rapid formation of PrP-res was scrapie strain-independent and could be initiated in both neuronal and nonneuronal cells. However, acute formation of PrP-res was not always indicative of a persistent scrapie infection. Only fibroblast cells that had been exposed to the mouse scrapie strain 22L persistently produced PrP-res and infectivity. Quantitative and qualitative changes in newly formed PrP-res accompanied the establishment of a persistent infection and could be indicative of the scrapie agent localizing to a cellular compartment suitable for efficient PrP-res formation. Thus, our data suggest that whereas acute formation of PrP-res may occur in a variety of cell types, initiation of persistent PrP-res formation and scrapie infection requires additional cell- and/or scrapie strain-specific factors for the scrapie agent to adapt to and persistently infect a cell. Cell Lines—The mouse neuroblastoma cell line Mo3F4-MNB and the mouse fibroblast cell cultures Mo3F4-Ψ2C2 and Mo3F4-Ψ2/PA317 have been described previously (16Vorberg I. Raines A. Story B. Priola S.A. J. Infect. Dis. 2004; 189: 431-439Crossref PubMed Scopus (108) Google Scholar, 19Vorberg I. Priola S.A. J. Biol. Chem. 2002; 277: 36775-36781Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. All cell cultures express high levels of mouse PrP-sen containing the epitope to the hamster PrP-specific mouse monoclonal antibody 3F4 (Mo3F4). The 3F4 epitope had previously been inserted into mouse PrP by two amino acid substitutions at positions 108 and 111 (20Bolton D.C. Seligman S.J. Bablanian G. Windsor D. Scala L.J. Kim K.S. Chen C.M. Kascsak R.J. Bendheim P.E. J. Virol. 1991; 65: 3667-3675Crossref PubMed Google Scholar). Antibodies—The mouse monoclonal antibody 3F4 was used to distinguish exogenous PrP molecules from the endogenous mouse PrP present in both mouse brain homogenate and mouse cell lines. The rabbit polyclonal antiserum R30 was used to detect mouse PrP-sen expressed in wild-type mouse neuroblastoma cells (21Raymond G.J. Hope J. Kocisko D.A. Priola S.A. Raymond L.D. Bossers A. Ironside J. Will R.G. Chen S.G. Petersen R.B. Gambetti P. Rubenstein R. Smits M.A. Lansbury Jr., P.T. Caughey B. Nature. 1997; 388: 285-288Crossref PubMed Scopus (238) Google Scholar). Scrapie Strains and Preparation of Inocula—The mouse-adapted scrapie strains ME7, 87V, and 22L were the kind gift of Dr. James Hope (Lasswade Veterinary Laboratory, Penicuik, Midlothian, UK) and were passaged in C57Bl/10 (ME7, 22L) or VmDk (87V) mice. Scrapie strain RML was passaged in RML mice. Brain homogenates were prepared in Dulbecco's phosphate-buffered saline (10%, w/v) using the brains of terminally sick animals. Brain tissue was homogenized and sonicated for 5 min. Cell debris was removed by low speed centrifugation (10 min, 4 °C, 2000 × g), and the resultant brain homogenates were stored at –70 °C. For cell inoculation, brain homogenate was diluted into Opti-MEM medium (Invitrogen) and sonicated for 2 min prior to use. The level of PrP-res and infectivity present in the brain homogenates used were similar for each strain. The titers of the different strains used were based upon the amount of inoculum needed to infect 50% of animals that had been injected intracranially (IC) with an infected brain homogenate (ID50). In C57Bl/10 mice, ME7 had an IC ID50 of 2.2 × 107.9 infectious units/g of brain tissue, whereas 22L had an IC ID50 of 2 × 108.8 infectious units/gram of brain tissue. Scrapie strain RML had an IC ID50 of 3.3 × 107.8 infectious units/gram of brain tissue when titered in RML mice. The titer of 87V in VmDk mice is ongoing. Exposure of Cells to Scrapie Agent and Detection of Newly Formed PrP-res—Experiments were performed in 24-well microtiter plates. Cells (0.5 × 105/well) were overlaid with different dilutions of 10% brain homogenate in Opti-MEM. For kinetics studies, different cell numbers were plated to ensure comparable levels of cells at the time point of harvest (4 h, 4 × 105; 24 h, 4 × 105; 48 h, 2 × 105; 72 h, 1 × 105; 96 h, 0.5 × 105 