Abstract
In transmissible encephalopathies (TSEs), it is commonly believed that the host prion protein transforms itself into an infectious form that encodes the many distinct TSE agent strains without any nucleic acid. Using a Ф29 polymerase and chromatography strategy, highly infectious culture and brain preparations of three different geographic TSE agents all contained novel circular DNAs. Two circular “Sphinx” sequences, of 1.8 and 2.4 kb, copurified with infectious particles in sucrose gradients and, as many protected viruses, resisted nuclease digestion. Each contained a replicase ORF related to microviridae that infect commensal Acinetobacter. Infectious gradient fractions also contained nuclease-resistant 16 kb mitochondrial DNAs and analysis of >4,000 nt demonstrated a 100% identity with their species-specific sequences. This confirmed the fidelity of the newly identified sequences detailed here. Conserved replicase regions within the two Sphinx DNAs were ultimately detected by PCR in cytoplasmic preparations from normal cells and brain but were 2,500-fold less than in parallel-infected samples. No trace of the two Sphinx replicases was found in enzymes, detergents, or other preparative materials using exhaustive PCR cycles. The Sphinx sequences uncovered here could have a role in TSE infections despite their apparently symbiotic, low-level persistence in normal cells and tissues. These, as well as other cryptic circular DNAs, may cause or contribute to neurodegeneration and infection-associated tumor transformation. The current results also raise the intriguing possibility that mammals may incorporate more of the prokaryotic world in their cytoplasm than previously recognized.
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References
Aiken JM, Williamson JL, Marsh RF (1989) Evidence of mitochondrial involvement in scrapie infection. J Virol 63:1686–1694
Aiken JM, Williamson JL, Borchardt M, Marsh RF (1990) Presence of mitochondrial D-loop DNA in scrapie-infected brain preparations enriched for prion protein. J Virol 64:3265–3268
Akowitz A, Sklaviadis T, Manuelidis L (1994) Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt–Jakob disease. Nucleic Acids Res 22:1101–1107
Alais S, Simoes S, Baas D, Lehmann S, Raposo G, Darlix J, Leblanc P (2008) Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles. Biol Cell 100:603–615
Arjona A, Simarro L, Islinger F, Nishida N, Manuelidis L (2004) Two Creutzfeldt–Jakob disease agents reproduce prion protein-independent identities in cell cultures. Proc Natl Acad Sci USA 101:8768–8773
Baker CA, Martin D, Manuelidis L (2002) Microglia from CJD brain are infectious and show specific mRNA activation profiles. J Virol 76:10905–10913
Bian J, Napier D, Khaychuck V, Angers R, Graham C, Telling G (2010) Cell-based quantification of chronic wasting disease prions. J Virol 84:8322–8326
Bruce ME, Dickinson AG (1987) Biological evidence that scrapie has an independent genome. J Gen Virol 68:79–89
Couzin-Frankel J (2010) Prion diseases: no accomplice needed. ScienceNOW. Available at http://news.sciencemag.org/sciencenow/2010/01/28-03.html
Davidson I, Shulman L (2008) Unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus. Virus Res 137:1–15
Dean F, Nelson J, Giesler T, Lasken R (2001) Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11:1095–1099
Diringer H, Gelderblom H, Hilmert H, Ozel M, Edelbluth C, Kimberlin RH (1983) Scrapie infectivity, fibrils and low molecular weight protein. Nature 306:476–478
Dron M, Manuelidis L (1996) Visualization of viral candidate cDNAs in infectious brain fractions from Creutzfeldt–Jakob disease by representational difference analysis. J Neurovirol 2:240–248
