Polyoma virus middle T antigen: meddler or mimic?

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57,159-163 29 Douvas, G.S. et al. (1985) Infect. lmmun. 50,1-8 30 Flesch, I. and Kaufmann, S.H.E.(1991) Infect.Immun. 59,3213-3218 31 Chan, J. et al. (1992) 1. Exp. Med. 175,

(1978)]. Gen. Micro&o/. 104,37-45 26 Sturgill-Koszycki, S. et al. (1994) Science 263,678-681 27 Denis, M. (1991) Clin. Exp. Immunol. 84,200-206 28 Rook, G.A.W. et al. (1986) Immunology

1111-1122 32 Lowenstein, C.J., Dinerman, J.L. and Snyder, S.H. (1994) Ann. Intern. Med. 120,227-237 33 Geller,D.A. et al. (1993) Proc. Nuti Acud. Sci. USA90,3491-3495

Polyomavirus middleT antigen: meddleror mimic? Stephen M. Dilworth olyoma virus is a small, doublestranded, closed circular DNA

Polyoma virus middle T antigen duplicates the actions of growth-factor receptors in binding the signaliing molecules phosphatidyliuositol3’-OH kinase and She. These properties indicate that middle T is mitogenic and may be required to overcome inhibition of DNA replication during the lytic life cycle of the virus.

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virus that is endemic in wild mouse populations, where it causes few harmful effects. Because of its small genome size and ease of propagation in culture, polyoma virus and its simple lytic cycle have been studied extensively, and its genome organization has been established (Fig. 1). The virus adsorbs to the cell surface by interacting with membrane receptors, enters the cell nucleus and then uncoats. One half of the genome (the early region) is then transcribed and, through multiple splicing events, generates mRNA species that code for three proteins, large (LT), middle (MT) and small (ST) T antigens. LT is a DNA-binding protein that interacts with the viral origin of DNA replication and, through interaction with DNA polymerase a and other factors, promotes initiation of bidirectional replication. As this progresses, transcripts from the other half of the genome (the late region) accumulate and are translated to produce the virion proteins VPl, VP2 and VP3. The replicated DNA is compacted by interaction with host histones to form chromatin, and then assembled with the capsid proteins into virions within the nucleus. The cell then ruptures and releases the mature viral particles (see Ref. 1 for early reviews). When cells of species closely related to mouse, such as rat or hamster, are infected with polyoma

S.M. Dilworth is in the Dept of Metabolic Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London, UK W12 ONN. tel: +44 181 7432030 x215.5, fax: +44 181 7461159, e-mail: [email protected]

virus, DNA replication fails to occur. Most cells lose the viral DNA at this point, but, in a small number, the viral genome becomes integrated into the host DNA and the T antigens continue to be synthesized. These cells change dramatically in phenotype, becoming fully transformed and tumorigenic. Injection of large quantities of the virus into neonatal mice can also generate multiple tumours within the animal (hence the name polyoma), presumably by similar mechanisms. It is now clear that this transforming effect is caused primarily by MT, a 5.5 kDa protein that is associated with cellular membranes. Closely related viruses are found in other animal species, notably simian virus 40 (SV40) in monkeys and BK virus in man, but MT is unique to the mouse and hamster variants. As 0

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with many other oncogenes of DNA viruses, MT does not have any known intrinsic enzymatic activity, but instead binds host regulatory proteins and alters their function. Proteins associated with MT A growing number of proteins have been identified bound to MT, and more are sure to be characterized in the future. The A and C components of protein phosphatase 2A (PP2A), a major soluble serine/threoninespecific phosphatase that is found in most cells, are associated with both MT and ST (Refs 2,3). It is not yet clear what effects these interactions have on cells, although it is thought that, in SV40, the PP2A-ST interaction may prevent dephosphorylation and hence inactivation of the mitogen-activated protein kinase (MAP kinase) signal transduction pathwaf. Members of the 14-3-3 protein family have also been shown to interact with MT (Ref. 5). These proteins are found in most eukaryotic cells and have been implicated in regulating protein-kinase-C activity, as well as other reaction@. Very recently, some 14-3-3 proteins have been shown to bind to c-Raf-1 and to stimulate its protein-kinase activity7-9. The c-Raf-1 protein is an important signal transduction component that associates with the active GTP-bound form of p21”. This brings c-Raf-1 to the membrane, which is sufficient to activate the MAP kinase pathway’*. It is feasible, therefore, that association of

