Role of JC Virus Agnoprotein in DNA Repair

Armine Darbinyan

Khwaja Siddiqui

Dorota Slonina

Nune Darbinian

Shohreh Amini

Martyn White

Kamel Khalili

Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania 19122

^{Corresponding author. Mailing address: Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 N. 12th St., 015-96, Room 203, Philadelphia, PA 19122. Phone: (215) 204-0678. Fax: (215) 204-0679. E-mail:}ude.elpmet@ililahk.lemak.

Received 2004 Jan 19; Accepted 2004 Mar 23.

Abstract

The late region of human neurotropic JC virus encodes a small 71-amino-acid agnoprotein that is also found in the polyomaviruses simian virus 40 and BK virus. Several functions of agnoprotein have been identified, including roles in regulating viral transcription and virion maturation. Earlier studies showed that agnoprotein expressed alone induced p21/WAF-1 expression and caused cells to accumulate in the G₂/M stage of the cell cycle. Here we report that agnoprotein expression sensitized cells to the cytotoxic effects of the DNA-damaging agent cisplatin. Agnoprotein reduced the viability of cisplatin-treated cells and increased chromosome fragmentation and micronucleus formation. Whereas cisplatin-treated control cells accumulated in S phase, cells expressing agnoprotein did not, instead becoming aneuploid. Agnoprotein expression correlated with impaired double-strand-break repair activity in cellular extracts and reduced expression of the Ku70 and Ku80 DNA repair proteins. After agnoprotein expression, much of the Ku70 protein was located in the perinuclear space, where agnoprotein was also found. Results from binding studies showed an interaction of agnoprotein with Ku70 which was mediated by the N terminus. The ability of agnoprotein to inhibit double-strand break repair activity when it was added to cellular extracts was also mediated by the N terminus. We conclude that agnoprotein inhibits DNA repair after DNA damage and interferes with DNA damage-induced cell cycle regulation. Since Ku70 is a subunit of the DNA-dependent protein kinase that is responsible both for double-strand break repair and for signaling damage-induced cell cycle arrest, the modulation of Ku70 and/or Ku80 by agnoprotein may represent an important event in the polyomavirus life cycle and in cell transformation.

Abstract

JC virus (JCV) is a human polyomavirus that was first isolated from the brain of a patient suffering from progressive multifocal leukoencephalopathy (PML) (26). Polyomavirus is a genus of nonenveloped DNA viruses with icosahedral capsids containing small, circular, double-stranded DNA genomes (4). JCV is the etiologic agent of PML. The virus is widespread throughout the population, with >80% of adults exhibiting JCV-specific antibodies. Infection is thought to take place during early childhood and is usually subclinical. However under conditions of immunosuppression, e.g., in patients with AIDS, JCV can emerge from latency to cause PML (12, 30). PML is a neurodegenerative disease of the central nervous system that is characterized by multiple regions of demyelination caused by a lytic infection of oligodendrocytes by JCV. The destruction of myelin-producing cells leads to brain lesions and death (30). The occurrence of PML was very rare until the advent of the AIDS pandemic, but now it is much more prevalent and affects ∼5% of human immunodeficiency virus-infected persons (2). JCV is one of the few opportunistic infections that continues to occur with some frequency in patients with AIDS despite the widespread use of highly active antiretroviral therapy (2, 3).

JCV can transform cells in culture and is oncogenic in laboratory animals (8, 19). The transforming ability of JCV appears to be limited to specific cell types, particularly those of neural origin, and this property maps to the noncoding regulatory sequence at the origin of DNA replication. JCV DNA sequences have been detected in several kinds of human cancer, including glial tumors (7), medulloblastoma (9), and colon cancer (10). The role of JCV in human malignancies has been reviewed recently (8, 19).

The genome of JCV is organized in a similar fashion to that of the other two primate polyomaviruses, simian virus 40 (SV40) and BK virus (BKV). The double-stranded circular DNA of JCV contains the following three functional regions: the early and late coding genes and the noncoding regulatory sequence (4). The early region encodes the large T and small t antigens, while the late region encodes the viral capsid proteins VP1, VP2, and VP3 and a small regulatory protein known as agnoprotein encoded near the 5′ end of the primary late transcript. Agnoprotein is produced late in the infectious cycle, although it is not incorporated into virions (4, 16). In cells infected by JCV, the 8-kDa agnoprotein is found mainly in the cytoplasm, especially in the perinuclear region, while a small amount may also be found in the nucleus (25). A similar localization was observed in cells transfected with a plasmid encoding agnoprotein (6). Agnoprotein may have regulatory roles in viral transcription and translation as well as in virion assembly and maturation, and these roles were reviewed recently (30). JCV agnoprotein can interact with the large T antigen and can downregulate viral gene expression and DNA replication (28). It also interacts with YB-1, a cellular transcription factor that contributes to JCV gene expression in glial cells, and negatively regulates YB-1-mediated JCV gene transcription (29, 31).

Previous studies demonstrated that the expression of JCV agnoprotein dysregulates cell cycle progression in the absence of other viral proteins (6). NIH 3T3 mouse fibroblasts that constitutively expressed JCV agnoprotein accumulated at the G₂/M stage of the cell cycle, and a decline in cyclin A- and B-associated kinase activity was observed in these cells. Agnoprotein showed the ability to augment the activity of the p21/WAF-1 promoter and increased the level of p21/WAF-1 protein in cells. In addition, agnoprotein was shown to bind p53. The activation of p21/WAF-1 gene expression in cells expressing agnoprotein may be mediated, at least in part, through cooperation with p53 (6). Results for a p53 null cell line revealed that agnoprotein could induce p21/WAF-1 transcription, but to a much lesser extent than in p53-expressing cells, indicating the existence of a p53-independent mechanism for p21/WAF-1 activation by agnoprotein (6).

Since cell cycle progression is linked to the process of DNA repair through p53, we were interested in looking for a possible effect of agnoprotein in the response of cells to DNA damage. DNA damage was induced by the treatment of cultured cells with the cytotoxic antitumor drug cisplatin, which interacts with DNA to form DNA adducts, primarily intrastrand cross-linked adducts, which activate signaling pathways that arrest the cell cycle, including the p53 pathway (33). Data will be presented indicating that agnoprotein expression renders cells more prone to the effects of DNA damage and less able to signal cell cycle arrest.

Acknowledgments

We thank past and present members of the Center for Neurovirology and Cancer Biology for their insightful discussions and for the sharing of ideas and reagents. We also thank C. Schriver for editorial assistance.

This work was supported by grants awarded by the NIH to K.K.

Acknowledgments

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