Oncogene (2005) 24, 7729–7745 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc SV40 large T antigen targets multiple cellular pathways to elicit cellular transformation Deepika Ahuja1,2, M Teresa Sa´ enz-Robles1,2 and James M Pipas*,1 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA DNA tumor viruses such as simian virus 40 (SV40) three proteins that are structural components of the express dominant acting oncoproteins that exert their virion (VP1, VP2, VP3) and two nonstructural proteins effects by associating with key cellular targets and termed large T antigen (T antigen) and small T antigen altering the signaling pathways they govern. Thus, tumor (t antigen). In addition, some members of the Polyoma- viruses have proved to be invaluable aids in identifying viridae encode additional proteins that are virus specific proteins that participate in tumorigenesis, and in under- or are encoded by a subset of viruses in the family. For standing the molecular basis for the transformed pheno- example, SV40 encodes three such proteins: agnopro- type. The roles played by the SV40-encoded 708 amino- tein, 17K T, and small leader protein. The genomes of acid large T antigen (T antigen), and 174 amino acid small all polyomaviruses can be divided into three elements: T antigen (t antigen), in transformation have been an early coding unit, a late coding unit, and a regulatory examined extensively. These studies have firmly estab- region (Figure 1). In a productive infection, expression lished that large T antigen’s inhibition of the p53 and Rb- of the early coding unit is effected by the cellular family of tumor suppressors and small T antigen’s action transcription apparatus and results in expression of the on the pp2A phosphatase, are important for SV40-induced large, small and 17K T antigens. Expression of the late transformation. It is not yet clear if the Rb, p53 and pp2A coding unit, which encodes VP1, VP2, VP3, and the proteins are the only targets through which SV40 trans- agnoprotein requires an active T antigen in conjunction forms cells, or whether additional targets await discovery. with the cellular transcriptional apparatus. In addition Finally, expression of SV40 oncoproteins in transgenic to activating transcription from the viral late promoter, mice results in effects ranging from hyperplasia to invasive T antigen also functions in viral DNA replication and carcinoma accompanied by metastasis, depending on the virion assembly. tissue in which they are expressed. Thus, the consequences Little is known about the natural history of SV40 of SV40 action on these targets depend on the cell type infection in the wild. In its natural host, the Rhesus being studied. The identification of additional cellular macaque, SV40 is thought to infect the terminally targets important for transformation, and understanding differentiated, growth-arrested epithelial cells of the the molecular basis for the cell type-specific action of the kidney. Since these cells are quiescent they do not viral T antigens are two important areas through which express proteins involved in DNA replication, nucleo- SV40 will continue to contribute to our understanding of tide metabolism, or chromatin assembly, proteins that cancer. are used by SV40 to replicate viral DNA and assemble Oncogene (2005) 24, 7729–7745. doi:10.1038/sj.onc.1209046 new virions. However, the large and small T antigens cooperate to drive infected cells into S-phase thus Keywords: SV40; polyomavirus; transformation; cell- providing the raw materials needed for progeny virus type specificity production. Cells often respond by activating multiple defense systems that guard against abnormal cell proliferation and/or virus infection. The T antigen proteins and virus-encoded micro-RNAs act to neutra- lize or ameliorate these defenses allowing productive SV40 infection to proceed. Shortly after its discovery SV40 was shown to induce Simian virus 40 (SV40) is a member of the Polyoma- tumors when injected into newborn hamsters or viridae, a family of viruses characterized by small immunocompromised mice (Eddy et al., 1962; Girardi icosahedral virions and a circular double-stranded et al., 1962). The ability to induce tumors and the types DNA genome of about 5 kb. The SV40 genome consists of tumors observed depended on the route of infection, of 5243 bp and like other members of the family encodes amount of virus, and immune status of the test animals. SV40 was shown to induce an array of different tumor types including osteosarcoma, mesothelioma, lymphoma, *Correspondence: JM Pipas, Department of Biological Sciences, University of Pittsburgh, 559 Crawford Hall, Pittsburgh, PA 15260, and choroid plexus neoplasia (reviewed by Arrington USA; E-mail: [email protected] and Butel, 2001). SV40 also induces neoplastic trans- 2These two authors contributed equally to this work formation in cell culture as assessed by a number of SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7730 death and the production of about 300 infectious progeny virions per infected