Journal of NeuroVirology

, Volume 17, Issue 2, pp 131–145 | Cite as

Nuclease resistant circular DNAs copurify with infectivity in scrapie and CJD



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.


Prion Phi 29 polymerase Acinetobacter plamids Neurodegeneration Cancer Circovirus 



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.


  1. Aiken JM, Williamson JL, Marsh RF (1989) Evidence of mitochondrial involvement in scrapie infection. J Virol 63:1686–1694PubMedGoogle Scholar
  2. 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 64:3265–3268PubMedGoogle Scholar
  3. Akowitz A, Sklaviadis T, Manuelidis L (1994) Endogenous viral complexes with long RNA cosediment with the agent of Creutzfeldt–Jakob disease. Nucleic Acids Res 22:1101–1107PubMedCrossRefGoogle Scholar
  4. 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 100:603–615PubMedCrossRefGoogle Scholar
  5. 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 101:8768–8773PubMedCrossRefGoogle Scholar
  6. Baker CA, Martin D, Manuelidis L (2002) Microglia from CJD brain are infectious and show specific mRNA activation profiles. J Virol 76:10905–10913PubMedCrossRefGoogle Scholar
  7. Bian J, Napier D, Khaychuck V, Angers R, Graham C, Telling G (2010) Cell-based quantification of chronic wasting disease prions. J Virol 84:8322–8326PubMedCrossRefGoogle Scholar
  8. Bruce ME, Dickinson AG (1987) Biological evidence that scrapie has an independent genome. J Gen Virol 68:79–89PubMedCrossRefGoogle Scholar
  9. Couzin-Frankel J (2010) Prion diseases: no accomplice needed. ScienceNOW. Available at
  10. Davidson I, Shulman L (2008) Unraveling the puzzle of human anellovirus infections by comparison with avian infections with the chicken anemia virus. Virus Res 137:1–15PubMedCrossRefGoogle Scholar
  11. 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 11:1095–1099PubMedCrossRefGoogle Scholar
  12. Diringer H, Gelderblom H, Hilmert H, Ozel M, Edelbluth C, Kimberlin RH (1983) Scrapie infectivity, fibrils and low molecular weight protein. Nature 306:476–478PubMedCrossRefGoogle Scholar
  13. Dron M, Manuelidis L (1996) Visualization of viral candidate cDNAs in infectious brain fractions from Creutzfeldt–Jakob disease by representational difference analysis. J Neurovirol 2:240–248PubMedCrossRefGoogle Scholar
  14. 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 107:14402–14406PubMedCrossRefGoogle Scholar
  15. Elsner C, Dörries K (1992) Evidence of human polyomavirus BK and JC infection in normal brain tissue. Virology 191:72–80PubMedCrossRefGoogle Scholar
  16. Falsig J, Nilsson K, Knowles T, Aguzzi A (2008) Chemical and biophysical insights into the propagation of prion strains. HFSP J 2:332–341PubMedCrossRefGoogle Scholar
  17. 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 10:59CrossRefGoogle Scholar
  18. Franklin R (1956) X-ray diffraction studies of cucumber virus and three strains of tobacco mosaic virus. Biochim et Biophys Acta 19:203–211CrossRefGoogle Scholar
  19. 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 282:36341–36353PubMedCrossRefGoogle Scholar
  20. 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 90:648–653PubMedCrossRefGoogle Scholar
  21. Li J, Browning S, Mahal S, Oelschlegel A, Weissmann C (2010) Darwinian evolution of prions in cell culture. Science 327:869–872PubMedCrossRefGoogle Scholar
  22. Liu Y, Sun R, Chakrabarty T, Manuelidis L (2008) A rapid accurate culture assay for infectivity in transmissible encephalopathies. J NeuroVirol 14:352–361PubMedCrossRefGoogle Scholar
  23. 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 84:461–464PubMedCrossRefGoogle Scholar
  24. 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 65:418–422PubMedCrossRefGoogle Scholar
  25. Manuelidis L (1994) Dementias, neurodegeneration, and viral mechanisms of disease from the perspective of human transmissible encephalopathies. Ann NY Acad Sci 724:259–281PubMedCrossRefGoogle Scholar
  26. Manuelidis L (1997) Beneath the emperor's clothes: the body of data in scrapie and CJD. Annales de L’Institute Pasteur 8:311–326Google Scholar
  27. Manuelidis L (2003) Transmissible encephalopathies: speculations and realities. Viral Immunology 16:123–139PubMedCrossRefGoogle Scholar
