Transforming Functions of Simian Virus 40

Transforming functions of Simian Virus 40

M Teresa SaÂ enz-Robles1, Chris S Sullivan1 and James M Pipas*,1

1Department of Biological Sciences. University of Pittsburgh, Pittsburgh, Pennsylvania, PA 15260, USA

Oncogene (2001) 20, 7899 ± 7907. SV40 and the polyomaviruses

Keywords: SV40; tumor suppressor; chaperone; tumor SV40 is a prototype member of the polyomavirus virus group (for a complete review, see: Fields, 1996), having a 45 nm diameter virion containing a single molecule of closed circular double-stranded DNA of 5243 bp. Introduction There are seven known virus-encoded proteins gener- ated by dierential splicing of two transcription units Cancer is thought to progress through multiple stages (Figure 1). The viral T antigens, large T antigen (LT), with each stage giving rise to cells showing increased small T antigen (ST) and 17K T antigen (17KT) are malignant characteristics. Progression through these expressed soon after infection from a transcript stages is driven by genetic events such as mutations in initiating at the early promoter. Gene expression is tumor suppressor genes or oncogenes, or the acquisi- accomplished using the cellular transcription apparatus tion of speci®c viral transforming sequences. These and, in fact, the SV40 early promoter is used to drive tumor suppressor genes and oncogenes encode proteins expression of transgenes in a number of eucaryotic that are key components of regulatory pathways that expression vectors. The functions of the viral T govern genomic stability, cell proliferation, and antigens are discussed below. apoptosis. Three key questions in cancer research are: The SV40 transcript initiated from the viral late (1) what speci®c genetic pathways, and which speci®c promoter is dierentially spliced to result in mRNAs proteins within each pathway must be altered at each encoding the major capsid protein VP1, the minor stage of tumor progression?; (2) how is cellular capsid proteins VP2 and VP3, or the viral agno behavior changed by altering each speci®c pathway? protein. VP1, VP2, and VP3 are the only virus-encoded and (3) are the cellular consequences the same for proteins found in mature virions. The agno protein altering a given pathway in every cell type, or does a seems to function at some stage of virion assembly or single pathway govern multiple cellular properties virus exit from infected cells. depending on the cell-type? All polyomaviruses share the same basic genome DNA tumor viruses are important both because they structure, a circular DNA with the T antigens and the provide us with powerful tools to address mechanisms capsid proteins expressed from separate transcription of tumorigenesis, and because, in some cases, they units, and an approximately 400 bp noncoding reg- contribute directly to cancer. Rather than alter cellular ulatory region containing the origin of viral DNA pathways by mutation, DNA tumor viruses encode replication and the early and late promoters. All dominant acting transforming proteins. Each of these polyomaviruses encode LT, ST, VP1, VP2, and VP3. transforming proteins target one or more key regula- Murine polyomavirus and hamster polyomavirus both tory proteins within a cell, thus altering the cell's encode a middle T antigen (MT) as well. Like LT and growth/survival properties. Because the viral trans- ST, MT is produced by dierential splicing of the early forming proteins must directly associate with their region primary transcript. target, they serve as `proteo-divining rods,' guiding SV40 is one of 14 characterized polyomaviruses, of investigators through a milieu of cellular pathways and which 12 have been fully sequenced. These include two proteins irrelevant to the tumorigenic process and human viruses, JCV and BKV, and the well-character- directly to the key players involved in malignant ized murine polyomavirus (PyV). Some of these viruses transformation. In addition, insights into how these such as SV40 and BKV are rarely pathogenic in their key pathways are regulated can be gleaned from natural hosts, while others such as BFDV, PyV, or studies of the biochemical mechanisms by which the murine K virus are deadly (Pipas, 1992). viral transforming proteins alter their target. Finally, The host of SV40 is the Rhesus macaque and many cell culture and transgenic mouse systems have allowed of these animals harbor apparently harmless lifelong studies of the role that each regulatory pathway plays persistent infections of SV40. The kidney is thought to in controlling cellular behavior. be the primary target organ of SV40 infection. The virus undergoes productive infection in terminally dierentiated epithelial cells. This presents a problem *Correspondence: JM Pipas; E-mail: [email protected] for the virus in that because of its small genome size it Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7900

