Isolation and characterization of covalently closed circular proviral DNA molecules of several type D retroviruses isolated from human cell lines.

Infection of a human lymphoblastoid B cell line (Raji cells) with type D retroviruses, originally isolated either from subhuman primates (MPMV, LV) or from permanent human cell lines (PMFV, HeLaV, HEp-2V) led to the production of type D retrovirus particles. Subsequent cocultivation of uninfected and virus-producing Raji cells was employed for the generation of sufficient amounts of covalently closed circular DNA molecules (cccDNA). Highest amounts of cccDNA were obtained after cocultivation of virus-producing Raji cells and homologous uninfected cells at a ratio of 1 to 3 for 72 hr. The cccDNAs of type D retroviruses migrated at about 4.3 kbp compared to lambda DNA/HindIII markers. Digestion of cccDNAs with restriction endonucleases which have one recognition site generated molecules of approximately 8 kbp. The restriction endonuclease site analysis of the cccDNA of type D retroviruses revealed a genomic heterogeneity among the different isolates.