Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.
We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.
Dunn
M M
MM
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7295.
Olsen
J C
JC
Swanstrom
R
R
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002645:Chickens; D004279:DNA, Viral / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012313:RNA / Q000032:analysis; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D016662:Virus Integration
D004279:DNA, Viral; D012313:RNA