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Under permissive conditions of growth in tissue culture, the retrovirus visna multiplies over the course of a few days to high titer and kills the host cell. We show that in this lytic life cycle, viral DNA is tightly associated with, but not covalently linked to, chromosomal DNA. This finding provides explanations for a number of the unusual properties of the lentivirus subfamily of retroviruses, and suggests potential mechanisms for the block in virus gene expression in vivo responsible for the slow infection in nature.Visna virus is the prototype of the subfamily of retroviruses that cause slow infections in sheep characterized by pathological changes in the lungs and central nervous system (1,2). Both the persistence of virus in the face of the immune response mounted by the host and the slow evolution of disease can be explained by restriction in virus gene expression imposed at the transcriptional level (3, 4). Most of the cells that harbor virus genomes have insufficient antigen to be detected and destroyed by immune surveillance, and limitation in synthesis of virus gene products allows the host cell to survive for the extended periods characteristic of slow infections.The mechanism of the block in transcription is unknown but has generally been assumed to be related to a lysogenic state, since visna virus is a retrovirus, and viral DNA is associated with high molecular weight host DNA (5-7). However, we recently found that transcription in vitro is governed by the extent of early DNA synthesis and suggested that extrachromosomal DNA might be the template for viral RNA (8). Moreover, Panganiban and Temin (9) have shown that production of spleen necrosis virus, an avian C-type retrovirus, can occur from unintegrated viral DNA. For these reasons, we reexamined the role of integration in the visna life cycle in vitro and found that in the vast majority of cells unintegrated DNA must serve as the template for transcription and for virus production. MATERIALS AND METHODSInfection of Cells and Isolation of DNA. Confluent cultures of sheep choroid plexus (SCP) cells were infected at a multiplicity of 3 plaque-forming units per cell as described (8,10). At 60-70 hr after infection, cells were removed by trypsinization and either replated under agarose for infectious center assay or separated into nuclear and cytoplasmic fractions (11). High molecular weight DNA was isolated by the Hirt fractionation procedure (12). In some experiments, high molecular weight DNA was purified further by electrophoresis in 0.5% low-gelling temperature agarose; the portion of the gel containing DNA 20 kilobase pairs (kbp) or greater in size was excised and melted, and DNA was isolated by phenol/chloroform extraction.Cloning of Visna DNA. Viral DNA for probes and for reconstruction experiments was obtained by cloning. The relevant restriction enzyme sites in visna DNA (9) are shown in Fig. 1. DNA from the Hirt supernatant fraction from SCP cells infected for 60 hr was digested with Sst I, and the fragments were inserted into ...

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