  28. Manuelidis L (2007) A 25 nm virion is the likely cause of transmissible spongiform encephalopathies. J Cell Biochem 100:897–915PubMedCrossRefGoogle Scholar
  29. Manuelidis L (2010) Transmissible encephalopathy agents: virulence, geography and clockwork. Virulence 1(2):101–104PubMedCrossRefGoogle Scholar
  30. Manuelidis L, Manuelidis EE (1981) Search for specific DNAs in Creutzfeldt–Jakob infectious brain fractions using nick translation. Virol 109:435–443CrossRefGoogle Scholar
  31. Manuelidis L, Ward DC (1984) Chromosomal and nuclear distribution of the Hind III 1.9 kb repeat segment. Chromosoma (Berl) 91:28–38CrossRefGoogle Scholar
  32. 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 84:871–875PubMedCrossRefGoogle Scholar
  33. Manuelidis L, Murdoch G, Manuelidis E (1988) Potential involvement of retroviral elements in human dementias. Ciba Found Symp 135:117–134PubMedGoogle Scholar
  34. 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 92:5124–5128PubMedCrossRefGoogle Scholar
  35. 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 104:1965–1970PubMedCrossRefGoogle Scholar
  36. 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 106:13529–13534PubMedCrossRefGoogle Scholar
  37. 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 106:220–231PubMedCrossRefGoogle Scholar
  38. Merz PA, Somerville RA, Wisniewski HM, Manuelidis L, Manuelidis EE (1983) Scrapie associated fibrils in Creutzfeldt–Jakob disease. Nature 306:474–476PubMedCrossRefGoogle Scholar
  39. Mizuta R, Mizuta M, Kitamura D (2003) Atomic force microscopy analysis of rolling circle amplification of plasmid DNA. Arch Histol Cytol 66:175–181PubMedCrossRefGoogle Scholar
  40. 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 111:239–247PubMedCrossRefGoogle Scholar
  41. 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 152:112–116PubMedCrossRefGoogle Scholar
  42. Nicoll A, Collinge J (2009) Preventing prion pathogenicity by targeting the cellular prion protein. Infect Disord Drug Targets 9:48–57PubMedGoogle Scholar
  43. Nishida N, Katamine S, Manuelidis L (2005) Reciprocal interference between specific CJD and scrapie agents in neural cell cultures. Science 310:493–496PubMedCrossRefGoogle Scholar
  44. Oesch B, Groth DF, Prusiner SB, Weissmann C (1988) Search for a scrapie-specific nucleic acid: a progress report. Ciba Found Symp 135:209–217PubMedGoogle Scholar
  45. Oleszak E, Manuelidis L, Manuelidis EE (1986) In vitro transformation elicited by Creutzfeldt–Jakob infected brain material. J Neuropathol Exp Neurol 45:489–502PubMedCrossRefGoogle Scholar
  46. Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216:136–144PubMedCrossRefGoogle Scholar
  47. Prusiner S, Baldwin M, Collinge J, DeArmond S, Marsh R, Tateishi J, Weissmann C (1995) Prions. Springer, WienGoogle Scholar
  48. Safar J, Kellings K, Serban A, Groth D, Cleaver J, Prusiner S, Riesner D (2005) Search for a prion-specific nucleic acid. J Virol 79:10796–10806PubMedCrossRefGoogle Scholar
  49. 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 98:9289–9294PubMedCrossRefGoogle Scholar
  50. 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 26:241–254PubMedCrossRefGoogle Scholar
  51. Spelbrink J (2010) Functional organization of mammalian mitochondrial DNA in nucleoids: history, recent developments, and future challenges. IUBMB Life 62:19–32PubMedGoogle Scholar
  52. Sun R, Liu Y, Zhang H, Manuelidis L (2008) Quantitative recovery of scrapie agent with minimal protein from highly infectious cultures. Viral Immunol 21:293–302PubMedCrossRefGoogle Scholar
  53. Supattapone S (2010) Biochemistry. What makes a prion infectious? Science 327:1091–1092PubMedCrossRefGoogle Scholar
  54. Taruscio D, Manuelidis L (1991) Integration site preferences of endogenous retroviruses. Chromosoma 101:141–156PubMedCrossRefGoogle Scholar
  55. 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 100:251–257PubMedCrossRefGoogle Scholar
  56. 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 115:388–398PubMedCrossRefGoogle Scholar
  57. Zou W, Gambetti P (2007) Prion: the chameleon protein. Cell Mol Life Sci 64:3266–3270PubMedCrossRefGoogle Scholar

Copyright information

© Journal of NeuroVirology, Inc. 2010

Authors and Affiliations

  1. 1.Yale University Medical SchoolNew HavenUSA

Personalised recommendations