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Fig. 1. The circular genome of polyoma virus, together with the regions transcribed into mRNA species either early or late in infection. The sequences present in mature mRNA are indicated by solid lines, those removed from the primary transcript by splicing events by dotted lines. The coding regions for all six known proteins are indicated by shaded boxes. The sequences required to form the core element of the DNA replication origin and the transcriptional enhancer element required for early transcription are also shown. The solid box shows a region that has no sequence similarity to simian virus 40, BK virus or JC virus. This region of the genome encodes two different proteins from the same DNA sequence using alternative reading frames. Base-pair numbers are shown on the inside of the map in kb and the position of the coding regions is indicated in each box. Base-pair numbering is as in GenBank Accession no. PLY2CG.

the 14-3-3 proteins with MT could relocate both them and c-Raf to membrane sites, and also activate this pathway. MT also binds the non-receptor tyrosine kinases pp60‘-‘“, p~62~-~~” and ppSSc-fy”, and these interactions are closely involved in MT-induced cell transformation (reviewed in Refs 11,12). The amino-terminal half of MT is involved in binding both pp60c-‘” and PP2A. Mutations in this region can disrupt binding of both proteins, as well as abolishing transforming properties (reviewed in Ref. 13). An additional sequence present only in MT is also required to bind ~~60’.‘“, but not PP2A (Ref. 14) (Fig. 2). Surprisingly,

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little is known about the MTpp60c-‘” interaction at the molecular level, mainly because the complex cannot yet be re-created in vitro. The reasons for this difficulty are not clear, but it is possible that the interaction is not direct, requiring either an intermediary protein that may not have been identified yet, or post-translational modification of either species. In support of this idea, there is some evidence that phosphorylation of MT may be required for the association to form. Once bound, the tyrosine-kinase activity of pp60c”” is stimulated, possibly by MT preventing the inhibitory phosphorylation of pp60’“” Tyr.527 (Refs 11,12). Presumably,

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this causes cellular proteins to become phosphorylated, although only a small number of such proteins have been identified, and there is little overall increase in cellular phosphotyrosine levels. MT itself, however, is known to be phosphorylated by the activated ~~60’~‘” on tyrosines in the carboxyterminal region of the molecule. When phosphorylated, Tyr315 of MT acts as a binding site for either of the two SH2 domains in the 85 kDa component of phosphatidylinositol 3’-OH kinase (P13K)‘5-‘8. This 85 kDa subunit is, in turn, tyrosine phosphorylated, presumably by pp60‘-‘“. Cells expressing MT have increased levels of the products of PI3K, but it is unclear whether the 85 kDa component binding to MT and/or phosphorylation are responsible for this increase. Another major site of phosphorylation in MT, Tyr250, also acts as a binding site for a protein containing an SH2 domain, in this case, the She oncoprotein 1v,20. Again, She is phosphorylated, probably by pp60‘-“‘, but this time the consequences are clear. Tyrosine-phosphorylated She acts as a binding protein for the SH2 domain of the adapter protein Grb2, which is already associated via its SH3 domains with the guanine nucleotide exchange factor mammalian Sos (mSos). Locating mSos to membrane sites through indirect association with MT brings it into close proximity with, and thereby activates, p21”‘, by catalysing the exchange of GDP for GTP (Refs 21,22). The downstream events that this initiates are also now becoming clear10,23. Interestingly, mutants that disrupt She binding to MT also affect the increase in PI3K products, suggesting that a degree of crosstalk may occur between these two pathways23,24. Comparison with growth-factor receptors (GFRs) Stimulation of mitogenic tyrosinekinase-associated GFRs also initiates events similar to those outlined above (Fig. 3). Although the nature of the kinase and substrate molecules differ between receptor types, there is a common principle. Signals stimulate a tyrosine-kinase activity that phosphorylates sev-

