The T-antigens of Simian virus 40 and polyoma virus: their role in transformation

T I B S - February 1 980

2 Andersen, K. and Shanmugam, K. T. (1977) J. Gen. MicrobioL 103,107-122 3 Stretcher,S. L., Gumey, E. and Valentine, R. C. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1174--1177 Acknowledgements 4 Shanmugam, K. T., Loo, A. S. and Valentine, R. C. (1974) Biochim. Biophys. Acta 338,545--553 This report was prepared with the sup5 MacNeil, T., MacNeil, D., Roberts, G. P., port of the National Science Foundation Supiano,M. A. and Brill,W. J. (1978)Z Bacteriol. (Grant Number PCM 76-82766 and A E R 136, 253-266 77-07301), the Science and Education 6 Merrick, M., Filser, M., Kennedy,C. and Dixon, Administration of the US Department of R. (1978) Molec. Gen. Genet. 165, 103-111 7 Kennedy, C. (1977) Molec. Gen. Genet. 157, Agriculture under Grant No. 5901-0410- 8 199-204 -0142-0 from the Competitive Research 8 Elmerich, C., Houmard, J., Sibold, L., ManGrants Office. Any opinions, findings, heimer, I. and Charpin, N. (1978) Molec. Gen. conclusions, or recommendations expresGenet. 165, 181-189 sed in this publication are those o f the 9 Roberts, G. P., MacNeil, T., MacNeil, D. and Brill, W. J. (1978)J. BacterioL 136, 267-279 authors and do not necessarily reflect the 10 Postgate, J. R. (9171) in Chemistry and views of these granting agencies. Biochemistry of Nitrogen Fixation, pp., 161-190, Plenum Press, London References 11 Shanmugam,K. T., O'Gara, F., Andersen, K. and 1 Bums,R. C. and Hardy, R. W. F. (1975)Nitrogen Valentine, R. C. (1978)Annu. Rev. Plant Physiol. Fixation in Bacteria and ttigher Plants, Springer29, 263-276 Verlag, New York 12 Scott, D. B., Hennecke, It. and Lira, S. T. (1979) must be upheld. These goals are among the most exciting challenges facing the world today.

39 Biochim. Biophys. Acta (in press) 13 Lim,S. T., ltennecke, H. and Scott, D. B. (1979) J. Bacteriol. (in press) 14 Lim,S. T. and Shanmugam,K. T. (1979)Biochim. Biophys. Acta 584, 479--492 15 Hardy, R. W. F. and Havelka, U. D. (1975) Science 188, 633--643 16 Schubert, K. R. and Evans, It. J. (1976) Proc. Natl. Acad. ScL U.S.A, 73, 1207-1211 17 Lira, S. T. (1978) Plant Physiol. 62,609-611 18 Pootjes, C. F. (1977) Biochem. Biophys. Res. Commun. 76, 1002-1006 19 Andrews,T. J. and Lorimer,G. H. (1978) FEBS Lett. 90, 1-9 20 Andersen, K. (1978) Biochim. Biophys. Acta 585, 1-I1 21 Flowers,T. J., Troke, P. F. and Yeo, A. R. (1977) Annu. Rev. Plant PhysioL 28, 89-121 22 Measures, J. C. (1975) Nature (London) 257, 398--400 23 Christian, J. M. B. (1955) Aust. Z Biol. Sci. 9, 75---82 24 Tetsuya, T. and Lewis, I. P. (1971) J. Bacteriol. 106, 972-982 25 Delwiche,C. C. and Bryan, B. A. (1976) Annu. Rev. MicrobioL 30, 241-262