cells/ml). Kinetic studies performed with the same number of cells plated (0.5 × 105/well) were also done and gave similar results when compared with experiments where different numbers of cells were plated. After 4 h, 400 μl of DMEM plus 10% fetal bovine serum was added. Cells were either directly lysed or incubated for up to 96 h before being lysed in lysis buffer (1 mm Tris-HCl, pH 7.4, 140 mm NaCl, 5 mm EDTA, 0.5% sodium deoxycholate, and 0.5% Triton X-100). Cell lysates were cleared of cell debris by centrifugation (5 min, room temperature, 16,000 × g). For Mo3F4 PrP-sen detection, one-hundredth of the cell lysate was reserved. In order to detect PrP-res, the remaining cell lysate was incubated with 20 μg/ml Proteinase K (PK) for 40 min at 37 °C. The reaction was stopped by the addition of phenylmethylsulfonyl fluoride to a final concentration of 3 mm, and samples were centrifuged at 200,000 × g for 1 h at 4 °C. Pellets were sonicated into sample buffer (2.5% SDS, 3 mm EDTA, 2% β-mercaptoethanol, 5% glycerol, 0.02% bromphenol blue, and 63 mm Tris-HCl, pH 6.8) and assayed on 14% NOVEX precast gels (Invitrogen). PrP-sen and newly formed PrP-res were detected by Western blot using the mouse monoclonal antibody 3F4 and enhanced chemiluminescence or enhanced chemifluorescence according to the manufacturer's instructions (Amersham Biosciences). Metabolic Labeling and Immunoprecipitation of PrP-sen—Confluent monolayers of cells were labeled with trans-[35S]methionine/cysteine (PerkinElmer Life Sciences) as previously reported (21Raymond G.J. Hope J. Kocisko D.A. Priola S.A. Raymond L.D. Bossers A. Ironside J. Will R.G. Chen S.G. Petersen R.B. Gambetti P. Rubenstein R. Smits M.A. Lansbury Jr., P.T. Caughey B. Nature. 1997; 388: 285-288Crossref PubMed Scopus (238) Google Scholar). Briefly, cells were rinsed in phosphate-buffered balanced salt solution and incubated in methionine/cysteine-free DMEM for 1 h. Trans-[35S]methionine/cysteine was added to a final concentration of 15 μCi, and cells were incubated for 2 h at 37 °C. Monolayers were lysed, and cell debris was removed by low speed centrifugation (10 min, 4 °C, 2000 × g). Proteins were precipitated in 4 volumes of methanol and sonicated into detergent lipid protein complex buffer (4.2 mg of l-α-phosphatidylcholine per ml, 123 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% N-lauroylsarcosine). Samples were divided into two fractions, and PrP-sen was immunoprecipitated with either the polyclonal antiserum R30 to detect total PrP or the monoclonal antibody 3F4 to detect epitope-tagged PrP. Proteins were separated on 14% NOVEX Precast gels (Invitrogen), and radiolabeled proteins were visualized by autoradiography. Detection of Scrapie Infectivity in Cell Cultures Exposed to Scrapie Agent—Following exposure to brain homogenate, cells were continuously cultured and harvested for inoculation at passage 12 or 13 postinfection. Cells were resuspended in DMEM plus 10% fetal bovine serum. Following five consecutive cycles of freeze/thawing, lysates were sonicated, and ∼5 × 105 cells/mouse were inoculated IC into C57Bl/10 weanling mice. Since the scrapie strain 87V had been propagated in VmDk mice, lysates prepared from cells exposed to 87V brain homogenate were inoculated into VmDk mice. The mouse scrapie strain 87V has an incubation time of >700 days in C57Bl/10 mice but a shorter incubation time in VmDk mice, which express a mouse PrP allele that differs from the C57Bl/10 allele at positions 108 and 189 (22Westaway D. Goodman P.A. Mirenda C.A. McKinley M.P. Carlson G.A. Prusiner S.B. Cell. 1987; 51: 651-662Abstract Full Text PDF PubMed Scopus (371) Google Scholar). As controls, 50 μl of 1% brain homogenates from uninfected mice or mice infected with scrapie strain 87V, RML, 22L, or ME7 were also inoculated IC into the appropriate mouse strain. Mice were observed at least twice a week for the onset of neurological disease, and mice showing signs of scrapie disease were sacrificed. All animal experiments were approved by the Rocky Mountain Laboratory Animal