cell (Figure 2a). As with all viruses, many factors determine whether or not the outcome of infection is successful. First, exposure of the cell to virus particles must result in the attachment of virions to the cell surface. For this to occur the cell must express the appropriate receptors for the virus. Thus, cell types lacking a functional receptor cannot be infected or transformed by SV40 virus particles. Following attachment, virions must penetrate the plasma membrane and be transported to the nucleus. Upon arrival in the nucleus SV40 chromatin is released and transcription from the early promoter is initiated. This results in expression of the large and small T antigen proteins, which act to drive the cells into S-phase. Large T antigen also functions to initiate and maintain viral DNA replication and to activate tran- scription from the late promoter. As the level of the structural proteins rises, progeny virions are assembled. In culture, cell death occurs about 96 h postinfection. SV40 infection of rodent cells does not result in cell death and no progeny virions are produced (Figure 2b). The basis for this restriction is poorly understood but the consequences are a failure to initiate viral DNA replication and to activate transcription from the late promoter. On the other hand the early events of virus infection appear to occur normally in these cells. Virus particles attach to the cell surface, are transported to the nucleus and uncoated, and the T antigens are produced. Furthermore, T antigen synthesis results in the infected cells entering S-phase. For example, when quiescent mouse embryo fibroblasts (MEFs) are infected with a high multiplicity of infection of SV40 they are driven into S-phase and the entire cell population appears to become transformed. However, since viral DNA cannot replicate in these cells T antigen expression is eventually Figure 1 Structure of SV40. (a) SV40 genomic DNA is composed lost due to dilution as the cells divide or due to of three elements: the early and late coding units and the regulatory region. The early unit encodes large T antigen (LT), small t antigen degradation of viral DNA. Thus, descendents of these (sT), 17K T antigen (17KT). The late unit encodes the three infected cells have a nontransformed phenotype and do structural proteins (VP1, VP2, and VP3) and the agnoprotein not contain viral DNA. (agno) and a pre-microRNA (miRNA). The regulatory region (ori) Sublines of stably transformed cells do arise from contains sequences for the early and late promoter and the origin of replication. (b) Several domains and motifs make up the SV40 T infections of nonpermissive cells, but with a low antigens: J domain (J), Rb-protein-binding motif (LXCXE), frequency (Figure 2c). The rate-limiting step to stable nuclear localization signal (NLS), phophatase pp2A-binding transformation is thought to be the integration of viral domain (pp2A binding), origin-binding domain (OBD), Zn domain DNA into the cellular genome by nonhomologous (Zn), ATPase domain (AAA þ ), variable region (VR), and host- recombination. Integration occurs at random sites with range domain (HR). Residue numbers are indicated below the domain structure respect to both the cellular chromosome and the viral DNA. If this integration occurs such that the early coding sequences are intact and expressed, the cell and different assays. Genetic analysis has shown that its subsequent descendants are transformed. expression of the large T antigen protein is necessary and often sufficient for transformation by SV40. Under many circumstances both large and small T antigen What is transformation? expression is required for transformation. Neoplastic transformation refers to the acquisition of expanded proliferation and/or survival potential by a SV40 transforms cells that are nonpermissive for viral cell. Several different assays are used to distinguish a productive infection ‘normal’ cell from transformed cells. In each of these assays cells are placed in an environment that is growth Infection of permissive cells, such as the CV1 or BSC40 restrictive for normal cells whereas transformed cells lines of African green monkey kidney cells, results in cell proliferate and survive. In these assays, to score as Oncogene SV40 large T antigen targets multiple cellular pathways D Ahuja et al 7731 Figure 2 SV40 effects in different cellular environments. (a) Infection of permissive cells results in cell death and virion production. (b) SV40 infection of rodent cells induces S-phase but does not result in cell death or virus production. (c) Integration of viral DNA occurs in a very low percentage of nonpermissive cells, which then become stably transformed transformed requires that the rate of cell proliferation be covered with a monolayer of cells. Growth arrest is greater than the rate of cell loss. Thus, each assay thought to be initiated by contact with adjacent cells so measures the ability of cells to escape specific growth that when a monolayer is obtained most cells exit the and/or survival restrictions.