Detection, purification, and characterization of two species of covalently closed circular proviral DNA molecules of bovine leukemia virus

S V Kashmiri, R Mehdi, J F Ferrer
1983 Journal of Virology  
Cocultivation of uninfected and bovine leukemia virus-producing bat cells yielded, in addition to the unintegrated linear DNA duplex, DNA molecules that migrated as 4.4-and 4.8-kilobase-pair DNA fragments in gel electrophoresis. These DNA molecules were purified by acid-phenol extraction and cleaved with restriction endonucleases EcoRI, and HindIII, which have one recognition site each on the bovine leukemia virus proviral DNA. Such cleavage generated DNA molecules of approximately 10.0 and 9.4
more » ... kilobase pairs, thus indicating the existence of two species of covalently closed circular molecules of bovine leukemia virus proviral DNA. Insertion of retroviral DNA sequence into the genome of the host is an essential step for both productive infection and morphological transformation of cells in culture. After infection, the incoming genomic RNA is copied into a DNA intermediate by the virion-associated, RNA-dependent DNA polymerase. As a result, several copies of viral linear DNA duplex appear in the cytoplasm of infected cells (22, 24) . Subsequently, the double-stranded linear DNA finds its way into the nucleus, and two major species of covalently closed circular DNA molecules appear in the nucleus (9, 17, 24) . It is not yet known whether the linear form or one of the circular forms of DNA is the immediate precursor of the integrated viral genome. The linear DNA molecule is longer than the genomic RNA, because each of its termini carries copies of unique sequences of both the 3' and the 5' ends of viral RNA (4, 9, 17) . These terminal se-
doi:10.1128/jvi.45.3.1172-1176.1983 fatcat:cfqy2uype5hd7ppmjvx7eo2cgq