Organization and Replication of Chromosome-Associated Polyoma Virus DNA and Flanking Cellular Sequences in Polyoma-Transformed Rat Cells

Abstract

In this chapter we review studies on the organization and replication of polyoma virus (Py) DNA sequences which have integrated into chromosomal DNA, and of cellular DNA flanking the Py integration site, in an inducible line of polyoma-transformed rat cells designated the LPT line. We also discuss the relationship between replication of the chromosome-associated viral DNA and excision of free viral DNA molecules in LPT cells treated with virus-inducing agents. These studies have shown that LPT cells contain multiple copies of Py DNA integrated into a single chromosomal site. These copies are not evenly distributed among the cells. Instead, various Py insertions include 1, 2, 3,…whole viral genomes arranged in a direct tandem repeat. A 3.0 kilobase pairs (kb) deletion of cellular DNA was discovered next to the Py integration site. LPT cells were found to be heterozygous with respect to the viral insertions and the cellular deletion. In LPT cultures treated with mitomycin c, the most effective inducing agent in this system, replication of Py DNA is enhanced after lag periods which vary in individual cells between 9 and 20 hours. DNA synthesis is repeatedly initiated within the viral insertions and the replication forks proceed in opposite directions. The leftward moving forks were analyzed in detail and found to cross the left viral DNA-cell DNA boundary and to proceed into the flanking cellular DNA sequences. Fork movement is halted within a 0.30 kb segment which maps about 2.0 kb away from the boundary. This segment may include a termination site of a normal cellular replicon. Similar results were obtained in LPT cells exposed to bromodeoxyuridine and ultraviolet light. Extrachromosomal circular Py DNA monomers are excised and replicated in induced LPT cells after the lag phase. The viral DNA monomers are encapsidated and converted into infectious virions. Excision of free viral DNA molecules and virus synthesis do not occur in an LPT subclone which contains chromosome-associated tandemly repeated viral DNA, but in which replication of the viral DNA cannot be induced. Models of papovavirus integration and induction based on studies of the LPT line and of other Py and SV40 transformants are discussed.