Journal of General Virology (1993), 74, 125-128. Printed in Great Britain 125 Distinct segments of the hamster polyomavirus regulatory region have differential effects on DNA replication Christophe de La Roche Saint Andr6 and Jean Feunteun* Laboratoire d'Oncologie Mol6culaire, Institut Gustave Roussy, 94805 Villejuif Cedex, France The replication ofplasmids containing various fragments of the hamster polyomavirus (HaPV) DNA non-coding region was tested in a permissive hamster cell line. We first investigated the importance of some methodological parameters including the time course and the amount of transfecting plasmid DNA and have shown that these factors can greatly influence the relative amount of newly replicated DNA accumulated within the trans- fected cells. Taking these into account, quantitative comparisons could be made showing the effect of various parts of the regulatory sequence on the HaPV DNA replication. Functional analysis of the regulatory region of the polyomavirus genome has defined three distinct elements involved in viral DNA replication and transcription (DePamphilis, 1989): an upstream transcriptional enhancer, an origin of replication and a downstream region containing large T antigen-binding sites. The sequence of the hamster polyomavirus (HaPV) regu- latory region suggests a similar organization (Delmas et al., 1985). Conserved motifs are recognized in the region in which viral DNA replication putatively initiates. They include a GC-rich palindrome that contains a putative binding site for the large T antigen surrounded on its late side by an AT-rich sequence and on its early side by an inverted repeat sequence. Together, these three conserved motifs are referred to as the core component of the replication origin (ORI in the figures), that is required for replication under all conditions. Thus, small deletions within the GC-rich palindrome or the adjacent AT-rich sequence inactivate the HaPV origin of replication in permissive cells in vitro (de La Roche Saint Andr6 et al., 1989). The remainder of the non-coding region, con- taining promoter or enhancer elements, constitutes the auxiliary component of the replication origin, possibly dispensable under some conditions. The aim of the experiments reported here was to characterize functionally some of the sequences in the non-coding region that might influence the replication of the HaPV genome. The replication capacity of recombinant plasmids containing various DNA fragments of the HaPV regulatory region was measured after their introduction into the permissive hamster cell line BOH. BOH cells are BHK cells transformed by an origin-defective mutant of HaPV. This mutant represents the wild-type HaPV genome carrying a 4 bp deletion of the single ApaI site located in the middle of the GC-rich palindrome (de La Roche Saint Andr6 et al., 1989). BOH cells constitutively express the viral early genes and provide a permissive context for the replication of recombinant plasmids containing an HaPV origin. The replication of virus origin-carrying vectors is usually measured at a fixed time after the transfection of a standard amount of plasmid DNA. The amount of unmethylated DpnI-resistant DNA species accumulated in the transfected cells is taken as a measure of the replication activity (Katinka & Yaniv, 1983; Muller et al., 1983; Veldman et al., 1985; Li et al., 1986; Hertz & Mertz, 1986; Deb et al., 1986). Quantitative data (average results from several experiments) obtained under these conditions are used to express the relative replication capacities of plasmids. The influence on replication of sequences flanking the polyomaviruses' origin cores has been studied by several laboratories, but even when the experimental protocols used were similar the quantitative assessment of the effects of these sequences varied considerably. For example, there is a considerable variation in the magnitude of stimulation reported for the simian virus 40 origin auxiliary sequences (see Guo et al., 1989, for references). This led us to evaluate the influence of some parameters involved in the assay on the quantitative results obtained, to optimize the comparative analysis of various plasmids in our experimental system. The replication time course of the HaPV-carrying vectors was the first parameter investigated. The kinetic experiment presented in Fig. 1 compares the replication activities of two representative plasmids. The reference plasmid Ha-CAT121, which contains the HpaI-EcoRI fragment (nucleotides 4854 to 199) spanning the entire non-coding region of the wild-type HaPV DNA cloned 0001-1183 © 1993SGM