Insulin receptor substrate 1 (IRS-1) is the major signaling molecule for the insulin and insulin-like growth factor I receptors, which transduces both metabolic and growth-promoting signals, and has transforming properties when overexpressed in the cells. Here we show that IRS-1 is translocated to the nucleus in the presence of the early viral protein-T-antigen of the human polyomavirus JC. Nuclear IRS-1 was detected in T-antigenpositive cell lines and in T-antigen-positive biopsies from patients diagnosed with medulloblastoma. The IRS-1 domain responsible for a direct JC virus T-antigen binding was localized within the N-terminal portion of IRS-1 molecule, and the binding was independent from IRS-1 tyrosine phosphorylation and was strongly inhibited by IRS-1 serine phosphorylation. In addition, competition for the IRS-1-T-antigen binding by a dominant negative mutant of IRS-1 inhibited growth and survival of JC virus T-antigen-transformed cells in anchorageindependent culture conditions. Based on these findings, we propose a novel role for the IRS-1-T-antigen complex in controlling cellular equilibrium during viral infection. It may involve uncoupling of IRS-1 from its surface receptor and translocation of its function to the nucleus. Insulin receptor substrate 1 (IRS-1)1 is a 160-kDa cytosolic protein implicated in insulin and IGF-I signal transduction. IRS-1 plays an essential role in IGF-I-mediated cell proliferation (1, 2), and has transforming properties when overexpressed in different cell types (3, 4). The structure of IRS-1 reveals two conserved regions within the N-terminal portion of the protein (5, 6). The first one is called PH for its similarity to a pleckstrin homology domain (7), and the second shows similarity to a putative phosphotyrosine-binding (PTB) domain present in Shc and other proteins (6). The PTB domain recognizes phosphorylated tyrosine within NPXY motifs, providing a mechanism to couple IRS-1 with the Tyr 950 in the juxtamembrane region of the IGF-IR (8). PH domains contain a positively charged binding pocket that may mediate interaction with phospholipids (9) and with proteins containing acidic motifs (10). Following activation, over 20 phosphorylation sites on the IRS-1 docking molecule can recruit a variety of proteins equipped with Src homology domains (11). Independent from its tyrosine phosphorylation, IRS-1 interacts with ␣ v  3 (12) and ␣ 5  1 (13, 14) integrins, with typical nuclear proteins such as the SV40 large T-antigen (3) and nucleolin (10) and is constitutively phosphorylated in v-Src transformed cells (15). Transforming properties of IRS-1 were suspected for quite some time even before the first convincing evidence was furnished by utilizing R Ϫ cells (3T3-like fibroblasts derived from mice with targeted disruption of IGF-IR gene) (3, 4). Although R Ϫ cells are remarkably resistant to transformation (15, 16), co-expression of IRS-1 and SV40 T-antigen induced R Ϫ transformation, a phenotype efficiently reversed by antisense IRS-1 mRNA (4). Importantly, overexpressio...
Our data provide evidence that the JCV late gene encoding the auxiliary agnoprotein is expressed in tumor cells. The finding of agnoprotein expression in the absence of T-antigen expression suggests a potential role for agnoprotein in pathways involved in the development of JCV-associated medulloblastomas.
BACKGROUND The human polyomavirus JC virus (JCV) causes progressive multifocal leukoencephalopathy. Subclinical infection with JCV occurs in 85–90% of the population worldwide. The virus usually remains latent but can reactivate under immunosuppressive conditions, resulting in progressive multifocal leukoencephalopathy. JCV is oncogenic in experimental animals and is associated with human brain tumors. JCV is found in normal mucosa of the gastrointestinal tract, and some colon carcinomas express the oncogenic JCV T‐antigen protein. The objective of this study was to examine the presence of JCV DNA sequences and JCV protein expression in normal and malignant human esophageal tissues. METHODS The authors examined the presence of JCV DNA sequences and protein expression in normal and malignant human esophageal tissues. Seventy well characterized biopsy specimens from patients with a spectrum of esophageal disorders were studied by immunohistochemistry, and 18 specimens were analyzed further by polymerase chain reaction amplification. RESULTS JC viral DNA was isolated from 11 of 13 normal esophageal biopsy specimens (85%) and from 5 of 5 esophageal carcinomas (100%). Using immunohistochemistry, JCV T antigen was detected in 10 of 19 carcinomas (53%), agnoprotein was detected in 8 carcinomas (42%), p53 tumor suppressor was detected in 11 carcinomas (58%), and β‐catenin was detected in 4 carcinomas (21%). Zero of 51 normal, benign, and premalignant esophageal samples expressed viral proteins. Laser‐capture microdissection verified the presence and specificity of JCV DNA sequences. β‐Catenin and p53 were colocalized with JCV T‐antigen in the nuclei of neoplastic cells. CONCLUSIONS The results provide evidence for infection of gastrointestinal tract cells by JCV and suggest a potential role of JCV in the development of upper digestive tract carcinomas. Cancer 2005. © 2005 American Cancer Society.
Cell-type-specific transcription of the JC virus (JCV) promoter in glial cells initiates a series of events leading to viral replication in the brain and the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in patients with neurologic complications due to infection with human immunodeficiency virus type 1. Here we employed an in vitro infection of primary cultures of human astrocytes to compare the transcriptional profile of cellular genes after JCV infection by using an oligonucleotide-based microarray of 12,600 genes. Transcription of nearly 355 genes was enhanced and expression of 130 genes was decreased to various degrees. Many transcripts that were increased upon JCV infection were found to encode proteins with properties that suggest their involvement in cell proliferation, including cyclin A and cyclin B1; signaling pathways, such as transforming growth factor  receptor 1, platelet-derived growth factor receptor and fibroblast growth factor family receptor; and other regulatory events, such as inflammatory responses, including cyclo-oxygenase-2 (Cox-2). Microarray-based data for several cell cycle-regulatory genes were further examined by using Western blot analysis of in vitro infected astrocytes harvested early and late during the infection. Results demonstrate that protein levels of all upregulated genes were found to increase at some point during the infection time course. In parallel, immunohistochemical assessment of cell cycle proteins, including cyclins A, B1, E, and Cdk2, showed positive staining of astrocytes within PML lesions of brain tissue from patients with neuro-AIDS. Microarray analysis was found to be a useful predictor of gene expression in infected cells; however, it may not directly correlate with protein levels during infection with JCV.The human polyomavirus, JC (JCV), is the etiologic agent for the fatal demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy (PML) (for review see references 5 and 11). Once a rare disease seen primarily in patients with impaired immune systems due to myeloproliferative and lymphoproliferative disorders, the incidence of PML has been significantly increased due to the AIDS epidemic (27). Clinical studies show that Ͼ4% of AIDS patients with neurologic problems are diagnosed with PML (1,2,6,7,10,18,19,20,26,31,32). JCV infects Ͼ65% of the human population by early childhood with no major clinical symptoms (16,23,28,30). Reactivation of the viral genome that preferentially occurs in oligodendrocytes and astrocytes leads to cytolytic destruction of the myelin-producing oligodendrocytes and degeneration of the myelin sheath in white matter. The chief pathological features of PML are the presence of altered oligodendrocytes whose nuclei are two to three times the size of normal cells and are filled with virions and viral antigens (34-36); giant, bizarre astrocytes with pleomorphic, hyperchromatic nuclei; and occasional mitotic figures. Early studies revealed that expre...
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