Site-specific in Situ amplification of the integrated polyomavirus genome: A case for a context-specific over-replication model of gene amplification¹

Abstract

The fate of the genome of the polyoma (Py) tumor virus following integration in the chromosomes of transformed rat FR3T3 cells was re-examined. The viral sequences were integrated at a single transformant-specific chromosomal site in each of 22 transformants tested. In situ amplification of the viral sequences was observed in 24 of 34 transformants analyzed. Large T antigen, the unique viral function involved in initiating DNA replication from the viral origin, was essential for the amplification process. There was an absolute requirement for a reiteration of viral sequences and the extent of the reiteration affected the degree of amplification. The reiteration may be important for homologous recombination-mediated resolution of in situ amplified sequences. Among 11 transformants harboring a 1 to 2 kb repeat, the degree of amplification was transformant-specific and varied over a wide range. At the high end of the spectrum, the genome copy number increased 1300-fold at steady state, while at the low end, amplification was below twofold. Some aspect of the host chromatin at the site integration that affected viral gene expression, also directly or indirectly modulated the amplification.

Use of high-resolution electrophoresis for the analysis of the integrated amplified sequences revealed a recurring novel pattern, consisting of a ladder with numerous bands separated by a constant distance approximately the size of the Py genome. We suggest that this pattern was generated by conversion of the amplified viral genomes to head to tail linear arrays with cell to cell variations in the number of genome repeats at single, transformant-specific, chromosomal sites. In light of the known “out of schedule” firing of the Py origin, we propose an “onion skin” structure intermediate and present a homologous recombination model for the conversion from onion skins to linear arrays. The relevance of the in situ amplification of the Py genome to cellular gene amplification is discussed. Finally, these results clarify our understanding of the integration of the Py genome in rat cells. They suggest that, in most cases, the multiple bands previously described in Py-transformants are likely to reflect genome amplification rather than multiple independent integration events, as assumed in the past. This interpretation is congruent with the accepted view that the integration of the Py genome is a rare and rate-limiting event in transformation.

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.