grated into the cellular genome and in some cases it has been shown that the expression of some virus gene(s) is needed to maintain the transformed phenotype of the cell. Integration does not seem to be site specific and can occur at many different places in the cell DNA. The virus D N A is about one million times smaller than that of L. V. Crawford the host and it is difficult to see how so little viral DNA, carrying so little genetic inforSimian virus 40 and p o l y o m a virus code for two and three, respectively, major t u m o r [~'ntimation, can cause so many changes in the gens. These are involved in the initiation and maintenance o f transformation. In S V 4 0 phenotype of the cell that it has transtransformed cells there is a complex o f the vin~s-coded large-T antigen with a host-coded formed. protein (53,000 daltons). This protein m a y play a central role in transformation, since cells The double-stranded circular genomes transformed by other carcinogens and viruses also appear to contain this 53 K protein. of SV40 and polyoma virus comprise about Simian virus 40 has been the subject of virus replication and gene expression 5250 base pairs. The entire nucleotide intensive study since its isolation in 1960 occur. Semi-permissive cells give an inter- sequence of both viral DNAs has now been from monkey cells being used for the pro- mediate response, with some virus produc- determined [4-7], a very impressive duction of polio vaccine. Polyoma virus tion but also some cell survival and trans- achievement and the basis for much of was detected in mice even earlier, in 1953 formation. Among the cells which survive what is described here. Slightly less than by Gross [1] and isolated and characterized abortive infection, a small fraction are half of this is devoted to specifying the proby 1960 (see reviews [2] and [3]). Both vir- stably transformed and can be selected by teins that make up the shell of the virus paruses appeared as passenger viruses in their making use of their altered properties. All ticle. The rest of the DNA, starting from natural hosts. In vitro they grow in thecells the activities of the virus, including tumor near the origin of D N A replication, codes of these hosts and cell death accompanies production and transformation are also for the functions expressed from early the production of progeny virus. In addi- carded by the naked virus DNA, although times after lytic infection throughout the tion, cells of other species are transformed the D N A is less infectious than the intact growth cycle. These same functions are expressed in transformed cells and it is by these viruses. The transformed cells virus particle. have altered growth control properties and Transformed cells are different in many amongst them that we must search for an resemble cells cultured from the tumors ways from their normal counterparts understanding of the role of the virus in these viruses cause in rodents. (Table II). They contain virus D N A inte- initiating and maintaining the transformed state of the cell into which the virus D N A Examples of the two types of virus-cell has become integrated. Transformed cells interactions are given in Table I. Permis- TABLE 1 The effect of species of origin on response to virus contain all or most of the early region of sive cells give a full lytic response with pro- infection these viruses, although much of the rest of duction of progeny virus. Non-permissive the D N A is often missing or not expressed. cells give only an abortive infection, in SV4O Polyoma vints At first it seemed that the early region which the early, but not the late, events in Monkey Permissive coded for just one protein - T-antigen Mouse Non-permissive Permissive which was found in all transformed cells Lionel Crawford is ttead of the Department of Molecu- tlantrter Semi-permissive Semi-permissive and must account for their being translar Virology at the Imperial Cancer Research Fund, Rat . Semi-permissive Semi-permissive London, U.K. formed. At the 1974 Cold Spring Harbor

The T-antigens of Simianvirus 40 and polyoma virus: their role in transformation

9 Elscvier/Nor~-Holland Biomedical Press 1950

40 TABLE I1 Some altered properties of transformed cells used in isolation of transformants Higher saturation density Reduced serum requirement Growth in agar or Me,bocci suspension Changes in colonial growth patterns Growth on top of normal cell monolayers

Symposium it was possible to talk about the transforming'protein and the transforming gene, both in the singular. There were many puzzling features of the situation, in particular how a single protein could accomplish all the many and varied functions it did both in lytic growth and in the transformed cell. But at least the mystery was concentrated in a single entity. This all changed with the advent of RNA splicing, first discovered in adenovirus replication [8] and then rapidly shown to occur during the production of almost all eucaryotic messengers. The long RNA molecules transcribed from the cellular or viral DNA are not only processed at both ends, but in addition, internal sequences ('introns') are also removed in a precise and specific way to give rise to 'spliced' messenger RNAs whose sequences are no longer colinear with that of the DNA. In the context we are discussing here, it was not so much the realization that messenger RNAs are specified by non-contiguous regions of DNA that was important, but rather the realization that alternative splicing pathways make it possible to generate more than one mRNA from any given region of the virus DNA. It was possible to envisage several proteins from an amount of DNA which had previously been thought to code for only one. The overall layout of the genome of SV40 and polyoma is shown diagrammatically in Fig. 1. The late region shows several fascinating features, including multiple usage of the DNA in the same and in different reading frames, to give rise to the three capsid proteins. Since, however, these are not involved in transformation we shall concentrate on the other part of the genome, the early region. In SV40 the way in which the two major early proteins, large-T and small-t antigens fit in is relatively simple. A model for the arrangement of this region, derived from studies of deletion mutants and characterization of the two antigens, was put forward soon after the discovery of splicing [9]. The splices which were implied by this arrangement corresponded very well with those actually found when early mRNA was analysed [10] and a great deal of more precise information has now been published (Fig. 2). This gives us a detailed pic-

