Integration of avian sarcoma virus DNA in chicken cells

Abstract

We have used restriction endonucleases and hybridization reagents specific for various regions of the avian sarcoma virus (ASV) genome to assess the structure and sites of integration of proviruses found in chicken cells, the natural host for thiss viris, after infection in culture. Colonies of ASV-transformed chicken fibroblasts were shown to contain new proviruses located at from one to several sites in the host genome; the sites of integration appeared to differ in each of the nine colonies studied. Fractionation of chromosomes from lymphoblastoid cells infected in mass culture with ASV also failed to reveal preferred sites for insertion of ASV proviruses. Thus, in chicken cells, as well as in heterologous cells[ Hughes S. H. et al. (1978) . Cell 15, 1397–1410; Sabran, J. L., et al. (1979) . J. Virol. 29, 170–185; Martin, S. et al. (1979) . Virology, 96, 530–546; Gilmer, T., and Parsons, J. T. (1979). J. Virol. 32, 762–770], many regions of cellular DNA can accomodate an ASV provirus, and these regions are distributed over several chromosomes. Although proviruses are found at many sites in cellular DNA, the structure of proviral DNA appears to be invariant in infected chicken cells. Viral DNA is linked to cellular DNA near or at the extremities of a ca. 330-nucleotide sequence repeated at the ends of unintegrated linear DNA. The repeated structure is composed of sequences derived from both the 3′ and the 5′ ends of viral RNa. The resulting structure for ASV DNA can be written “cell DNA-3′5′- gag-pol-env-src-3′5′-cell DNA,” as similarly deduced from studies of ASV proviruses in nonpermissive, heterologous host cells[ Hughes, S. H., et al. (1978) . Cell 15, 1397–1410]. The colonies examined in detail were obtained by suspending cells in agar 3 to 6 days after infection. Some but not all of these colonies appeared to be clonal, as judged by the physical mapping of integrated proviruses. Colonies obtained from cells placed in agar directly after infection did not appear clonal by this criterion; explanations for this anomalous behavior are considered.