Nuclease resistant circular DNAs copurify with infectivity in scrapie and CJD

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.

Replication and spread of CJD, kuru and scrapie agents in vivo and in cell culture

“…The GT1 culture model provides an inexpensive and stable source of unique TSE agents that can be rapidly titered with only ~3-fold less sensitivity than 500 d mouse assays. Infectivity assays are essential for discovery of agent-specific molecules, 6,27 and in analyzing various TSE agents we uncovered profound agent differences by serial end-point titrations. The current extensive end-point determinations for many mouse brains and infected GT1 cells was not practical by animal assay, and GT1 assays showed reproducible end-point titers for independent samples.…”

“…24,25 Moreover, when these infectious particles are disrupted, they lose their infectivity and release nucleic acids of viral size, as well as protective nucleic acid binding proteins. 24,26 The recent discovery of new circular DNAs, designated Sphinx elements in many different infectious samples that can encode agent-specific properties, 27 further undermines the presumption of a protein-only agent.…”

“…We have discovered several new circular DNAs of viral size (1.8 kb and 2.4 kb) in various TSE infectious particle preparations that resist detergent lysis and nuclease digestion. 27 Interestingly, these DNA sequences have links to environmental endosymbiotic pathogens that are widespread in soil and water. Such prevalent and possibly ancient agents may infrequently cause disease.…”

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms

“…Although the vast majority of host nucleic acids were removed in the p18 (line D), residual mitochondrial and nuclear DNA could still be detected by gel analysis of >5e6 cell equivalents (CE) on a Sybr Gold stained agarose gel lane, as well as by PCR of long mitochondrial DNA as previously detailed. 45 Since there are 3 TCID/cell of the FU-CJD agent prior to nucleic acid extraction, this represents a load of e7 TCID on a lane, far more than others have loaded on a gel to conclude that there are no nucleic acids of >50nt in infectious preparations.…”

“…13 Non-PrP proteins that are particle-associated 20,46 may also be needed to effectively transmit an agent genome from among the many nucleic acids present in all highly infectious preparations. 41,45 Thiourea (ThU) alone, or combined with urea, is very effective for destroying >4 logs of TSE infectivity within 1 hr at 22 C, and ThU has not been used previously for TSE agent disruption. Notably, this chemical combination disrupts keratins, 56 and a keratin-like protein structure(s) may protect TSE agents.…”