TIBS - February 1980

ture of the splicing patterns of the mRNAs and consequent amino acid sequences which would be predicted for the proteins. Small-t terminates just before the 66 nucleotide splice in the small-, messenger, whereas large-T comes from a spliced messenger which lacks 346 nucleotides, relative to the DNA sequence. The pattern in polyoma virus is more complex (Fig. 3). The existence of mutants of polyoma virus which were defective for transformation and grew well only in certain host cells, was the first indication that the virus had two early functions. Until the discovery of splicing it was easier to envisage them as separable activities of the same protein, rather than different proteins from the same region of the DNA as they are now known to be. When the polyoma early proteins were analysed in detail it turned out that there were three major proteins, large-T, small-t and middle-T antigens, all occuring in different places in the infected cell [11,12]. Instead of two alternative splices with a common 3' end, there are at least two alternatives at both the 5' and the 3' ends. The splices all occur within the coding region for the proteins, in contrast to SV40 small-t where the splice comes immediately after the termination codon 9for the protein. The usage of the three possible reading frames is also more complex. All three probably start off from the same AUG codon, with the same N-terminal sequence and are read in the same frame up to the splice. After the splice they are in different reading frames so that the C-terminal part of each protein has its own unique amino acid sequence. DNA $ V 40

The properties of the different T-antigen species are remarkably different, considering how much N-terminal sequence they share with each other. Large-T is associated with the chromosomal DNA in the nucleus of the cell and the antigen binds to DNA in vitro. It binds particularly strongly to the origin of DNA replication on the virus DNA as e:~pected of a protein required for the initiation of virus DNA synthesis. It also affects synthesis of virus messenger RNAs, inhibiting transcription of early mRNA and stimulating transcription of late mRNA. Its involvement in transformation is clear; cells transformed by temperature-sensitive mutants of SV40, which have alterations in large-T, lose some of their transformed properties at the non-permissive temperature. But this is by no means always the case. Some transformed cells of this type remain transformed at high temperature and some polyoma-transformed cells have only part of the normal large-T, so it does not seem to be absolutely required for transformation. Nor does it seem to be sufficient, since there are cells that have normal large-T but lack the other antigens and are not fully transformed. The situation with small-, is rather similar, in that it does not always seem to be absolutely required but is nevertheless involved in transformation. Small-t antigen is found largely in the cytoplasm, in contrast to large-T, and it does not seem to bind to DNA. Boyh large-T and small-t antigens inside cells seem to mimic the action of growth factor hormones acting from outside the cell. Polyoma virus middle-T antigen is difMAPS PC)LYOMA

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Fig. 1. The genomes of SV40 and polyoma virus. Each of, he DNAs is oriented with the origin of DNA replication (0) at the top, the termin us at the bottom ( T), the early region on the lefi and the late region on the right. The restriction enzyme fragments used in producing the physical map are indicated by the letters A-4 (SV40, llind H + HI) and numbers 1--8 (polyoma, Hpa lI). Within the circle the EcoR, site is shown. This is conventionally used as the 01100point on the maps. The locations shown in Figs 2 and 3 are measured from this site and expressed as percentages of the genome. Reproduced from: Cold Spring Harbor Symposia on Quantitative Biology 39, Fried et al. (1974), 45--52.

