JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity Brigitte Bollag . , Catherine A. Hofstetter., Marta M. Reviriego-Mendoza, Richard J. Frisque* Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America Abstract Background: The human polyomavirus, JC virus (JCV) produces five tumor proteins encoded by transcripts alternatively spliced from one precursor messenger RNA. Significant attention has been given to replication and transforming activities of JCV’s large tumor antigen (TAg) and three T9 proteins, but little is known about small tumor antigen (tAg) functions. Amino-terminal sequences of tAg overlap with those of the other tumor proteins, but the carboxy half of tAg is unique. These latter sequences are the least conserved among the early coding regions of primate polyomaviruses. Methodology and Findings: We investigated the ability of wild type and mutant forms of JCV tAg to interact with cellular proteins involved in regulating cell proliferation and survival. The JCV P99A tAg is mutated at a conserved proline, which in the SV40 tAg is required for efficient interaction with protein phosphatase 2A (PP2A), and the C157A mutant tAg is altered at one of two newly recognized LxCxE motifs. Relative to wild type and C157A tAgs, P99A tAg interacts inefficiently with PP2A in vivo. Unlike SV40 tAg, JCV tAg binds to the Rb family of tumor suppressor proteins. Viral DNAs expressing mutant t proteins replicated less efficiently than did the intact JCV genome. A JCV construct incapable of expressing tAg was replication-incompetent, a defect not complemented in trans using a tAg-expressing vector. Conclusions: JCV tAg possesses unique properties among the polyomavirus small t proteins. It contributes significantly to viral DNA replication in vivo; a tAg mutant failed to display detectable DNA replication activity, and a tAg substitution mutant, reduced in PP2A binding, was replication-defective. Our observation that JCV tAg binds Rb proteins, indicates all five JCV tumor proteins have the potential to influence cell cycle progression in infected and transformed cells. It remains unclear how these proteins coordinate their unique and overlapping functions. Citation: Bollag B, Hofstetter CA, Reviriego-Mendoza MM, Frisque RJ (2010) JC Virus Small t Antigen Binds Phosphatase PP2A and Rb Family Proteins and Is Required for Efficient Viral DNA Replication Activity. PLoS ONE 5(5): e10606. doi:10.1371/journal.pone.0010606 Editor: Wang-Shick Ryu, Yonsei University, Republic of Korea Received March 15, 2010; Accepted April 19, 2010; Published May 12, 2010 Copyright: 2010 Bollag et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Public Health Service grant CA115771 from the National Cancer Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rjf6@psu.edu . These authors contributed equally to this work. Introduction JC virus (JCV) belongs to the Polyomaviridae family of small double-stranded DNA tumor viruses that includes four other human polyomaviruses: BKV, WUV, KIV and MCV. These five viruses are distributed globally among the human population with seroprevalence ranging from 39% to 82% among healthy adult blood donors [1]. Some studies have suggested that simian virus 40 (SV40), the prototype member of the primate polyomavirus subgroup, also circulates in humans as a consequence of exposure to virus present in early preparations of poliovirus vaccine. The JCV, BKV and SV40 genomes share a high degree of sequence homology (69–75%) and organization of the viral genes is nearly identical [2], but the viruses do exhibit distinct biological differences. For example, JCV exhibits restricted growth and oncogenic potential in cell culture, in part due to highly tissue- specific transcriptional signals and early regulatory proteins that appear to be less robust than those of SV40 [reviewed in 3–6]. A comparison of the primate polyomavirus genomes indicates that promoter-enhancer sequences have diverged to the greatest extent. JCV produces five early proteins, the large tumor antigen (TAg), small tumor antigen (tAg), and three T9 proteins, which share overlapping N-terminal sequences and exhibit common and unique replication and transforming functions [7,8]. The SV40 genome encodes three early proteins, TAg, tAg, and 17KT that, like the JCV early regulatory proteins, are encoded by alterna- tively spliced early transcripts. TAg is the major polyomavirus tumor protein. At least three distinct TAg domains contribute to oncogenic transformation. The J domain, named for functional and sequence similarities to cellular DnaJ co-chaperones, binds to the molecular chaperone Hsc70. This domain, in cooperation with a second motif, the LxCxE domain, regulates cell cycle progression, in part, by interacting with the Rb family of proteins, activating the intrinsic ATPase activity of Hsc70 and effecting the release of members of the E2F family of transcription factors from their Rb partners PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10606