Care and Use Committee. The Rocky Mountain Laboratories are fully accredited by the American Association for Laboratory Animal Care. Determination of the Number of PrP-res-accumulating Cells—To determine the number of cells accumulating PrP-res upon infection with scrapie strain 22L, Mo3F4-Ψ2C2 cells were exposed to 1% 22L scrapie brain homogenate in 24-well plates. Cells from three wells were pooled and cloned by limiting dilution after 48, 72, or 96 h of exposure. Additionally, cells were exposed for 96 h to scrapie brain homogenate and then continuously passaged in the absence of brain homogenate. After five and 10 passages, these cells were also cloned. Single cell clones were expanded, and cell lysates of clones were tested for their PrP-res content as described above. Statistical analysis of the resulting data was done using the χ2 test. Acute PrP-res Formation following Exposure of Neuroblastoma and Fibroblast Cells to Different Scrapie Strains—In order to study the early events that occur during infection with a TSE agent, we have used a cellular assay to detect newly formed, fully glycosylated PrP-res (19Vorberg I. Priola S.A. J. Biol. Chem. 2002; 277: 36775-36781Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This assay utilizes a cloned mouse neuroblastoma cell line (Mo3F4-MNB) that produces high levels of mouse 3F4-positive PrP-sen when compared with the endogenous mouse PrP-sen expressed in these cells (Fig. 1A). New PrP-res derived from mouse 3F4-positive PrP-sen can be distinguished from the PrP-res present in the infectious inoculum by simple Western blot analysis using the mouse monoclonal antibody 3F4 (18Korth C. Kaneko K. Prusiner S.B. J. Gen. Virol. 2000; 81: 2555-2563Crossref PubMed Scopus (52) Google Scholar, 19Vorberg I. Priola S.A. J. Biol. Chem. 2002; 277: 36775-36781Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Upon exposure to different dilutions of RML scrapie brain homogenate for 4 days, Mo3F4-MNB cells produced 3F4-positive PrP-res in a brain homogenate concentration-dependent manner (Fig. 1B). By contrast, no PrP-res was present in cells exposed to normal brain homogenate (Fig. 1B, Mock). Furthermore, 3F4-positive PrP-res was also generated in Mo3F4-MNB cells upon exposure to scrapie strains 22L, ME7, and 87V (Fig. 1C) (19Vorberg I. Priola S.A. J. Biol. Chem. 2002; 277: 36775-36781Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These experiments demonstrate that new (i.e. acute) formation of PrP-res in neuroblastoma cells can be driven by multiple scrapie strains. Whereas the presence of PrP-res in the lymphoreticular tissues of infected mice can vary between scrapie strains, PrP-res is almost always found within the central nervous system, and neuronal vaculation (i.e. spongiform change) is a central hallmark of TSE diseases. As a result, it is often assumed that it is primarily cells within the central nervous system that accumulate PrP-res and are susceptible to scrapie infection. To test whether acute PrP-res formation could also be detected in nonneuronal cells following exposure to different scrapie strains, we used a mixture of Ψ2/PA317 fibroblast cells expressing Mo3F4 PrP-sen (Mo3F4-Ψ2/PA317), a cell culture previously shown to be susceptible to 22L scrapie (16Vorberg I. Raines A. Story B. Priola S.A. J. Infect. Dis. 2004; 189: 431-439Crossref PubMed Scopus (108) Google Scholar). PrP-res formation was detected in cells exposed to RML, 22L, ME7, and 87V scrapie, although differences in the amount of PrP-res produced were observed (Fig. 1D). The in vivo titers and PrP-res content of the 22L, ME7, and RML scrapie brain homogenates used were similar (see "Experimental Procedures"), suggesting that significant differences in scrapie agent titer or input PrP-res cannot explain the observed differences in the amount of new PrP-res formed. Therefore, acute PrP-res is formed upon exposure to different scrapie strains and can be initiated in cells of both neuronal and nonneuronal origin. Acute PrP-res Formation Does Not Always Lead to Sustained