Summary
Scrapie and Creutzfeldt-Jakob disease (CJD) are caused by infectious agents that are defined phenomenologically. No agent-specific molecules or particles have been identified. Biological properties, such as exponential agent replication and strain variation, as well as physical characteristics of infectivity indicate a protected viral structure. A host membrane glycoprotein of 34 kDa (“prion” protein) that aggregates at end stages of disease is clearly important in pathology and susceptibility to infection, but has no demonstrable infectivity in any purified or recombinant form. Thus a characterization of more viral-like molecules is important. In order to identify viral-like nucleic acids we previously developed methods to substantially purify the human CJD agent from experimentally infected hamster brains, and demonstrated selected retro-viral-like LTR bands at pg levels that were insufficient for sequencing. To further define these and other viral-like sequences we cloned nucleic acids from highly infectious CJD fractions, and tested the efficacy of our methods using a selected retroviral probe. RNA extracted from an infectious 120 S Gaussian peak, which is reproducibly purified ∼ 100,000 fold with respect to starting nucleic acids, and contains ∼ 20% of the initial brain infectivity, was used to generate a cDNA library in a sequence independent amplification strategy for low levels of RNA (<6 ng). Reconstituted strong stop experiments using several retroviral tRNA primers had indicated that Syrian hamster IAP (SHIAP) sequences should be present in both CJD and uninfected control fractions. Because SHIAP particles are extremely resistant to denaturation, their representation in a cDNA library would imply adequate extraction of other protected RNAs of viral origin. At least 900 bases of the Syrian hamster retroviral IAP genome were unambiguously identified in the cDNA library, and in independent PCR walks with selected primers, all of which were based on our cloned sequences. Sequencing confirmed the presence of protected LTR and adjacent retroviral motifs. Because these sequences were also present in control preparations they may represent normal endogenous viral contaminants that cosediment with infectivity in size and density gradients. On the other hand, LTRs can drive the expression of many diverse sequences, and it remains to be seen if CJD specific sequences are either transduced, or copackaged with, protected IAP complexes. The effective extraction and amplification of highly protected SHIAP nucleic acids of significant length sets the stage for identifying additional protected viral elements that may specify the CJD agent.
Similar content being viewed by others
References
Aiken JM, Williamson JL, Borchardt M, Marsh RF (1990) Presence of mitochondrial D-loop DNA in scrapie-infected brain preparations enriched for the prion protein. J Virol 64: 3265–3268
Akowitz A, Manuelidis L (1989) A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA. Gene 81: 295–306
Akowitz A, Sklaviadis T, Manuelidis EE, Manuelidis L (1990) Nuclease resistant polyadenylated RNAs of significant size are detected by PCR in highly purified Creutzfeldt-Jakob disease preparations. Microb Pathog 9: 33–45
Baker HF, Ridley RM, Crow TJ (1985) Experimental transmission of an autosomal dominant spongiform encephalopathy: does the infectious agent originate in the human genome? Br Med J 291: 299–302
Bellinger-Kawahara C, Cleaver JE, Diener TO, Prusiner SB (1987) Purified scrapie prions resist inactivation by UV-irradiation. J Virol 61: 159–166
Bruce ME, Dickinson AG (1987) Biological evidence that scrapie has an independent genome. J Gen Virol 68: 79–89
Bruce ME, McConnell I, Fraser H, Dickinson AG (1991) The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol 72: 595–603
Canaani E, Dreazen O, Klar A, Rechavi G, Ram D, Cohen JB, Givol D (1983) Activation of the c-mos oncogene in a mouse plasmacytoma by insertion of an endogenous intracisternal A-particle genome. Proc Natl Acad Sci USA 80: 7118–7123
Czub MB, Braig HR, Diringer H (1986) Pathogenesis of scrapie: study of the temporal development of clinical symptoms of infectivity titers and scrapie-associated fibrils in brains of hamsters infected intraperitoneally. J Gen Virol 67: 2005–2009
