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.