PrP-res Formation—To test whether acute PrP-res formation led to persistent PrP-res formation, Mo3F4-MNB and Mo3F4-Ψ2/PA317 cells were exposed to mouse scrapie infectivity and continuously passaged. In subsequent passages (passages 1–13) of Mo3F4-MNB cells inoculated with any of the scrapie strains, no 3F4-positive PrP-res was detected. Multiple passages of Mo3F4-Ψ2/PA317 cells exposed to the strain RML, 87V, or ME7 (Fig. 2, A and B) (data not shown) were also negative for 3F4-positive PrP-res. However, as reported previously (16Vorberg I. Raines A. Story B. Priola S.A. J. Infect. Dis. 2004; 189: 431-439Crossref PubMed Scopus (108) Google Scholar), Mo3F4-Ψ2/PA317 fibroblasts inoculated with 22L brain homogenate accumulated high levels of 3F4-positive PrP-res upon continuous passage (Fig. 2B). Thus, whereas multiple scrapie strains could initiate PrP-res formation in both neuronal and nonneuronal cells, detectable PrP-res formation was not sustained in these cultures except for fibroblast cells exposed to the scrapie strain 22L. These data suggest that the requirements for sustained PrP-res formation are both cell type- and scrapie strain-dependent. Acute PrP-res Formation Does Not Always Lead to Infection—Previous studies have demonstrated that cell cultures that do not accumulate detectable levels of PrP-res upon exposure to TSE agent can still propagate scrapie infectivity (23Race R.E. Fadness L.H. Chesebro B. J. Gen. Virol. 1987; 68: 1391-1399Crossref PubMed Scopus (161) Google Scholar). To determine whether mouse neuroblastoma cells or mouse fibroblast cells exposed to different scrapie strains generated scrapie infectivity, cell lysates from cells passaged at least 12 times in vitro were inoculated intracranially into either C57Bl/10 (cells exposed to scrapie strain RML, 22L, or ME7) or VmDk mice (cells exposed to scrapie strain 87V) (Table I). As controls, cells exposed to normal mouse brain homogenate ("None" in Table I) as well as brain homogenates from scrapie-infected or uninfected mice were also inoculated.Table IInoculation of C57BL/10 mice and VmDk mice with cells exposed to different scrapie strainsScrapie strainInoculum (brain homogenate or cell lysate)MiceClinical scrapie/TotalIncubation time ± S.E.NoneBrain homogenateC57Bl/100/6>755Mo3F4-MNB0/5>755Mo3F4-ψ2/PA3170/4>75522LBrain homogenateC57Bl/107/7161.9 ± 3.7Mo3F4-MNB0/6>755Mo3F4-ψ2/PA3178/8284.6 ± 39.6RMLBrain homogenateC57Bl/108/8164.4 ± 3.9Mo3F4-MNB0/4>753Mo3F4-ψ2/PA3171/7624,aOne mouse that died 624 days postinfection was positive for PrP-res. All other nonclinical mice sacrificed up to 755 days postinfection were negative for PrP-res. >753ME7Brain homogenateC57Bl/108/8187.9 ± 7.8Mo3F4-MNB0/7>753Mo3F4-ψ2/PA3170/5>75387VBrain homogenateVmDk8/8320.8 ± 5.5Mo3F4-MNB0/4>755Mo3F4-ψ2/PA3170/6>755a One mouse that died 624 days postinfection was positive for PrP-res. All other nonclinical mice sacrificed up to 755 days postinfection were negative for PrP-res. Open table in a new tab Whereas mice inoculated with 22L-infected fibroblast cells succumbed to disease within 284.6 ± 39.6 days, mice inoculated with neuroblastoma cells exposed to the 22L scrapie strain did not show any signs of scrapie up to >755 days postinfection (Table I). Furthermore, mice inoculated with either neuroblastoma cells or fibroblast cells exposed to ME7, RML, or 87V mouse scrapie did not display any signs of disease. The brains of all nonclinical animals sacrificed up to 755 days postinfection (when the experiment was terminated) were analyzed for PrP-res. All were negative except for one mouse inoculated with fibroblast cells exposed to RML scrapie that accumulated PrP-res in the brain 624 days postinoculation (Table I). This suggests that a very low amount of RML infectivity (<1 ID /5 × 10550 cells) was present in the cell culture. Overall, these experiments demonstrate that whereas mouse neuroblastoma cells and mouse fibroblasts were capable of forming detectable PrP-res upon acute exposure to scrapie brain homogenate, this did not lead to persistent infection