Figure 1 Genomic Organization of SV40 virus (5243 bp). Arrows indicate the mRNAs encoding the capsid proteins VP1, VP2 and VP3 (found in mature virions) originated from the late promoter (PL), and the viral antigens large T antigen (LT), small T antigen (ST) and 17K T antigen (17KT) (involved in cellular transformation and originated from the early promoter PE). Origin of replication is indicated (Ori)

relies primarily on cellular proteins to eect viral DNA Why rodent cell lines? They were beautifully amenable replication. Since the expression of most genes to these studies because of a weird aspect of arcane encoding replication-related proteins is restricted to virology. Rodent cells are nonpermissive for SV40 late G1 or S phases of the cell cycle, SV40 must ®nd a productive infection. That is, infection of established way to activate the expression of these genes in growth- rodent ®broblast cell lines such as BalbC/3T3 with arrested, dierentiated kidney cells. SV40 does not result in the production of progeny SV40 undergoes ecient productive infection in virions. Careful observation of such nonpermissive established cell lines such as CV1, BSC, or VERO, infections reveals a remarkable observation. SV40 all of which were derived from the kidneys of African attaches, penetrates, and uncoats more or less normally green monkeys. In these cell lines productive infection in these cell lines and the viral T antigens are expressed is complete about 96 hours post-infection, resulting in on schedule. However, neither viral DNA replication about 300 infectious progeny virions per cell. nor transcription from the late promoter occur. Thus, The program of SV40 productive infection initiates the infection is blocked after T antigen production but with attachment of a virion to a permissive cell and is before components of the progeny virions are followed by penetration, transport to the nucleus, and expressed. Like permissive monkey cells expressing virion uncoating. Release of the viral chromatin is the viral T antigen, the infected rodent cells are followed by activation of the early promoter by the stimulated to progress through the G1/S transition cellular transcription machinery and by subsequent and cellular DNA replication does occur. Infection of a synthesis of LT, ST, and 17KT. This results in a rodent cell line at a high multiplicity-of-infection (moi) change in the pattern of cellular gene expression. The of SV40 results in all of the cells acquiring transformed expression of many genes associated with cellular DNA characteristics. However, as cell proliferation proceeds synthesis is enhanced and the cells are in fact driven the viral DNA driving T antigen expression is diluted through G1 and enter S phase. Shortly afterwards, so fewer and fewer daughter cells are transformed. viral DNA replication begins resulting in the produc- Eventually the viral DNA is lost and the cell tion of progeny viral DNA. Concomitant with the population regains the properties of normal, growth- initiation of viral DNA replication, the late promoter regulated cells. This phenomenon has been termed becomes active resulting in the expression of VP1, VP2, `abortive transformation.' VP3, and agno protein. This results in the assembly of When abortively transformed cell populations in progeny virions and eventually in cell death. culture are maintained continuously, a few `mounds' of multilayered, transformed cells overgrow the normal cell monolayer. These are termed foci and represent a SV40 and cellular transformation true stable transformation event since cells picked and expanded from them continue to exhibit the trans- Shortly after SV40 was discovered it was found to formed phenotype through many generations. All such induce tumors when inoculated into newborn baby foci-derived cells express the SV40 T antigens and all hamsters. This observation, coupled with the fact that contain SV40 DNA integrated at a random point it is relatively easy to grow in cell culture, catalyzed an within the cellular DNA. In these cases, a portion of impressive burst of activity in SV40 research. At about the viral DNA integrates into the host genome by the same time, cell culture assays that mimic or nonhomologous recombination at some point in the measure aspects of oncogenic transformation were abortive infection. If such an integration event leaves being developed. Although the variations are many, the viral early promoter and T antigen coding ®ve basic assays are used to assess cellular transforma- sequences intact, then daughter cells derived from this tion: (1) growth in low serum; (2) growth to high event will continuously express the T antigens. Since saturation density; (3) focus formation; (4) anchorage these cells are not growth-arrested by contact inhibi- independent growth and (5) tumorigenicity in animals. tion or reduced availability of serum, they continue to SV40 induces cellular transformation by all of these proliferate when the normal cells surrounding them criteria. It is important to note that most of these have arrested and thus, they form the foci. studies have been performed on established rodent The isolation of temperature-sensitive mutants in LT ®broblast cell lines. We discuss below more recent (tsA mutants) and deletion mutants that eliminate studies on SV40 transformation of multiple cell types. expression of ST allowed an assessment of the dierent

