Brazilian Journal of Medical and Biological Research (1999) 32: 861-865 Polyomavirus transformation 861 ISSN 0100-879X

Mechanisms of cell transformation induced by polyomavirus

M.L.S. Oliveira, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brasil S.M. Brochado and M.C. Sogayar

Abstract

Correspondence Polyomavirus is a DNA tumor virus that induces a variety of tumors in Key words M.C. Sogayar mice. Its genome encodes three , namely large T (LT), middle · Signal transduction · Instituto de Química, USP T (MT), and small T (ST) antigens, that have been implicated in cell Malignant transformation Caixa Postal 26077 · transformation and tumorigenesis. LT is associated with cell immor- Polyomavirus T antigens 05599-970 São Paulo, SP · Polyomavirus-induced talization, whereas MT plays an essential role in cell transformation by Brasil transcriptional control Fax: +55-11-818-3820 binding to and activating several cytoplasmic proteins that participate E-mail: [email protected] in growth factor-induced mitogenic signal transduction to the nucleus. The use of different MT mutants has led to the identification of MT- Presented at the I International binding proteins as well as analysis of their importance during cell Symposium on “Signal Transduction transformation. Studying the molecular mechanisms of cell transfor- and in Cell mation by MT has contributed to a better understanding of cell cycle Proliferation and Differentiation”, São Paulo, SP, Brasil, regulation and growth control. August 31-September 2, 1998.

Publication supported by FAPESP. Introduction Cells infected by Py express three pro- teins that have been implicated in cell trans- DNA tumor viruses have proved to be formation, the so-called tumor (T) antigens:

Received November 27, 1998 important tools in the study of cell growth large T (LT), middle T (MT) and small T Accepted January 11, 1999 control and neoplasia. The relatively small (ST). T antigens are coded by the early re- size of their genomes, together with the fact gion of the genome and are expressed in the that they efficiently and reproducibly induce early phase of the virus cycle, before replica- tumors when injected in animals and with tion of viral DNA. Three other proteins, the advances in Molecular Biology tech- namely VP1, VP2 and VP3, that are ex- niques, has facilitated the identification and pressed after viral DNA replication, are coded characterization of the viral proteins that are by the late region of the genome and are responsible for cellular transformation and essential for viral assembly (3). tumor induction (1). T antigens have partially overlapping cod- Polyomavirus (Py), a nuclear icosahedral ing sequences and are generated by alterna- virus containing a circular genome of double- tive splicing of a single RNA precursor. All stranded DNA, belongs to the papovavirus T antigens share a common N-terminal re- family and was first discovered as a tumor gion, but differ in the C-terminal region as agent in 1953 by Ludwig Gross. Its ability to the result of a frameshift reading. induce tumors in adult mice is relative low, A large number of early region mutants, but polyomavirus, as the name indicates, as well as cDNA cloning and expression of causes a wide variety of tumors in newborn each T antigen, have contributed to the anal- mice (2). ysis of their role during cell transformation

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(4). Py mutants that are unable to induce interaction with or inactivation of the tumors in animals display mutations that tumor suppressor . Through the ex- map in the early region of their genome. pression of a temperature-sensitive p53, Doherty and Freund (15) have shown that The large T antigen LT is able to overcome the growth suppres- sive activity of p53 despite its failure to bind The LT antigen is a 98-kDa protein (5) p53, but binding to pRb is essential for this that binds to specific sequences in the viral effect. DNA, regulating both DNA replication and In Py-transformed cells, there is no sig- transcription. It acts as an important negative nificant accumulation of p53 protein, as op- and positive transcriptional regulator of the posed to SV40-transformed cells, in which early and the late regions of the genome, the amount of steady-state p53 protein is respectively (6-8). The amino-terminal por- elevated. However, accumulation of p53 is tion of LT contains two observed following exposure of Py-trans- sites, at residues T187 and T278 formed cells to UV or X rays. Rapid induc- which are potential substrates for cyclin- tion of p21/WAF1 is also observed under dependent kinases (CDKs). Li and colleagues these conditions, suggesting that Py does not (9) have reported that mutations in T278, but interfere with the p53 DNA damage-induced not in T187 abolish LT DNA replication activities (16). functions. In contrast to the wild-type and T187 mutant, the T278 mutant is weakly The small T antigen phosphorylated by the cyclin B-cdc2 com- plex, suggesting the involvement of S and The ST antigen is a 22-kDa protein local- G2 phase-specific CDKs in viral replication. ized in the cytoplasm of cells infected with The importance of LT phosphorylation by Py, where it binds to cellular proteins such as this cyclin/CDK complex may explain in phosphatase PP2A (17,18). part why DNA tumor viruses require ac- Together with MT, ST appears to have a tively cycling host cells. Although this phos- role in viral DNA synthesis, since viruses phorylation site is involved in viral DNA expressing mutated ST and MT show a 100- replication, it is not important for the ability fold defect in genome accumulation during of LT to drive cellular DNA replication (10). infection of NIH-3T3 cells when compared LT is expressed in the nucleus of infected to wild type viruses (19). cells, where it binds to the product of the Although the expression of ST is not pRb (11,12). Mutant required for cell transformation, ST is able viruses which express LT antigen defective to augment the saturation density and to in pRb binding, but normal MT antigen, are induce changes in the cytoskeleton (20). unable to immortalize primary fibroblast cultures, but are still able to transform cells The middle T antigen established in culture (12,13). The immortality function of LT appears The MT antigen is a 55-kDa phosphopro- to be due to a block in apoptosis, even though, tein associated with the plasma membrane of in contrast to SV40, polyomavirus LT does cells infected with Py through a mirystyl not bind the product of the tumor suppressor anchor attached to the C-terminus of the gene p53. In this case, alternative pathways protein. Its function in the viral cycle ap- may be involved (14). pears to be related to virus assembly, since In fact, Py transforms cells in culture and MT induces phosphorylation of the VP1 induces tumors in mice without an apparent protein (21).