Czub M, Braig HR, Diringer H (1988) Replication of the scrapie agent in hamsters infected intracerebrally confirms the pathogenesis of an amyloid-inducing virosis. J Gen Virol 69: 1753–1756
Diedrich JF, Minnigar H, Carp RI, Whitaker JN, Race R, Frey W, Haase AT (1991) Neuropathological changes in scrapie and Alzheimer's disease are associated with increased expression of Apoliopoprotein E and cathepsin D in astrocytes. J Virol 65: 4759–4768
Diringer H (1984) Sustained viremia in experimental hamster scrapie. Arch Virol 82: 105–109
Dickinson AG, Fraser H (1977) Scrapie: pathogenesis in inbred mice; an assessment of host control and response involving many strains of agent. In: ter Meulen V, Katz M (eds) Slow virus infections of the central nervous system. Springer, Wien New York, pp 3–14
Duguid JR, Rohwer RG, Seed B (1988) Isolation of cDNAs of scrapie-modulated RNAs by subtractive hybridization of a cDNA library. Proc Natl Acad Sci USA 85: 5738–5742
Duguid JR, Bohmont CW, Liu N, Tourtellotte WW (1989) Changes in brain expression shared by scrapie and Alzheimer disease. Proc Natl Acad Sci USA 86: 7260–7264
Ehlers B, Diringer H (1984) Dextran sulphate 500 delays and prevents mouse scrapie by impairment of agent replication in spleen. J Gen Virol 65: 1325–1330
Ehlers B, Rudolph R, Diringer H (1984) The reticuloendothelial system in scrapie pathogenesis. J Gen Virol 65: 423–428
Eklund C, Hadlow NJ, Kennedy RC (1963) Some properties of the scrapie agent and its behaviour in mice. Proc Soc Exp Biol 112: 974–979
Hawley RG, Shulman MJ, Hozui N (1984) Transposition of two different intracisternal A-particle elements into an immunoglobulin kappa-chain gene. Mol Cell Biol 4: 2565–2572
Heidmann O, Heidmann T (1991) Retrotransposition of a mouse IAP sequence tagged with an indicator gene. Cell 64: 159–170
Kimberlin RH, Walker CA (1986) Pathogenesis of scrapie (strain 263 K) in hamsters infected intracerebrally, intraperitoneally or intraoccularly. J Gen Virol 67: 255–263
Kuff EL, Leuders KK (1988) The intracisternal A-particle gene family: structure and functional aspects. Adv Cancer Res 51: 183–276
Kuroda Y, Gibbs CJ, Gajdusek DC (1983) Creutzfeldt-Jakob disease in mice: persistent viremia and preferential replication of virus in low density lymphocytes. Infect Immun 41: 154–161
Leuders KK, Kuff EL (1983) Comparison of the sequence organization of related retrovirus-like multigene families in three evolutionary distinct rodent genomes. Nucleic Acids Res 11: 4391–4408
Manuelidis EE (1985) Creutzfeldt-Jakob disease. J Neuropathol Exp Neurol 44: 1–17
Manuelidis EE, Angelo J, Kim JH, Manuelidis L (1976) Serial propagation of Creutzfeldt-Jakob disease in guinea pigs. Proc Natl Acad Sci USA 73: 223–227
Manuelidis EE, deFigueiredo JM, Kim JH, Fritch WW, Manuelidis L (1988) Transmission studies from blood of Alzheimer's disease patients and healthy relatives. Proc Natl Acad Sci USA 85: 4898–4901
Manuelidis EE, Fritch WW, Kim JH, Manuelidis L (1987) Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent. Proc Natl Acad Sci USA 84: 871–875
Manuelidis EE, Gorgacz EJ, Manuelidis L (1978) Viremia in experimental Creutzfeldt-Jakob disease. Science 200: 1069–1071
Manuelidis EE, Gorgacz EJ, Manuelidis L (1978) Transmission of Creutzfeldt-Jakob disease to mice with scrapie-like syndromes. Nature 271: 778–779
Manuelidis EE, Gorgacz EJ, Manuelidis L (1978) Interspecies transmission of Creutzfeldt-Jakob disease to Syrian hamsters with reference to clinical syndromes and strains of agent. Proc Natl Acad Sci USA 75: 3432–3436
Manuelidis EE, Kim JH, Manuelidis L (1986) Serial transmission of Gerstmann-Straussel Scheinker disease to different strains of hamsters. In: Abstracts X Interim. Congress of Neuropatholy, Stockholm, Sweden, 1986, p 166
Manuelidis EE, Kim JH, Mericangas JR, Manuelidis L (1985) Transmission of Creutzfeldt-Jakob disease from human blood. Lancet ii: 896–897
Manuelidis EE, Manuelidis L (1979) Clinical and morphological aspects of transmissible Creutzfeldt-Jakob disease. Prog Neuropathol 4: 1–26
Manuelidis EE, Manuelidis L (1989) Suggested links between different types of dementias: Creutzfeldt-Jakob disease, Alzheimer's disease and retroviral CNS infections. Alzheimer Dis Assoc Disord 3: 101–109