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era1 sites in an acceptor molecule that is usually membrane bound. This is built on by proteins containing phosphotyrosine-motif-binding SH2 domains. These polypeptides can, in turn, be phosphorylated, and this, or their relocation close to the membrane, initiates a series of downstream events, including activation of the MAP kinase pathway again. MT stimulates at least two of these pathways (those involving PI3K and She) in the same way, and so acts, at least partially, as a functional homologue of a mitogenie activated GFR. It is likely that all of the SH2-domain-containing proteins that bind to MT or GFRs have not yet been uncovered, and so the similarities may be even greater. However, just as GFRs do not all bind the same group of SH2-domain-containing proteins, the full complement may not be required by MT. The reason why MT may also be able to bypass this route and activate the MAP kinase pathway directly (see above) is not yet clear. MT evolution and function The mechanisms outlined above also help to explain how polyoma virus acquired the MT activity. For many years, it had been assumed that the virus had acquired a cellular oncogene by integrating a piece of host DNA, and that this accounted for the region that has no sequence similarity to SV40 and BK virus (see Fig. 1). However, despite many DNA and antibody studies, no cellular homologues have been found. The model described above indicates that the key property that is required for MT action is the binding of ~~60’.“‘. Once this is achieved, the other elements, small sequences surrounding tyrosine residues, each of which is specific for a particular SH2 domain, could be picked up individually, or could have evolved separately. MT, therefore, need not have been acquired as a single package, but may instead have been assembled from separate elements, rather like a jigsaw puzzle. Each piece as it was added presumably supplied an evolutionary advantage to the virus, and so became stabilized. It is ironical that the key event, binding of pp60c-src,

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pp60c+rc-binding region Fig. 2. The linear middle T (MT) sequence, showing the mRNA splice junction (open triangle). The positions of the two tyrosine residues known to be phosphotylated by pp60c*“, Tyr250 and Tyr315, are also shown. The shaded boxes represent the sequences altered in a number of nontransforming mutants of MT. The regions, as deduced from these mutants, that are required to interact with cellular proteins are also shown. Abbreviation used: PP2A, protein phosphatase 2A.

is still the one that is understood least. Viruses that multiply in higher eukaryotic organisms face a common problem: the cells that they infect are usually quiescent and therefore have barriers to prevent initiation of DNA replication. In MT, polyoma virus has acquired a method of stimulating the proliferation of the cells it infects by activating cell-growth-signalling pathways internally. Presumably, then, MT helps to facilitate virus replication on infection of quiescent cells within the host organism. Furthermore, LT is mitogenic, and both these activities probably contribute to the effects achieved by the virus. Indeed, there are reports that, although many mutant viruses with disrupted MT functions are viable in the cosseted environment of tissue-culture cells? where the cells are normally dividing, when introduced back into mice they disappear rapidlyz5J6. Obviously, this may have other explanations, not least, interactions with the host immune system, but stimulation of cell proliferation is probably a factor in viral persistence. In addition, MT is required to broaden the range of cells in which polyoma virus can replicate2’ and is involved in the assembly of the virus after replication. It is not yet clear whether these activities are related to its role as a functional GFR homologue. MT may therefore activate growth-factor-mediated mechanisms of signal transduction to help overcome problems faced by

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polyoma virus when infecting a quiescent cell. Other viruses may achieve the same goal, but through different mechanisms. For example, vaccinia and its relatives encode a molecule similar to transforming growth factor a or epidermal growth factor that stimulates cell proliferation28, and fibropapillomaviruses encode the ES protein, which can activate the platelet-derivedgrowth-factor receptor29. These viruses, therefore, activate earlier steps of the same signal transduction pathway. SV40 does not have an MT-like molecule despite having a very similar genome structure (see Fig. 1). The LT of SV40, however, binds ~53 and also interacts well with plOSRB and related proteins; polyoma LT does not bind ~53, but does interact with plOSRB, and this is necessary for its mitogenic effect. It is tempting to speculate that these interactions of LT take on the MT function in SV40. Therefore, SV40 may achieve a similar growth effect to polyoma virus by working within the nucleus, rather than outside it. The end result, however, is not identical because cells transformed by polyoma virus have different phenotypic properties to those transformed by SV4O. The consequence for replication of the virus could, however, be largely the same. Adenoviruses also encode proteins that interact with ~10.5~~ and ~53, but this time the binding sites are on separate proteins, ElA and ElB, respectively. Hence, nuclear effects are common to several viruses.