in most cases. These data suggest that the cellular requirements for acute PrP-res formation may differ from those for sustained PrP-res formation and persistent scrapie infection. Acute PrP-res Formation Is Cell-associated and Requires Intact Cells—The foregoing results demonstrated that acute PrP-res formation could be initiated in two different cell types by multiple scrapie strains, but persistent infection did not necessarily occur. One possible explanation for these results is that de novo PrP-res formation was not cell-associated. PrP-res formation can occur in vitro without the context of a living cell (24Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, 25Soto C. Saborio G.P. Anderes L. Trends Neurosci. 2002; 25: 390-394Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), and a recent study suggested that removal of PrP-sen from the cell membrane facilitates new PrP-res formation (26Baron G.S. Wehrly K. Dorward D.W. Chesebro B. Caughey B. EMBO J. 2002; 21: 1031-1040Crossref PubMed Scopus (238) Google Scholar). Thus, it was possible that newly formed PrP-res was being generated solely from the conversion of secreted or non-cell-associated Mo3F4 PrP-sen present in the cell medium. To confirm that the newly formed PrP-res detected was cell-associated, Mo3F4-MNB culture supernatant was harvested after 96 h of incubation with brain homogenate from a mouse infected with the mouse scrapie strain ME7, and the supernatant as well as the cell lysate was checked for 3F4-positive PrP-res. Newly formed PrP-res was detectable in the cell lysate but not in the cell culture supernatant (Fig. 3A), demonstrating that the de novo PrP-res produced in this assay was cell-associated. To study whether PrP-res formation was dependent upon intact cells, Mo3F4-MNB cells were sonicated to rupture the cell membranes and mixed with brain homogenate in cell culture medium supplemented with protease inhibitors. No 3F4-positive PrP-res could be detected in samples containing either normal brain homogenate or brain homogenate from a scrapie-infected animal (Fig. 3B). These experiments are in agreement with previously published data, demonstrating that in vitro PrP-res formation driven by crude TSE brain homogenates is dependent on the presence of detergent (19Vorberg I. Priola S.A. J. Biol. Chem. 2002; 277: 36775-36781Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 25Soto C. Saborio G.P. Anderes L. Trends Neurosci. 2002; 25: 390-394Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). These results show that newly formed PrP-res is cell-associated and that its formation is dependent upon intact cells. Thus, the inability to establish a persistent infection in these cells was not due to the lack of cell-associated PrP-res formation. Acute PrP-res Formation Is a Rapid Process—The fact that de novo PrP-res formation was easily detectable just 96 h after exposure of uninfected cells to scrapie brain homogenate suggested that PrP-res formation was occurring rapidly in these cells. To determine how rapidly PrP-res was generated, Mo3F4-MNB cells were exposed for different time periods to scrapie strains known to replicate with either long (87V) or short (RML) incubation periods in C57Bl/10 mice (27Bruce M.E. Br. Med. Bull. 1993; 49: 822-838Crossref PubMed Scopus (213) Google Scholar). Maximum levels of 3F4-positive PrP-res were reached after 24 h of inoculation with either RML or 87V scrapie brain homogenates (Fig. 4). Interestingly, de novo formation of PrP-res was rapid regardless of whether the PrP-res in the infected brain homogenate was derived from a short or long incubation time strain. These data are consistent with recent data using hamster scrapie demonstrating that, regardless of the strain incubation time, the same amount of PrP-res is formed after 24 h (28Mulcahy E.R. Bessen R.A. J. Biol. Chem. 2004; 279: 1643-1649Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Thus, our results demonstrate that the acute formation of PrP-res followin
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