Edgeworth J, Gros N, Alden J, Joiner S, Wadsworth J, Linehan J, Brandner S, Jackson G, Weissmann C, Collinge J (2010) Spontaneous generation of mammalian prions. Proc Natl Acad Sci USA 107:14402–14406
Elsner C, Dörries K (1992) Evidence of human polyomavirus BK and JC infection in normal brain tissue. Virology 191:72–80
Falsig J, Nilsson K, Knowles T, Aguzzi A (2008) Chemical and biophysical insights into the propagation of prion strains. HFSP J 2:332–341
Fondi M, Bacci G, Brilli M, Papaleo M, Mengoni A, Vaneechoutte M, Dijkshoorn L, Fani R (2010) Exploring the evolutionary dynamics of plasmids: the Acinetobacter pan-plasmidome. BMC Evolutionary Biol 10:59
Franklin R (1956) X-ray diffraction studies of cucumber virus and three strains of tobacco mosaic virus. Biochim et Biophys Acta 19:203–211
Geoghegan J, Valdes P, Orem N, Deleault N, Williamson R, Harris B, Supattapone S (2007) Selective incorporation of polyanionic molecules into hamster prions. J Biol Chem 282:36341–36353
Kekarainen T, Martínez-Guinó L, Segalés J (2009) Swine torque teno virus detection in pig commercial vaccines, enzymes for laboratory use and human drugs containing components of porcine origin. J Gen Virol 90:648–653
Li J, Browning S, Mahal S, Oelschlegel A, Weissmann C (2010) Darwinian evolution of prions in cell culture. Science 327:869–872
Liu Y, Sun R, Chakrabarty T, Manuelidis L (2008) A rapid accurate culture assay for infectivity in transmissible encephalopathies. J NeuroVirol 14:352–361
Ma S, Sakugawa H, Makino Y, Tadano M, Kinjo F, Saito A (2003) The complete genomic sequence of hepatitis delta virus genotype IIb prevalent in Okinawa, Japan. J Gen Virol 84:461–464
Maggi F, Fornai C, Vatteroni M, Siciliano G, Menichetti F, Tascini C, Specter S, Pistello M, Bendinelli M (2001) Low prevalence of TT virus in the cerebrospinal fluid of viremic patients with central nervous system disorders. J Med Virol 65:418–422
Manuelidis L (1994) Dementias, neurodegeneration, and viral mechanisms of disease from the perspective of human transmissible encephalopathies. Ann NY Acad Sci 724:259–281
Manuelidis L (1997) Beneath the emperor's clothes: the body of data in scrapie and CJD. Annales de L’Institute Pasteur 8:311–326
Manuelidis L (2003) Transmissible encephalopathies: speculations and realities. Viral Immunology 16:123–139
Manuelidis L (2007) A 25 nm virion is the likely cause of transmissible spongiform encephalopathies. J Cell Biochem 100:897–915
Manuelidis L (2010) Transmissible encephalopathy agents: virulence, geography and clockwork. Virulence 1(2):101–104
Manuelidis L, Manuelidis EE (1981) Search for specific DNAs in Creutzfeldt–Jakob infectious brain fractions using nick translation. Virol 109:435–443
Manuelidis L, Ward DC (1984) Chromosomal and nuclear distribution of the Hind III 1.9 kb repeat segment. Chromosoma (Berl) 91:28–38
Manuelidis E, Fritch W, Kim J, Manuelidis L (1987) Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt–Jakob disease agent. Proc Natl Acad Sci 84:871–875
Manuelidis L, Murdoch G, Manuelidis E (1988) Potential involvement of retroviral elements in human dementias. Ciba Found Symp 135:117–134
Manuelidis L, Sklaviadis T, Akowitz A, Fritch W (1995) Viral particles are required for infection in neurodegenerative Creutzfeldt–Jakob disease. Proc Natl Acad Sci USA 92:5124–5128
Manuelidis L, Yu Z-X, Barquero N, Mullins B (2007) Cells infected with scrapie and Creutzfeldt–Jakob disease agents produce intracellular 25-nm virus-like particles. Proc Natl Acad Sci USA 104:1965–1970
Manuelidis L, Chakrabarty T, Miyazawa K, Nduom N-A, Emmerling K (2009a) The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt–Jakob disease and scrapie agents. Proc Natl Acad Sci USA 106:13529–13534