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Fig. 3. A comparison of the reactions occurring on activated growth-factor receptors (GFRs) and middle T (MT). (a) Shows some of the interactions initiated by activation of a model GFR containing an intrinsic autophosphorylating tyrosine-kinase activity. Similar reactions occur in other receptor types, where the kinase or the tyrosine substrates (acceptors) may be on separate molecules. (b) Shows the events known to occur to MT in cell membranes. In both cases, the kinase is activated, either by binding the growth factor to the receptor or by binding pp60C-Snto MT. The kinase activity then phosphorylates multiple tyrosines in an acceptor molecule, shown here as the GFR itself or MT. These sites bind a series of proteins containing SH2 domains, the exact number varying between receptors. These attached proteins are usually then phosphorylated and activated either directly or indirectly, which initiates a series of downstream events. Only the three known to be stimulated by MT are shown here. Abbreviations used: GAP, GTPase-activating protein; Pi, inorganic phosphate; PLCy, phospholipase C subtype y.

The suggestion that DNA tumour viruses influence signal transduction pathways to facilitate viral growth in quiescent cells poses another question, however. If tumour viruses need such a mechanism, do other viruses? It seems likely that the GFR signalling pathways are a universal means of stimulating cell growth, so perhaps most viruses that replicate to high levels within higher eukaryotic cells can also influence these reactions. The exact point at which each virus has an effect could vary, but increased cell pro-

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pression of the viral genome can occur may also play a part in determining the tumorigenic potential of a virus. Usually, an infected cell is destroyed by viral infection, but if viruses in general need to stimulate host-cell growth to replicate, many more viruses than the obvious DNA tumour viruses should be considered when cofactors in carcinogenesis are being sought. Therefore, the number of viruses implicated in tumorigenesis, despite having expanded greatly in recent years, may continue to rise.

liferation that is sufficient to allow the virus to replicate may be a common consequence. As many GFRsignalling constituents can function as oncoproteins when deregulated, the differences between DNA tumour viruses and other DNA viruses may not be so great as it seems at first sight. Although only the tumour viruses were thought to express an overt oncogene, in reality, all viruses may be able to produce similar effects, albeit with varying potency. Therefore, the ease with which integration and stable ex-

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167-176 3 Walter, G. et al. (1990) Proc. Nat/ Acad. Sci. USA 87,2521-2525 4 Sontag, E. et al. (1993) Cell 75, 887-897 5 Pallas, DC. et al. (1994) Science 265, 535-537 6 Aitken, A. et al. (1992) Trends Biochem. Sci. 17,498-SO1 7 Freed,E. et al. (1994) Science 265, 1713-1716 8 Irie, K. et al. (1994) Science 265, 1716-1719 9 Fantl, W.J. et al. (1994) Nature 371, 612-614 10 Hall, A. (1994) Science 264, 1413-1414 11 Courtneidge, S.A. (1986) Cancer Sure. 5, 173-182 12 Kaplan, D.R. et al. (1989) Biochim.