Oncogene Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7901 roles of these proteins in transformation. Foci derived SV40 encoded tumor antigens from infections with tsA mutants display a fully transformed phenotype at the permissive temperature, There are three tumor antigens encoded by SV40: large usually 328C, however, they revert to a normal T antigen (T antigen), small t antigen (t antigen) and phenotype when maintained at the nonpermissive 17K t antigen (see Figure 2, for a complete review, see temperature (418C). Similarly, infection of rodent cells Fields, 1996). All are expressed early after infection with tsA mutants at 328C leads to foci production, from a common precursor mRNA that is dierentially while infection at 418C fails to yield foci (Rassoulza- spliced such that the three proteins share an amino degan et al., 1978; Seif and Cuzin, 1977). Thus, T terminal domain but contain dierent carboxy terminal antigen function is necessary to initiate and to maintain regions. The common amino-terminal domain includes the transformed phenotype. Some cell lines expressing the ®rst 82 amino acids and is a J domain, a domain tsA T antigens maintain the transformed phenotype found in the DnaJ family of co-chaperone regulators of even at the nonpermissive temperature. This phenom- the Hsc70 class of molecular chaperones. T antigen has enon has not been fully explained. been shown to function as an authentic DnaJ Studies using constructs that express either a wild- chaperone both in vivo and in vitro (Campbell et al., type LT and no ST, or ST and no LT revealed that 1997; Srinivasan et al., 1997; Sullivan et al., 2001; ST is not sucient to transform cells but is sometimes Zalvide et al., 1998). necessary. In many circumstances, LT is necessary and The 708 amino acid T antigen is a multifunctional sucient for transformation. However, in some cell protein that is required for several aspects of types, or under certain conditions such as limited productive infection and viral tumorigenesis, including steady state-levels of LT or dense cell monolayers, both the initiation and elongation steps of viral DNA both LT and ST are required for transformation replication. T antigen binds the viral origin of (reviewed in Rundell and Parakati, 2001). In sum- replication (ori) through a sequence-speci®c DNA mary, a number of dierent types of studies have binding domain, forming a double-hexamer and shown that the full tumorigenic potential of SV40 inducing structural changes in adjacent sequences. T resides in both LT and ST. antigen directly binds DNA polymerase a, DNA

ATPase

Figure 2 Domain map of SV40 tumor antigens. (a) Large T antigen; (b) small t antigen and, (c) 17K T antigen. Known and possible interactions with cellular components are indicated. Structures depicted were obtained from the following references: J domain of murine polyomavirus T antigen (Berjanskii et al., 2000, PDB ID: 1FAF); SV40 T antigen DNA binding domain (Luo et al., 1996, PDB ID: 1TBD); structure of a single zinc ®nger DNA-binding domain (Lee et al., 1989, PDB ID: 1ZNF)

