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/cell. The DNA from the infected cells was fractionated by the Hirt procedure into low (Hirt supernatant) and high (Hirt precipitate) molecular weight DNAs, and each fraction was quantitated for infectivity by plaque assay using the calcium phosphate transfection technique of Graham and Van Der Eb (1973). The appearance of infectious DNA in the Hirt supernatant (low molecular weight viral DNA) was biphasic at all multiplicities, apparently reflecting two 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 molecular weight 6 × 106, which appeared to be present as double-stranded linear molecules. Infectious DNA in the Hirt precipitate, in contrast, had a molecular weight of 166 x 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.
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