Biophys. Acta 948,34S-364 13 Markland, W. and Smith, A.E. (1987) Biochim. Biophys. Acta 907,299-321 14 Dilworth, S.M. and Horner, V.P. (1993) 1. Virol. 67,2235-2244 15 Talmage, D.A. et al. (1989) Cell 59, 55-65 16 Cohen, B. et al. (1990) Proc. Nat/ Acad. Sci. USA 87,4458-4462 17 Auger, K.R. et al. (1992) 1. BioI. Chem. 267,5408-5415 18 Yoakim, M. et al. (1992) 1. Viral. 66, 5485-5491 19 Dilworth, S.M. et al. (1994) Nature 367, 87-90 20 Campbell, K.S. et al. (1994) Proc. Nat/ Acad. Sci. USA 91,6344-6348 21 Schlessinger, J. (1993) Trends Biochem. Sci. 18,273-275 22 Aronheim, A. et al. (1994) Cell 78, 949-961 23 Feig, L.A. and Schaffhausen, B. (1994) Nature 370,508-509 24 Ling, L.E. et al. (1992) 1. Virol. 66, 1702-1708 25 McCance, D.J. (1981) J. Viral. 39, 958-962 26 Freund, R. et al. (1992) Virology 191, 716-723 27 Garcea, R.L. et al. (1989) Virology 168, 312-319 28 King, C.S. et al. (1986) Mol. Cell. Biol. 6,332-336 29 Petti, L., Nilson, L.A. and DiMaio, D. (1991) EMBO ].10,84S-855

host. The probability of severe disease developing is a reflection of the genetic fitness of the microorganism for infection, balanced against the ability of the host to limit bacterial proliferation. The bacteria, for their part, attempt to negotiate the outcome of the interaction by altering virulencegene expression in response to host signals encountered during infection. How this is accomplished is explored in detail in a new book by Charles Dorman, the Gelzetics of Bacterial Virulence. The stated goal of the book is to ‘comprehensively review the gene regulatory mechanisms that underlie the pathogenic processes of bacteria’, and the author has succeeded in this aim. A full appreciation for the subject of virulence-gene regulation requires an understanding of the fundamental properties of the prokaryotic genome. Introductory sections of the book review the basics of DNA

structure, transcription and recombination, with an emphasis on virulence-gene regulation, and provide a strong foundation on which the later chapters are built. Abstract concepts, such as DNA supercoiling and recombination, are presented in a comprehensive straightforward manner illustrated with an ample number of diagrams. The concept that bacteria use extrachromosomal elements, such as phages and transposons, to acquire and exchange genetic information has been appreciated for many years. More recently, it has become clear that many other types of genomic rearrangements are used by pathogenic bacteria to regulate virulence-gene expression. Many of the best-known examples of this type of regulation (such as pilin variation in Neisseria gonorrhoeae) are reviewed briefly in Dorman’s book. The author’s intention was not to cover these systems exhaustively,

QuestIons l

What effect does the interaction of middle T (MT) and small T with protein phosphatase 2A have on the cell?

. What are the details of the association between MT and pp60c-src?Why is this complex not produced in vitro? What cellular proteins are then phospholylated by pp60”‘“? l

Are other proteins involved in growth-factor-receptor-mediated duction also bound to MT?

l

Do other viruses not presently associated with tumours influence signal transduction pathways in eukaryotic cells to facilitate their growth in quiescent cells? Can such viruses act as cofactors in carcinogenesis?

Acknowled@ments I am particularly grateful to Dr Nick Dibb and members of both the Metabolic Medicine Cell Transformation Laboratory and the Virology Dept at the RPMS for helpful discussions during the preparation of this review. Work in the author’s laboratory is supported by grants from the Cancer Research Campaign, UK. Owing to space constraints, it has been impossible to reference the field fully: I apologize to the many authors who have made valuable contributions to MT research, but have not been referred to here. Ref8r8tlC8S

1 Tooze, J., ed. (1981) DNA Tumor Viruses: Molecular Biology of Tumor Viruses, Cold Spring Harbor Laboratory Press 2 Pallas, D.C. et al. (1990) Cell 60,

Flesh-eating bacteria Genetics of Bacterial Virulence by Charles J. Dorman Blackwell Scientific Publications, 1994. L29.50 pbk (xix + 369 pages) ISBN 0 632 03662 1

A

recent outbreak of necrotizing fasciitis in the UK is a dramatic example of what can happen when a mutually nondestructive host-parasite interaction is thrown out of balance. The cause of the lethal ‘flesh-eating’ infection was a strain of group A streptococcus. In a less virulent form, the group A streptococci are opportunistic inhabitants of the human throat, and are commonly associated with streptococcal pharyngitis, or strep throat. Like many other bacterial pathogens, the group A streptococci have adapted to survive and multiply in a specific niche without causing overt harm to the

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