41

TIBS - February 1980

ferent again, in that it is to some extent found in the outer membrane of the cell. This makes it a prime candidate for acting as part of TSTA, the antigen involved in cell-mediated rejection of transformed cells by immunized animals. Also the cell surface seems to be the location where control over cell multiplication is exerted by growth factors, etc., and it is the change in growth control which is the essence of transformation. Its membrane location, and the effect of deletions which alter middle-T antigen strongly suggest that this protein plays a central role in transformation by polyoma virus. How then can SV40 transform cells since it lacks a virus coded middle-T? The DNA sequence makes it very unlikely that it could have one, since there is only one reading frame free of termination codons throughout the C-terminal, two-thirds of the region coding for large-T antigen, leaving no possibility for a second protein unless very frequent splicing is invoked. The functions of polyoma middle-T antigen which are essential for transformation must be carried out in some other way in SV40 transformed cells. Perhaps SV40 large-T antigen has some of the properties of polyoma middle-T, or induces an analogou s cellular protein to replace middle-T antigen, as discussed later. It seems unlikely that SV40 small-t has functions similar to those of polyoma middle-T, although deletions in small-t do have a considerable effect on the transforming efficiency of the virus in some'systems. One unanswered question throughout all the studies of virus-coded antigens. described so far is whether or not they are enzymes. Once it had been shown by Collett and Erikson [13] that the src gene product of RNA tumor viruses, p60 src, had protein phosphotransferase (kinase) activity it was natural to test other potential transformation proteins for this activity. The problem was initially to obtain pure T antigens in sufficient quantity to make such studies feasible; this was solved by taking advantage of the properties of one of the adeno-SV40 hybrid viruses. These viruses, in which all or pa~:t of SV40 has been inserted into the adenovirus genome, have an extended host range growing well in both human and monkey cells, in contrast to the parental adenovirus which grows well only on human cells. In one hybrid virus, Ad2*D2 the arrangement of the genomes is such that a protein closely related to SV40 large-T antigen is produced in the large amounts characteristic of the adenovirus late proteins. This D2 protein lacks the sequences shared by SV40 large-T and small-t antigens, which are

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replaced by adenovirus-coded polypeptide. Tjian and Robbins [13] were able to detect protein kinase activity associated with the highly purified D2 protein, but it seems unlikely that this is an intrinsic property of the large-T related protein, since some separation could be achieved in further purification. An activity which remains with the most highly purified D2 preparation is ATPase and this may be connected with its role in DNA replication and transcription [14]. Polyoma large-T antigen has also been found to have ATPase activity [15]. In addition to these activities Which are associated with large-T, polyoma virus also has protein kinase associated with its middle-T antigen [16--18]. This activity is capable of phosphorylating the IgG in immune complexes as well as the antigen itself. However, reservations about this kinase activity being intrinsic to the viruscoded antigen, rather than an associated enzyme activity remain. Although it may

be disappointing in some ways if the kinase activity of T-antigens proves to be that of associated cellular enzymes, this ability to form complexes with kinase and to be phosphorylated may still be very important. The role of protein phosphorylation in control of enzyme activity is so widespread that it remains an attractive possibility as a way of mediating the effect of virus transformation on control of cell growth and division. As mentioned already, polyoma virus codes for a middle-T antigen, and SV40 does not. This difference is really striking because the two viruses are similar in so many other ways. SV40 transformed cells do have a protein of intermediate size, 53,000 daltons, which precipitates with anti-T serum. It turns out, however, to be a host protein which is associated with large-T antigen to form a complex, which is why it precipitates with sera specific for large-T [19]. This 53,000 dalton (53 K) protein is also found in cells transformed by

42

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produce normal wild type large-T antigen still have reduced transforming activity. In short, large-T antigen does not seem to be entirely necessary or sufficient for transformation and this really rules out any mechanism involving simple and direct action on host cell DNA. The complex of large-T antigen with the 53 K host protein is, however, a graphic demonstration of the intimate interaction between virus and host. We now have to ask what effect the association with 53 K has on the activities of large-T antigen. Conversely, does the association change the rate of synthesis of the 53 K protein, its location in the cell or its state of modification, and thus mediate changes in control? If it is the amount of the 53 K protein in the cell that controls the rate of cell growth, these studies on virus transformation will have given us a very significant lead in our search for an understanding of the mechanism of transformation. References