Manuelidis L, Liu Y, Mullins B (2009b) Strain-specific viral properties of variant Creutzfeldt–Jakob Disease (vCJD) are encoded by the agent and not by host prion protein. J Cell Biochem 106:220–231
Merz PA, Somerville RA, Wisniewski HM, Manuelidis L, Manuelidis EE (1983) Scrapie associated fibrils in Creutzfeldt–Jakob disease. Nature 306:474–476
Mizuta R, Mizuta M, Kitamura D (2003) Atomic force microscopy analysis of rolling circle amplification of plasmid DNA. Arch Histol Cytol 66:175–181
Miyazawa K, Emmerling K, Manuelidis L (2010) Proliferative arrest of neural cells induces prion protein synthesis, nanotube formation, and cell-to-cell contacts. J Cell Biochem 111:239–247
Navidad P, Li H, Mankertz A, Meehan B (2008) Rolling-circle amplification for the detection of active porcine circovirus type 2 DNA replication in vitro. J Virol Methods 152:112–116
Nicoll A, Collinge J (2009) Preventing prion pathogenicity by targeting the cellular prion protein. Infect Disord Drug Targets 9:48–57
Nishida N, Katamine S, Manuelidis L (2005) Reciprocal interference between specific CJD and scrapie agents in neural cell cultures. Science 310:493–496
Oesch B, Groth DF, Prusiner SB, Weissmann C (1988) Search for a scrapie-specific nucleic acid: a progress report. Ciba Found Symp 135:209–217
Oleszak E, Manuelidis L, Manuelidis EE (1986) In vitro transformation elicited by Creutzfeldt–Jakob infected brain material. J Neuropathol Exp Neurol 45:489–502
Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216:136–144
Prusiner S, Baldwin M, Collinge J, DeArmond S, Marsh R, Tateishi J, Weissmann C (1995) Prions. Springer, Wien
Safar J, Kellings K, Serban A, Groth D, Cleaver J, Prusiner S, Riesner D (2005) Search for a prion-specific nucleic acid. J Virol 79:10796–10806
Shlomchik M, Radebold K, Duclos N, Manuelidis L (2001) Neuroinvasion by a Creutzfeldt–Jakob disease agent in the absence of B cells and follicular dendritic cells. Proc Natl Acad Sci USA 98:9289–9294
Sklaviadis T, Dreyer R, Manuelidis L (1992) Analysis of Creutzfeldt–Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation. Virus Res 26:241–254
Spelbrink J (2010) Functional organization of mammalian mitochondrial DNA in nucleoids: history, recent developments, and future challenges. IUBMB Life 62:19–32
Sun R, Liu Y, Zhang H, Manuelidis L (2008) Quantitative recovery of scrapie agent with minimal protein from highly infectious cultures. Viral Immunol 21:293–302
Supattapone S (2010) Biochemistry. What makes a prion infectious? Science 327:1091–1092
Taruscio D, Manuelidis L (1991) Integration site preferences of endogenous retroviruses. Chromosoma 101:141–156
van Tuyle G, Pavco P (1985) The rat liver mitochondrial DNA–protein complex: displaced single strands of replicative intermediates are protein coated. J Cell Biol 100:251–257
Vincent I, Carrasco C, Guzylack-Piriou L, Herrmann B, McNeilly F, Allan G, Summerfield A, McCullough K (2005) Subset-dependent modulation of dendritic cell activity by circovirus type 2. Immunology 115:388–398
Zou W, Gambetti P (2007) Prion: the chameleon protein. Cell Mol Life Sci 64:3266–3270
Acknowledgements
Supported by NINDS grant R01 012674 and NAID grant R21 A1076645. I thank John N. Davis and Kaitlin Emmerling for their interest, and discussions and suggestions on the manuscript.
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Manuelidis, L. Nuclease resistant circular DNAs copurify with infectivity in scrapie and CJD. J. Neurovirol. 17, 131–145 (2011). https://doi.org/10.1007/s13365-010-0007-0
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DOI: https://doi.org/10.1007/s13365-010-0007-0