JC virus large T antigen interacts with the F-box protein β-transducin repeat containing protein (βTrCP), thereby influencing viral DNA replication
JC virus (JCV) is a small DNA tumor virus that causes the fatal demyelinating disease progressive multifocal leucoencephalopathy (PML) in immunocompromised patients and has been detected in certain human cancers. Lytic infection and transformation of cultured cells by JCV require five tumor proteins, which interact with factors regulating critical cellular processes. In this study, I have identified an interaction between large T Antigen (TAg) and beta-transducin repeat containing protein 1 and 2 (βTrCP1/2), the substrate recognition factors of the Skp1-Cul1-F box (SCFβTrCP) ubiquitination complex. Immunofluorescence analyses revealed that TAg and βTrCP1, as well as βTrCP2, co-localize in the cytoplasm, and a functional SCF complex is essential for the cytoplasmic translocation of TAg and βTrCP1. These interactions involve a phosphodegron (DpSGX2-4pS) found in βTrCP substrates. TAg stability is unaltered, suggesting that TAg is a pseudo-substrate. Site-directed mutagenesis revealed that serine 640 within the destruction motif is essential for binding. βTrCP targets several proteins involved in distinct cellular pathways including Wee1, p53, PDCD4, β-catenin and IκB. I analyzed the effect of the TAg-βTrCP interaction upon β-catenin and p53. I was unable to demonstrate that TAg elevates β-catenin levels as previously reported, and a mutant TAg unable to bind βTrCP, also had no detectable effect on β-catenin stability. I did, however, observe that wild type and mutant TAgs differentially influence p53 levels. Cells expressing mutant TAg, yielded higher levels of p53 than wild type TAg expressing cells. However, control experiments did not support my prediction that βTrCP is responsible for the observed differences in p53 levels. DNA replication assays revealed that mutations within TAg's destruction motif exhibit defects at late times of the viral replication cycle, highlighting possible roles for βTrCP in JCV DNA replication and infectious virion production. For the first time, the JCV TAg has been linked to the cellular destruction machinery. Proteasomal degradation pathways are essential for the proper spatial and temporal regulation of cellular functions, including cell cycle regulation, differentiation and proliferation. Further analyses are necessary to identify βTrCP substrates that may be affected by the TAg-βTrCP complex essential to viral function.