Journal of Molecular Biology
Regular articleSite-specific in Situ amplification of the integrated polyomavirus genome: A case for a context-specific over-replication model of gene amplification1
Introduction
The genomes of small DNA tumor viruses occasionally integrate into the chromosomes of their hosts. Thereafter most of them apparently become passively replicated as part of the cellular sequences. In this as in many other aspects, the fate of the polyomaviruses genomes (e.g. murine polyomavirus (Py) and SV40) is of particular interest. This is because these genomes are packaged as cellular chromatin and use the cellular DNA replication machine. They encode a single protein, large T antigen, that is directly involved in DNA replication. Large T binds to specific viral sequences that are part of the viral origin, induces DNA melting around the binding sites, targets the host replication machine to the viral genomes and unwinds DNA ahead of the replication forks (reviewed by Hassell & Brinton, 1996). However, unlike host origins, the viral origin must fire many times during a single cell-cycle to generate high virus yields. Thus, when the viral genomes are integrated, origin overfiring might be expected. In situ amplification of the integrated genomes has rarely been demonstrated, and when so, to very moderate extents. However, overfiring has been proposed as a model to explain the presence of “excised” genomes present in low abundance in some transformants (Botchan et al., 1976).
One way to select for integrated viral genomes is provided by the ability of these viruses to induce neoplastic transformation. This is best observed in unnatural host cells, so-called “non-permissive”, that have a reduced capacity to replicate the viral genome and can survive the infection (e.g. rat cells for Py). In this case, integration is thought to be required to achieve stable neoplastic transformation, as a way to ensure the maintenance of the viral genome (and hence that of the transforming function) in the absence of efficient viral genome replication Stoker 1968, Stoker and Dulbecco 1969. Thus, a minimal estimate of the integration frequency is based on the stable transformation frequency. This frequency is low and varies depending upon a number of parameters (between 0.05 and 2% under optimal conditions for Py infection of rat FR3T3 cells). Since a higher frequency of transient “abortive” transformation is observed, it has been proposed that integration is the major rate-limiting step in transformation and a rare event (Stoker, 1968).
The current understanding of the integration of the genomes of Py and SV40 into host chromosomes is limited. A role for large T has been implied from the results of studies with temperature-sensitive mutants (ts-a/A type mutants). These have shown that a large T function is mediated at the stage of initiation of the transformed phenotype Fried 1965a, Fried 1965b, Fluck and Benjamin 1979. This domain of large T presumably facilitates integration Stoker and Dulbecco 1969, Hacker and Fluck 1989 and affects the topology of the integrated viral sequences (Della-Valle et al., 1981). The mechanism of action of large T in integration is still unknown; it has been proposed that genomes that have initiated replication present a topology that is favorable for integration Pellegrini et al 1984, Hacker and Fluck 1989.
The analysis of the integration patterns of the viral genome in neoplastically transformed cells (Botchan et al., 1976) has focused on counting the number of integration sites and on providing maps of the integrated viral sequences. These studies have shown that integration patterns vary from transformant to transformant. Hence it has been suggested that the integration of Py and SV40 genomes is a random process, occurring without apparent specificity with respect to either cellular or viral sequences. In situ amplification has not been studied systematically and has not been revealed as a major phenomenon in previous studies. However, other features have appeared that we now believe to be related to amplification (reviewed in Discussion).
Here, we have re-evaluated the integration pattern of the Py genome in non-permissive FR3T3 rat cells and examined the effect of viral DNA replication on these patterns, in particular with respect to the apparent number of integration sites. By using high-resolution electrophoresis, we have shown that the apparent number of cell DNA fragments containing an integrated viral genome in 20 of 34 transformants analyzed is much larger than that revealed in previous analyses, and that these fragments form a regular ladder-like banding pattern. We have examined the effect of viral DNA replication on the integration pattern in 22 independent transformants derived from infections with large T temperature-sensitive mutants. These transformants could be divided into two major groups, depending upon the appearance of their integration patterns in response to the temperature of incubation past the integration stage, i.e. in response to inhibiting or permitting viral DNA replication. One group showed a simple temperature-invariant pattern characterized by a single integration band of transformant-specific size. Transformants in the other group displayed simple patterns upon incubation at high temperature and patterns with transformant-specific complexities resulting from genome amplification, when incubated at low temperature. A recurring ladder-like pattern was observed for most transformants capable of amplification. The comparisons of the characteristics of the two groups of transformants allowed the definition of features essential for the formation of the ladder-like patterns. These include an intact viral DNA replication function and the presence of a 1 to 2 kb reiteration in the integrated viral sequences. The analysis of the properties of transformants with temperature-responsive patterns is of particular interest. Indeed, the amplification of the viral sequences can offer a model to study the amplification of cellular genes. Interestingly, the degree of viral amplification was highly variable and suggests a site-specific cellular control of amplification.
Section snippets
Integration site analysis of Py wild-type transformants using high-resolution electrophoresis
The analysis of viral integration sites in 12 recently derived Py wild-type FR3T3 transformants was carried out by separating BglII fragments using high-resolution electrophoresis as described in Materials and Methods and shown in Figure 1. Only one of these (lane 9) displayed a single band containing all the integrated viral sequences. In contrast, a ladder-like banding pattern was obtained with 11 transformants. Each ladder consisted of a “base band” of a size ranging between 7 and 15 kb, a
The pattern of integration sites of the Py genome in transformed rat FR3T3 cells: a re-evaluation
Here, we have examined the pattern of integration sites of the Py genome in transformed cells, derived from infection of the commonly used FR3T3 Fischer rat non-permissive cell line. We have made use of a conventional technique (Botchan et al., 1976), that is, the enumeration of the number of bands that contain viral sequences following treatment of cellular DNA with restriction endonucleases that do not recognize the viral genome. By improving the resolution of the electrophoresis, the
Cell lines and cell culture
The non-permissive FR3T3 cell line derived from Fischer rat embryo fibroblasts (Seif & Cuzin, 1977) was used and maintained at 37°C under 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) newborn calf serum and penicillin-streptomycin. The Py transformants were grown as monolayers in the same medium and passed every three to six days, whenever confluency was reached.
Virus strains
Py wild-type A2 (Griffin et al., 1974) and three different large T temperature-sensitive mutants were used:
Acknowledgements
The contributions of Drs Karen Friderici and David Hacker to initial observations on the ladder banding pattern, of Dr Karen Friderici for the data shown in Figure 1 and of Ms Diane Redenius for data shown in Figures 10(b) and 11 and help with preparing the Figures is gratefully acknowledged. The present studies were supported by grant CA29270 from the National Cancer Institute.
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Edited by M. Yaniv
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Present address: L.-J. Syu, Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, MI 48105, USA.