Manuelidis EE, Manuelidis L (1991) Search for a transmissible agent in Alzheimer's disease: studies of human buffy coat. In: Chesebro BW (ed) Transmissible spongiform encephalopathies, scrapie, BSE and related disorders. Springer, Berlin Heidelberg New York Tokyo, pp 275–280 (Current topics in microbiology and immunology, vol 172)
Manuelidis L, Murdoch G, Manuelidis EE (1988) Potential involvement of retroviral elements in human dementias. CIBA Found Symp 135: 117–134
Manuelidis EE, Rorke LB (1989) Transmission of Alper's disease (chronic progressive encephalopathy) produces experimental Creutzfeldt-Jakob disease in hamsters. Neurology 39: 615–621
Manuelidis L, Sklaviadis T, Manuelidis EE (1987) Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease. EMBO J 6: 341–347
Manuelidis L, Tesin DM, Sklaviadis T, Manuelidis EE (1987) Astrocyte gene expression in Creutzfeldt-Jakob disease. Proc Natl Acad Sci USA 84: 5937–5941
Marciani DJ, Kuff EL (1973) Isolation and partial characterization of the internal structural proteins from murine intracisternal A particles. Biochemistry 12: 5075–5083
Meyer N, Rosenbaum V, Schmidt B, Gilles K, Mirenda C, Groth D, Prusiner SB, Riesner D (1991) Search for a putative scrapie genome in purified prion fractions reveals a paucity of nucleic acids. J Gen Virol 72: 37–49
Murdoch GH, Sklaviadis T, Manuelidis EE, Manuelidis L (1990) Potential retroviral RNAs in Creutzfeldt-Jakob disease. J Virol 64: 1477–1486
Oesch B, Groth DF, Prusiner SB, Weissman C (1988) Search for a scrapie-specific nucleic acid: a progress report. CIBA Found Symp 135: 209–233
Oleszak EL, Manuelidis L, Manuelidis EE (1986) In vitro transformation elicited by Creutzfeldt-Jakob infected brain material. J Neuropathol Exp Neurol 45: 489–502
Ono M, Toh H, Miyata T, Awaya T (1985) Nucleotide sequence of the Syrian hamster intracisternal A-particle gene: close evolutionary relationship of type A particle gene to types B and D oncovirus genes. J Virol 55: 387–394
Ono M, Yasunaga T, Miyata T, Ushikubo H (1986) Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome. J Virol 60: 589–598
Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science 216: 136–144
Prusiner SB (1989) Scrapie prions. Annu Rev Microbiol 43: 345–374
Rubenstein R, Merz PA, Kascsak RJ, Scalici CL, Papini MC, Carp RI, Kimberlin RH (1991) Scrapie-infected spleens: analysis of infectivity, scrapie-associated fibrils, and protease-resistant proteins. J Infect Dis 164: 29–35
Shih A, Misra R, Rush MG (1989) Detection of multiple, novel reverse transcriptase coding sequences in human nucleic acids: relation to primate retroviruses. J Virol 65: 64–75
Sklaviadis T, Akowitz A, Manuelidis EE, Manuelidis L (1990) Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of a nucleic acid-protein complex. Arch Virol 112: 215–229
Sklaviadis T, Manuelidis L, Manuelidis EE (1989) Physical properties of the Creutzfeldt-Jakob disease agent. J Virol 63: 1212–1222
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
Taruscio D, Manuelidis L (1991) Integration site preferences of endogenous retroviruses. Chromosoma 101: 141–156
Tateishi J (1985) Transmission of Creutzfeldt-Jakob disease from human blood and urine into mice. Lancet ii: 1074
Tateishi J, Nagara H, Hikita K, Sato Y (1984) Amyloid plaques in the brains of mice with Creutzfeldt-Jakob disease. Ann Neurol 15: 278–280
Weiss R, Teich N, Varmos H, Coffin J (1984) RNA tumor viruses, 2nd edn. Cold Spring Harbor Laboratory, New York
Wietgrefe SM, Zupanic M, Hasse A, Chesebro B, Race R, Frey W, Rustan T, Friedmann RL (1985) Cloning of a gene whose expression is increased in scrapie and in senile plaques in human brain. Science 230: 1177–1179
Xi YG, Ingrosso L, Ladogana A, Masullo C, Pocchiari M (1992) Amphotericin B treatment dissociates in vivo replication of the scrapie agent from PrP accumulation. Nature 356: 598–601
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Akowitz, A., Manuelidis, E.E. & Manuelidis, L. Protected endogenous retroviral sequences copurify with infectivity in experimental Creutzfeldt-Jakob disease. Archives of Virology 130, 301–316 (1993). https://doi.org/10.1007/BF01309662
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01309662