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young mice and hamsters (Israel et al., 1979). The expression of (at least) ... is a case in point (Courtneidge and Smith, 1983; Bolen et al.,. 1984; Cartwright et al., ...

The EMBO Journal vol.5 no.3 pp.491-499, 1986

Subceliular localisation of the middle and large T-antigens of polyoma virus

Stephen M.Dilworth, Hans-Arne Hansson1, Catarina Darnfors2, Gunnar Bjursell1, Charles H.Streuli3 and Beverly E.GrifFin3 Department of Zoology, Downing Street, Cambridge, UK, 'Department of Histology, University of Gothenburg, 2Department of Medical Biochemistry, University of Gothenburg, and 3Virology Department, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS, UK Communicated by B.E.Griffin

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no saining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells. Key words: polyoma virus T-antigens/subcellular localisation/ endoplasmic reticulum

Introduction The transformation of rodent cells by the small DNA virus, polyoma, is a valuable model system for studying oncogenesis since all the information necessary to induce tumour formation resides solely within the viral genome. This is shown, for example, by the appearance of tumours following injection of viral DNA into young mice and hamsters (Israel et al., 1979). The expression of (at least) two viral early antigens, the middle and large T-antigens, are required for generating the malignant phenotype, and in established lines expression of a functional middle T-antigen alone is both necessary and sufficient to produce transformed cells (reviewed by Tooze, 1981; Benjamin, 1982; Griffin and Dilworth, 1983; Smith and Ely, 1983; Cuzin, 1984; Basilico, 1984). Recent studies have uncoupled the functions implicated in transformation and delineated a role for the large T-antigen in cellular immortalisation and a potential role(s) for middle Tantigen in biochemical changes at cellular membranes (Treisman et al., 1981; Rassoulzadegan et al., 1982, 1983; Land et al., 1983; Ruley, 1983). Whether the polyoma virus small T-antigen is involved in cellular transformation or tumourigenesis has yet © IRL Press Limited, Oxford, England

to be resolved, although there is at least one report on a role for this antigen (Asselin et al., 1983). A variety of functions have been specifically identified with expression of the early polyoma viral genes. In permissive (mouse) cells these include the induction of both cellular and viral DNA synthesis and the regulation of mRNA levels, as well as putative roles in encapsidation. In non-permissive systems, virus infection in vitro results in a lowering of serum requirements and production of cells able-to grow to high saturation density on plastic or in semi-solid media, as well as the other well-characterised phenotypic and biochemical changes which are associated with tumour formation (reviewed by Griffin and Dilworth, 1983). Several explanations can be evoked to account for the variety of functions that are attributed to the expression of such a small number of genes. One is that a few viral functions, by altering gene expression, concomitantly modulate many cellular events. This is an assumption that is being tested with regard to transformation by a related virus, SV40 (Linzer and Nathans, 1983; Scott et al., 1983); it is undoubtedly relevant in the case of lytically infected cells, since polyoma virus makes use of cellular enzymes for replication and gene expression. Alternatively, viral antigens may interact with a variety of cellular functions to modify them; the association of middle T-antigen with the cellular homologue of the transforming gene product of Rous sarcoma virus is a case in point (Courtneidge and Smith, 1983; Bolen et al., 1984; Cartwright et al., 1985; Courtneidge, 1985). Another hypothesis is that each of the individual viral antigens fulfills a variety of roles, some of which are inherent within the protein itself and some determined by precise, but diverse, locations within a cell; the latter raises the possibility of conformational diversity determined by cellular location. It may be relevant that recent studies on the subcellular localisation of the transforming function pp60v-src have shown it and its cellular homologue to be located not only at the plasma membrane but also in intracellular structures (Resh and Erikson, 1985). A fourth notion is that, either via modification or processing, no gene product is entirely a unique species but belongs rather to a 'family' of structurally related proteins, each with a different function. In this regard, we have identified a 60-kd polypeptide as well as a number of smaller polypeptides which are structurally related to the 55-kd middle T-antigen of polyoma virus in lytically infected cells, some of which are also present in transformed cells (Dilworth and Griffin, 1983; our unpublished data). In an attempt to distinguish among the various mechanisms by which polyoma virus antigens might express multiple functions and, in particular, to use alternative strategies to the cell

fractionation protocols adopted previously in several laboratories for localising the viral antigens, especially the middle T-antigen (Ito et al., 1977; Schauffhausen et al., 1982; Segawa and Ito, 1982; Ballmer-Hofer and Benjamin, 1985), we have employed monoclonal antibodies directed against the viral middle and large T-antigens (Dilworth and Griffin, 1982) to determine the ultrastructural localisation of the antigens in lytically infected and in 491

S.M.Dilworth et al.

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