Oncogene Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7902 primase, RPA, and topoisomerase, thus recruiting the express similar proteins (Bollag et al., 2000; Riley et cellular replication apparatus to the SV40 ori and al., 1997). eecting the initiation of viral DNA replication. Subsequently, each T antigen hexamer acts as a DNA helicase, a function evoking multiple domains T antigens target key cellular regulatory proteins to including the ATP-binding/ATPase region. The role of eect transformation T antigen in viral DNA replication is regulated by phosphorylation. Phosphorylation of the amino acid Genetic studies have clearly delineated three regions of T124 is required for the ecient cooperative assembly T antigen required for transformation. The ®rst region, of double hexamers at ori and T antigen that is not consisting of the ®rst 82 amino acids, is the J domain. modi®ed at this site is defective for the initiation of The J domain governs the interaction of T antigen with replication. In contrast, phosphorylation of several its DnaK partner, hsc70. Deletion of the J domain serine residues antagonizes the formation of double- results in a transformation-negative phenotype in a hexamers. number of cultured cell-types as well as in transgenic T antigen is also a transcriptional regulatory protein. mice. However, it is not clear if hsc70 is the cellular T antigen acts as a transcriptional repressor by binding target relevant to transformation. Amino acid substitu- to speci®c sequences near the viral early promoter thus tion mutations in the conserved HPD motif of the J inhibiting its own expression as well as that of t antigen domain are defective for interaction with hsc70 yet and 17K t antigen. On the other hand, T antigen retain the ability to induce dense foci in primary and activates transcription by binding speci®c components established cell lines. These mutants do show some of the cellular transcriptional machinery, including transformation-related defects such as the inability to TBP and several transcription factors. Although the induce primary cells to grow to a high saturation exact mechanisms remain to be elucidated, it is clear density. One possibility is that hsc70 association is that these interactions are at least partly responsible for critical for transformation but that T antigen has the activation of the SV40 late promoter and several redundant activities that aect the same cellular cellular genes. T antigen also plays roles in virion pathways targeted by hsc70. Another possibility is that assembly although the precise nature of its contribu- some target other than hsc70 associates with the J tion is unclear (Spence and Pipas, 1994a,b). domain and that, while deletions of the J domain In addition to its multiple roles in productive eliminate action on this target, amino acid substitu- infection, T antigen is also necessary and often tions within the HPD motif are only partially defective sucient for inducing viral tumorigenesis. At least for this interaction. The observation that constructs in three dierent T antigen domains contribute to cellular which the SV40 J domain is replaced by the J domain transformation. These include a bipartite region within from the E. coli DnaJ protein, or by the yeast protein the carboxy-terminal half of the molecule that governs Ydj1p J domain, are transformation-defective while interaction with the cellular tumor suppressor protein, retaining hsc70 interaction, suggest that additional p53; a short domain containing an LXCXE motif cellular proteins interact with this region of T antigen responsible for binding the retinoblastoma (Rb) family (Sullivan et al., 2000b). of tumor suppressors; and, the J domain. The roles of T antigen binds to the retinoblastoma family of each of these transforming regions in tumorigenesis are tumor suppressors through an LXCXE motif located discussed below. between the J domain and the NLS. T antigen binds all The 174 aa small t antigen is not essential for three members of this family, pRb, p107, and p130, productive infection in cell culture. However, t antigen and in each case, can relieve cellular growth-arrest does contribute to viral tumorigenesis, and in some mediated by these proteins (reviewed in DeCaprio, circumstances, the cooperation of large T antigen and 1999). One function of the Rb-family is to negatively small t antigen is necessary for transformation. The regulate the transcriptional activation function of the unique carboxy-terminal domain of t antigen contains E2F-family of proteins. Release of E2F from Rb- a Zn-®nger that binds the cellular phosphatase pp2A. mediated control results in the expression of S phase This interaction is essential for transformation. The speci®c genes and drives cells into the cycle. In theory, direct biochemical target of the t antigen J domain is this is necessary for viral productive infection in that unclear, although, like large T antigen, it can stimulate SV40, for the most part, uses cellular proteins for viral the ATPase activity of Hsc70 family members DNA replication. T antigen mutants that cannot (Srinivasan et al., 1997). The t antigen J domain is interact with the Rb-family are defective for transfor- not required for binding or action on pp2A but it is mation in most cell-types tested. Clearly, one role of T essential for t antigen-induced transcription of the antigen in this interaction is to inhibit the Rb-protein's cyclin A gene (Rundell and Parakati, 2001). ability to interact with and to block E2F-mediated Finally, the 17K t antigen contains the J domain, transcriptional activation. the adjacent LXCXE domain and nuclear localization Binding of the Rb-proteins by T antigen is signal (NLS), followed by four unique amino acids at insucient to activate E2F. This requires both a the carboxy-terminus. The role of this protein in functional LXCXE motif and a J domain (Harris et SV40 infection and transformation is unclear. How- al., 1998; Sheng et al., 1997; Zalvide et al., 1998). One ever, other members of the Polyomavirus family model is that T antigen ®rst binds to Rb/E2F

