SYNTHESIS AND STRUCTURE OF VISNA VIRUS-DNA

The synthesis of proviral DNA of visna virus was measured at various intervals after inoculation of sheep cell cultures at multiplicities of 0.3, 1, and 10 PFU[plaque forming units]cell. The DNA from the infected cells was fractionated by the Hirt procedure into low (Hirt supernatant) and high (Hirt precipitate) MW DNA and each fraction was quantitated for infectivity by plaque assay using the calcium phosphate transfection technique of Graham and Van Der Eb. The appearance of infectious DNA in the Hirt supernatant was biphasic at all multiplicities, apparently reflecting 2 rounds of synthesis of this DNA. The amount of infectious DNA in the Hirt precipitate fraction increased with time, reaching maximum levels in all cultures at the time of peak virus production. Hirt supernatant DNA consisted predominantly of molecules of MW 6 .times. 106, which appeared to be present as double-stranded linear molecules. Infectious DNA in the Hirt precipitate had a MW of 166 .times. 106, suggesting its association with cellular sequences. The network test strongly suggested that the viral sequences were covalently linked with or integrated into the cellular DNA.