Clonal analysis of the integration and expression of endogenous avian retroviral DNA acquired by exogenous viral infection.

Rous-associated virus-0 is one of several endogenous avian retroviruses that are transmitted vertically and that can be isolated from different inbred lines of chickens. These viruses, referred to here as induced-leukosis viruses bearing a subgroup E glycoprotein (ILV-E), are all closely related. Clonal populations of fibroblasts from line 15B and line 100 inbred chickens have been examined for the presence and expression of exogenously acquired ILV-E sequences. Restriction enzyme analysis of uniform populations of line 15B fibroblasts, prepared by cloning cells either before or after infection with ILV-E, indicates that viral sequences were inserted at multiple sites within the cell genome. Analysis of 49 clonal populations of line 100 fibroblasts containing between one and five copies of exogenous ILV-E sequences demonstrated that each clone was characterized by a unique set of viral DNA insertions within the cell genome. The expression of the exogenous ILV-E sequences within these fibroblast clones was examined by using reverse transcriptase activity as a measure of virus production. Some clones produced an amount of virus equivalent to that produced by an equal number of the uncloned ILV-E-infected parental fibroblasts. Other clones produced 5- to 10-fold less virus. Still other clones produced no detectable virus at all. Among nine clones derived from cells containing a single copy of the ILV-E provirus, the level of virus production differed more than 100-fold. DNA from these clones was analyzed with several different restriction endonucleases to characterize the location and arrangement of the ILV-E sequences. All nine clones consisted of cells that appeared to contain a complete provirus inserted (i) in a different site within the cellular DNA and (ii) in an orientation that was colinear with the viral genomic RNA. It was observed that several cleavage sites potentially affected by methylation were equally available for cleavage in all clones regardless of the level of viral production.