Oncogene Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7903 complexes through its LXCXE motif and then these apoptotic genes such as bax. DNA tumor viruses complexes are dissolved using energy derived from abrogate this response by blocking one or more of the hsc70-mediated ATP hydrolysis (Srinivasan et al., p53 functions. SV40 T antigen regulates the function of 1997). In fact, puri®ed T antigen has been shown to p53 in at least two ways (Pipas and Levine, 2001). First bind p130/E2F-4 and pRb/E2F-4 complexes in vitro T antigen directly binds to the DNA binding domain and the dissolution of these complexes requires ATP of p53, which results in the inactivation of its hydrolysis by hsc70 (Sullivan et al., 2000a). This transcriptional activation activity. This interaction explains why the J domain is required in addition to prevents the growth inhibitory induction of genes the LXCXE motif to eect E2F transcriptional downstream of p53, such as the cyclin/cdk inhibitor activation. In fact, the shortest fragment of T antigen p21. Secondly, T antigen regulates p53 independent of that induces transformation is the ®rst 121 amino direct association. The mechanism by which this occurs acids, consisting of the J domain followed by 39 amino is unclear but it requires both the J domain and the acids, including the LXCXE motif. Nevertheless, the pRb binding motif of T antigen (Jiang et al., 1993; whole story is far from being completely understood. Quartin et al., 1994). First, Tevethia et al. (1997a) have shown that a short There is also evidence for transforming function(s) peptide, including the LXCXE motif but missing the J in the carboxy terminus of T antigen in addition to the domain, promotes cellular growth to high density when binding of p53. First, Dickmanns et al. (1994) have fused to the carboxy-terminus of T antigen. It is not identi®ed a mitogenic activity aected by mutation of yet clear whether the construct achieves this eect by amino acid 189. This mutation does not aect LT disrupting Rb/E2F complexes or whether the LXCXE binding to p53 but, on the other hand, this mutant is motif and/or immediately surrounding sequences are unable to bind to the transcription factor Tef-1, thus able to aect cell growth independent of action on Rb. supporting a possible role for Tef-1/T antigen Second, Kohrman and Imperiale (1992) have identi®ed interaction in transformation. Second, Cavender et al. a protein, termed p185, that binds to the ®rst 121 (1995) have demonstrated that deletion of the amino amino acids of T antigen, and whose binding to T acid 400 yields a transformation-defective T antigen, antigen is independent of the J domain and the although this mutant binds p53 normally. Finally, co- LXCXE motif. It is not clear if the T antigen/p185 expression of the ®rst 136 amino acids of LT which interaction is required for transformation but this inactivates the Rb-family with a dominant-negative observation raises the possibility that the ®rst 121 allele of p53 does not elicit transformation, again amino acids of T antigen targets cellular proteins in suggesting the presence of T antigen transforming addition to the Rb-family. Third, the amino-terminus functions in addition to Rb and p53 inactivation of T antigen has been reported to bind a pro-apoptotic (Sachsenmeier and Pipas, 2001). protein, p193 (Daud et al., 1993). p193 induces p53- independent apoptosis and binding to T antigen blocks p193-dependent apoptosis (Pasumarthi et al., 2001; How does T antigen action on cellular targets contribute Tsai et al., 2000). Fourth, T antigen binds to the to tumorigenesis? transcription factor Tst-1/Oct6/SCIP through its amino terminus and has been shown to synergistically Transgenic mouse models have been widely used to enhance its transactivation activities in a J domain- study gene expression under physiological conditions in dependent manner (Sock et al., 1999). Again, the vivo. With tissue-speci®c promoters, it is possible to eects of T antigen's interactions with p193 or Tst-1/ target the expression of a protein exclusively to a Oct6/SCIP on its transforming ability have not been speci®c tissue or to particular cell types. In this context, determined. Finally, mutations in the amino terminus the DNA coding sequence of the SV40 T antigen has of T antigen aect its interaction with the transcrip- been placed under the control of dierent promoters, tional adaptor protein p300 (Eckner et al., 1996). T thus directing T antigen expression to selected organs, antigen binding to p300 has been mapped to both the tissues or cell types. The expression of T antigen alters amino terminal and carboxy terminal portions of the T cellular growth control in a broad range of tissues, antigen, and at present it is not clear if this association some of which are listed in Table 1. These experiments is direct (Eckner et al., 1996; Lill et al., 1997). T illustrate the importance of whole-animal assays in antigen alters the phosphorylation state of p300 and determining the molecular basis of transformation, additionally has been shown to down-regulate its since the eects of SV40 T antigen have been found to transactivation-inducing abilities (Eckner et al., 1996). vary considerably in dierent tissues and organs. Again, the role of the LT/p300 interaction in Furthermore, mutant analysis has revealed distinct transformation has not been clearly established. roles for each T antigen activity in contributing to A number of cellular stresses, including the neoplasia. inappropriate activation of the E2F-family, activate The eects of ectopic expression of T antigen varies the p53 tumor suppressor pathway. This leads to widely depending on several factors, in particular the elevated steady-state levels of p53 and the expression of tissue where the protein is expressed, the speci®c p53-dependent genes including p21, a universal cdK- promoter controlling that expression and the particular inhibitor. Thus, activation of p53 blocks the cell cycle. cell types involved in the transformation. Conse- In addition, p53 induces the expression of pro- quently, LT expression leads to hyperplasia and