1 Gross, L. (1953) Proc.Soc. Exp. Biol. Med. 83, 414---431 2 Stewart, S. E. (1960)Adv. Virus Res. 7, 61-90 M i d d l e T i! C N I ~ 3 Eddy, B. E. (1960)Adv. Virus Res. 7, 91-102 I !i ! 4 Fiefs, W., Contreras, R., Haegeman, G., Rogiers, i I I ! It I R., van de Voorde, A., van Heuversw~'n,H., van , , 3 I tterreweghe, J., Volckaert, G. and Ysebaert, M. I" 1 h I (1978) Nature (London) 273, 113--119 II I 5 Reddy, V. B., Thimmappaya, B., Dhar, R., Subl N 9 I ramanian, K. N., Zain, B. S., Pan, J., Ghosh, P. K., Celma, M. L. and Weissman,S. M. (1978) Science 70 1200, 494-502 6 Friedmann, T., Esty, A., LaPorte, P. and l:ig. 3. Splicing patterns o f polyoma early messenger RNAs. The overall layout and conventions are the same as Deininger, P. (1979) Cell 17, 715-724, and to be used in Fig. 2. Locations o f the splices are based on the estimates kindly provided by Dr R. Kamen (personal compublished munication) and the rest on [6,7]. 7 Soeda, E., Arrand, J. R., Smolar, N. and Griffin, B. E. (1979) Cell 17, 357-370, and to be pubagents other than SV40 and it may be very mally found in adult cells, then this necessilished widely associated with the transformed tates a change in approach. Different vir8 Berget, S. M., Moore, C. and Sharp, P. A. (1977) uses may elicit this change in ways which" state. Proc. Natl. A cad. ScL USA. 74, 3171-3175 Since viruses are by no means the only are quite different and involve the action of 9 Crawford, L. V., Cole, C. N., Smith, A. E., Paucha, E., Tegtmeyer, P., Rundell, K. and Berg, agents which transform cells, there can be different species of T-antigen. In some P.(1978)Proc. Natl.Acad.Sci. USA. 75, 117-121 no absolute requirement for any virus- cases host functions can apparently substicoded protein for transformation. Some tute for virus functions and perhaps the dif- 10 Berk, A. J. and Sharp, P. A. (1978) Proc. Natl. Acad. Sci. USA. 75, 1274-1278 cells transformed by chemical carcinogens ferent species of T-antigen can substitute 11 Ito, Y., Brocklehurst, J. R. and Dulbecco, R. also have a protein, p53, which is similar to for each other, for example, both large-T (1977) Proc. Natl. Acad. Sci. USA. 74, 4666---4670 the 53 K protein which we find in virus- and small-t seem to cause a general stimutransformed cells [20]. Little or noneof this lation of cell growth similar to that pro- 12 Smart, J. E. and lto, Y. (1978) Cell 15, 1427-1437 protein is detected in t h e untransformed duced by growth factors. Certainly our 13 Collett, M. S. and Erikson, R. L. (1978) Proc. Natl. Acad. ScL USA. 75, 2021-2024 parents of these chemically transformed conception of transformation has altered, 14 Tjian, R. and Robbins, A. (1979) Proc. Natl. cells. If it proves to be the case that the from its being a rather crisp qualitative Acad. Sci. USA. 76, 610.-614 53 K phosphoprotein is always present in change accomplished by the action of a 15 Gaudray, P., Clertant, P. and Cuzin, F. (1979) (in preparation) transformed ceils, or cells transformed in a single virus coded protein, to a change 16 Eckhart, W., Hutchinson, M. A. and tlunter, T. particular way, this will be a most exciting made up of several components each with a (1979) Cell 18,925-933 and unexpected outcome of these studies quantitative effect and together exerting a 17 Schaffhausen, B. S. and Benjamin, T. L. (1979) cumulative effect on growth control. on virus transformation. Cell 18,935-946 Up to now most of our attention has Whereas previously it was possible to 18 Smith, A. E., Smith, R., Griffin, B. and Fried, M. (1979) Cell 18,915-924 been concentrated on the virus-coded pro- envisage a direct action of a virus coded 19 Lane, D. P. and Crawford, L. V. (1979) Nature teins, but if transformation is mediated by a protein such as large-T antigen on the cellu(London) 278, 261-263 change in the amount or state of modifica- lar DNA with consequent changes in DNA 20 DeLeo, A. B., Jay, G., Appella, E., Dubois, G. C., tion of a host protein, for example replication this no longer seems likely, Law, L. W. and Old, L. J. (1979) Proc. Natl. A cad. Sci. USA 76, 2420--2424 derepression of a foetal antigen not nor- because deletion mutants which seem to

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