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Using cDNA injection in newborn mice, tein. Mutation in MT Ser 257 completely abol- Asselin and colleagues (22) have shown that ishes binding, but does not affect the ability of MT is able to induce tumors only in combina- MT to transform fibroblasts in vitro. tion with the N-terminal region of LT and ST. Activation of cellular proteins by MT Since MT alone is able to transform cells triggers a variety of signals in the cell that established in culture (23), it is believed that culminate with growth proliferation, even in it plays a central role during Py transforma- the absence of growth factors (32). tion and tumorigenesis. Thus, MT binding to Shc (through the The MT antigen can be phosphorylated 250 Tyr residue of MT) leads to its binding at serine residues located in the C-terminal to and activation of the Ras protein and, region of the molecule, as well as in threo- consequently, activation of the mitogen-ac- nine and tyrosine residues present in differ- tivated protein kinase (MAPK) pathway ent regions of the protein. Its ability to trans- (33,34). When activated, MAPKs translo- form cells is related to binding and activa- cate to the nucleus, where they phosphoryl- tion of a number of cytoplasmic proteins ate transcriptional factors, regulating the ex- related to cell growth control (24,25). In pression of genes that are essential for cell general, the interaction between MT and proliferation (35). cellular proteins occurs through phosphoryl- Activation of PI3K by MT occurs through ated tyrosine residues in MT and Src homol- association of MT Tyr 315 with the SH2 ogy-2 (SH2) domains present in different domain of the p85 regulatory subunit of signal-transducing proteins, as is the case for PI3K (36). Elevated levels of phosphoinosi- Src homology and collagen protein (Shc), tides phosphorylated at position 3 of the phosphatidylinositol-3-kinase (PI3K) and inositol ring are observed in MT-transformed g g phospholipase C (PLC ) (26,27). Mutations cell lines, but not in cell lines expressing the in any of these render MT defec- MT315 mutant (37). These molecules were tive for transformation (24). reported to bind to SH2 domains of cellular In addition to tyrosine-SH2 interactions, proteins such as Src and PI3K itself, suggest- a proline-rich domain present in the MT ing that they play a role in the regulation of molecule is important for binding to SH3- these (38). Following PI3K activa- containing proteins (27). Viruses expressing tion by MT, induction of Akt (the protein MT mutated in this proline-rich domain are coded by the cellular homologue of the AKT8 partially defective for transformation and ) serine/threonine kinase tumorigenesis and MT binding to PLCg is activity is observed. Using MT mutants that partially affected (28). are not able to bind to Shc or PI3K, Summers Analysis of other MT mutants has led to and colleagues (39) were able to localize Akt the identification of the N-terminal region as downstream relative to PI3K, but not to Shc. the binding site for phosphatase PP2A and Recent studies evidenced the role of PI3K Src (17). Recently, MT resi- activation in the blocking of apoptosis by dues 185 to 210 were reported to be essential MT in a p53-independent manner (40). for Src binding (29). Transformation-defec- Transgenic mice expressing MT mutants tive MT mutants that retain the ability to defective in binding to Shc or PI3K develop bind Src were reported, suggesting that acti- mammary epithelial hyperplasias, in con- vation of Src is not sufficient to induce trans- trast to rapid metastatic mammary tumors formation by MT (24,30). observed in strains expressing wild-type MT Cullere and colleagues (31) recently de- (41). The mammary epithelial hyperplasias scribed the first MT phosphoserine residue as expressing the MT mutant defective in re- an important binding site for the 14-3-3 pro- cruiting PI3K are highly apoptotic, suggest-

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ing that recruitment of PI3K by MT affects during MT cell transformation. cell survival. Tumor progression in both High expression of urokinase-type plas- mutant strains correlates with upregulation minogen activator, which is regulated by of the epidermal growth factor receptor fam- AP-1 consensus sequences present in its pro- ily members which are known to couple to moter, is observed in MT-transformed cell the PI3K and Shc signaling pathways (41). lines. Injection of MT-transformed endothe- lial cell lines leads to the formation of vascu- Transcription factors in Py-MT lar tumors in newborn mice. These cell lines transformation exhibit altered proteolytic activity that con- tributes to tumor growth (44). MT overexpressing fibroblasts display The contribution of polyomavirus to the constitutively high transcription factor AP-1 understanding of cell growth control and activity (42) as a result of constitutive over- neoplasia began with the important discov- expression of the cJun and JunB AP-1 com- ery of the role played by tyrosine phosphory- ponents (30). Induction of AP-1 activity by lation in cell proliferation (45). Since then, MT was shown to be dependent on MT studies of T antigens have led to new find- binding to PI3K, but does not correlate with ings in cell immortalization, apoptosis and cell transformation, indicating that additional transformation. In particular, MT studies have events are necessary (30). contributed to the mapping of a variety of Using glucocorticoid-inducible MT-over- signalling pathways related to cell growth expressing fibroblasts, Rameh and Armelin control. Thus, expression of both wild type (43) have reported induction of the c-myc and mutant MT in a variety of cell types has proto-oncogene at the mRNA level, parallel- become an important tool to understand the ing induction of MT with glucocorticoids. differences among signalling pathways in Taken together, these results indicate that different cell systems (46). cMyc and Jun proteins act cooperatively

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