Oncogene Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7904 Table 1 Proliferation and oncogenesis induced in murine tissues following transgenic expression of SV40 T antigen Tissue/organ References

Adipose tissue Fox et al., 1989; Zennaro et al., 1998; Le Menuet et al., 2000 Bone Behringer et al., 1988; Knowles et al., 1990; Jensen et al., 1993; Wilkie et al., 1994 Brain and nervous system Brinster et al., 1984; Reynolds et al., 1988; Chen et al., 1989; Theuring et al., 1990; Chen and Van Dyke, 1991; Perraud et al., 1992; Smith et al., 1992; Jensen et al., 1993; Chiu et al., 2000 Cartilage Cheah et al., 1995 Digestive system Li et al., 1995; Kim et al., 1994; Hauft et al., 1992; Asa et al., 1996 Eye Theuring et al., 1990; Penna et al., 1998; Beermann et al., 1999 Heart Behringer et al., 1988 Kidney Theuring et al., 1990 Liver Dyer and Messing, 1989; Sandgren et al., 1989; Sepulveda et al., 1989; Butel et al., 1990; Manickan et al., 2001 Lung Sandmoller et al., 1994, 1995; Wikenheiser et al., 1992 Lymphoid tissue Reynolds et al., 1988; Chen et al., 1989 Mammary gland Husler et al., 1998; Green et al., 2000; Schulze-Garg et al., 2000 Pancreas Hanahan 1985; Murphy et al., 1987; Ornitz et al., 1987; Efrat et al., 1988; Dyer and Messing, 1989; Bell et al., 1990; Ceci et al., 1991; Smith et al., 1992; Asa et al., 1996 Pituitary Murphy et al., 1987 Prostate Garabedian et al., 1998; Green et al., 2000 Salivary gland Pascall et al., 1994; Sandmoller et al., 1994; Ewald et al., 1996 Smooth muscle Wilkie et al., 1994; March et al., 1999; Herring et al., 1999 Stomach Ceci et al., 1991; Thompson et al., 2000

carcinoma in some tissue types, but not in others (Lee the viral product is no longer expressed (Ewald et al., et al., 1992, Lopez et al., 1995). For instance, SV40 T 1996). antigen has been expressed in several dierentiated cell As a general rule, cells induced to proliferate in subpopulations of the small intestine and, although all response to LT expression seem to retain their of them originate from common precursor stem cells, dierentiation markers. For instance, expression of full the consequences of this expression are extremely length SV40 LT in postmitotic, villus-associated dierent. While hyperplasia and dysplasia of the small enterocytes causes them to reenter the cell cycle intestine result from the production of T antigen in without an apparent eect on their state of dierentia- enterocytic cells (Hauft et al., 1992; Kim et al., 1994), tion (Hauft et al., 1992) and a similar phenomenon is no apparent pathology is detected in the small bowel in observed upon expression of LT in secretory cells of transgenic mice expressing the T antigen in secretory the colon (Asa et al., 1996; Lopez et al., 1995), cells (Asa et al., 1996; Lee et al., 1992, Lopez et al., pancreas (Lopez et al., 1995), retinal (Penna et al., 1995). Expression of T antigen in Paneth cells results in 1998) and smooth muscle cells (March et al., 1999; a loss of the mature cell type and leads to an Herring et al., 1999). However, some exceptions have ampli®cation of cells showing an intermediate mor- been noticed: in adult animals where brain cells were phology between Paneth and goblet cells (Garabedian forced to express LT under the control of the FGF1 et al., 1997). In contrast, goblet cells expressing T promoter, tumors were produced which lacked term- antigen enter S phase but fail to progress through M inal dierentiation markers for astrocytes or neurons, phase, instead undergoing apoptosis (Gum et al., but expressed high levels of markers for proliferating 2001). Finally, some cell types seem refractory to cells. This phenotype is consistent with the tumor being transformation by T antigen in vivo, as its sole at an early stage of dierentiation (Chiu et al., 2000). expression does not result in tumor development (Efrat Similarly, pancreatic endocrine cells expressing T et al., 1988; Reynolds et al., 1988). antigen only show some low levels of particular In cases where T antigen expression is followed by dierentiation markers, resembling an intermediate malignant transformation, a general correlation is state of dierentiation (Asa et al., 1996). observed between the production of higher levels of In most cases tested, the amino terminal region of T antigen and the appearance of tumors, in terms of LT (comprising the J domain and Rb binding motif) is both severity and frequency (Husler et al., 1998; Kim necessary and sucient to induce tumors in various et al., 1994; Van Dyke et al., 1987). In fact, through murine tissues, in a manner that absolutely requires an the use of conditional promoters (e.g. tetracycline- intact Rb-interaction domain (Bennoun et al., 1998; responsive), it has been shown that continuous Chandrasekaran et al., 1996; Chen et al., 1992; Kim et expression of LT is required to maintain cellular al., 1994; SaÂ enz Robles et al., 1994). The role of the J transformation, as a reversion of the malignant domain has been less studied in transgenic models, and phenotype is observed if LT expression is suppressed the reports so far indicate an important -although (Ewald et al., 1996; Manickan et al., 2001). However, tissue-speci®c- dependency on the J domain to induce after prolonged LT expression these transformed cells tumors (Chen et al., 1992; Ratineau et al., 2000; lose their dependence on LT for the maintenance of the Symonds et al., 1993). In each of these cases, the transformed state, and remain tumorigenic even after amino-terminal fragment of LT is sucient to drive

Oncogene Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7905 dierentiated cells to reenter the cell-cycle resulting in T antigen function located in the carboxy terminal responses varying from hyperplasia to frank tumor- portion of the molecule. igenesis. Most of the transgenic constructs driving expression In contrast, the importance of the contribution of of large T also encode the small t antigen from SV40. p53 inactivation by T antigen to tumorigenesis varies In most of these cases, the possible role of small t depending on the cell-type examined. Some tissue antigen in cooperating with large T antigen or in types require a functional T antigen/p53 binding producing tumors by itself has not been addressed. One domain for oncogenic transformation (McCarthy et recent report has evaluated the possible role of small t al., 1994; SaÂ enz-Robles et al., 1994), while in other antigen in inducing proliferation of several secretory cases the binding of T antigen to p53 is not required cell types (Ratineau et al., 2000). This study revealed for induction of tumors (Bennoun et al., 1998; Chen that small t antigen is not necessary to produce et al., 1992; Tevethia et al., 1997b). In the case of insulinomas or lymphomas, but that a functional choroid plexus epithelium, the J domain coupled with cooperation between intact large T and small t the Rb-binding motif induces hyperplasia as eciently oncoproteins might be required to produce neoplasms as full length T antigen. However, while tissue of intestinal secretory cells. Clearly, this is an area that expressing wild-type T antigen rapidly expands and requires more intensive study. kills the animals in a few weeks, the overall expansion Many animal models expressing T antigen in mouse, of the hyperplastic choroid plexus expressing a hamsters and rats closely resemble speci®c human truncated T antigen missing the p53 interaction cancers (e.g. Green et al., 2000 for mammary gland; domain is much slower. Inactivation of p53 by T Thompson et al., 2000 for stomach) or particular antigen binding, the genetic removal of p53 or the aspects of the disease, such as hormone dependency interference with the normal p53 function by expres- and response (e.g. androgen dependency of prostate sing a p53-dominant negative mutant causes an carcinomas, Asamoto et al., 2001). These models are accelerated tumor growth which correlates with a therefore incredibly useful tools to ascertain molecular decrease in the apoptotic level (Bowman et al., 1996; markers as indicators of tumor progression, as well as SaÂ enz-Robles et al., 1994; Symonds et al., 1994). On to test preventive or corrective therapies, such as the the other hand, T antigen-induced carcinogenesis of use of chemotherapy or cytokines (e.g. Baratin et al., pancreatic beta cells is reduced when the expression of 2001 gamma interferon treatment of hepatomas). p53 is abolished in this tissue, perhaps in response to Although the consequences of T antigen expression the lower levels of T antigen found in cells lacking are normally restricted to the tissues in which it is p53 compared to wild-type tumor cells (Herzig et al., expressed, there have been reports of eects aecting 1999). Finally, the loss of p53 function caused by other organs and/or cell-types. For instance, a general- interaction with T antigen only has only a moderate ized peripheral neuropathy was observed in transgenic eect on hepatic tumor formation, while it remains mice even though the SV40 early genes were expressed completely dependent on the disruption of the Rb only in pancreas and liver, but not in the peripheral or pathway (Bennoun et al., 1998). Similarly, the central nervous systems (Dyer and Messing, 1989). In presence or absence of p53 do not seem to aect the this case the secondary consequences of T antigen in T antigen-induced transformation of the intestinal the nervous system could be related to alterations in epithelium (Coopersmith et al., 1997; Kim et al., the circulating glucose levels resulting from the direct 1993). In this case, one possibility is that the eects of damage to the islet cells. Similarly, expression of T T antigen inhibition of p53-independent apoptosis antigen in neuroendocrine cells of the prostate leads to contribute to tumor progression (Coopersmith et al., the development of prostatic neoplasia which pro- 1997; Kim et al., 1993; Li et al., 1995). gresses rapidly to local invasion and metastases One complication in trying to determine the roles of (Garabedian et al., 1998). In this case, the endocrine TAg in tumorigenesis is that carboxy terminal cells from the prostate secrete a variety of growth truncations of TAg produce very unstable products, factors that may aect development and maintenance and attempts to generate transgenic mice expressing of this tissue, and their alteration by tumorigenesis only the p53-binding domain of TAg have been could aect other contiguous and/or distant tissues. dicult to pursue. Nevertheless, only the production Other cases have been reported where expression of T of such transgenic mice, either alone or in combination antigen results in apparent metastases to other tissues with other mutant TAg transgenics, will indeed address (Asa et al., 1996; Fox et al., 1989; Garabedian et al., the role of the interaction between TAg and p53 in 1998). However, the expression of T antigen in these tumorigenesis, and the speci®city of the pathway in the putative metastatic nodes was not evaluated. Thus, it particular cell types under study. remains unclear whether these distant tumors represent In summary, SV40 T antigen-mediated induction of true clonal descendents of the original neoplasia, or if tumorigenesis in transgenic mice requires an intact Rb- they are the consequence of secondary events derived binding function (probably in combination with a from the eects of T antigen in cells from the primary functional J domain) to commit cells to further tumor. Thus, T antigen expression can result in both proliferation and exit from the resting state. Further cell autonomous and non-autonomous eects. The progression towards a more malignant phenotype non-autonomous eects could result either from an sometimes requires inactivation of p53, or some other expansion of a cell-type that naturally produces

Oncogene Transforming functions of Simian Virus 40 MT SaÂenz-Robles et al 7906 hormones or growth-factors that act on distant tissues the Rb-family and on p53 clearly contribute to and/or alter systemic metabolism, or from the T neoplasia. However, it is also clear that both of antigen-induced expression of such factors in cells these molecules possess additional, as yet unchar- where they are normally not transcribed. acterized functions that contribute to cellular transformation. Among the challenges facing investigators studying these proteins is to: (1) identify the Summary additional cellular targets relevant to transformation; (2) elucidate the biochemical basis for large and SV40 has provided investigators with very powerful small T antigen action on these targets, and ®nally tools for probing and understanding molecular (3) understand how altering each of these targets mechanisms contributing to tumorigenesis. The large changes cellular behavior and contributes to the and small T antigens of SV40 exert their eects by tumorigenic phenotype. Thus, the DNA tumor binding to and by altering the activity of key viruses continue to play a central role in uncovering cellular targets. Thus, small t antigen targeting of the mysteries of tumorigenesis, and SV40 continues phosphatase pp2A, and large T antigen action on to be an important contributor to these eorts.

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