Regulation of Virus Replication by BK Polyomavirus Small T Antigen.

Polyomavirus small T antigen (tAg) plays important roles in regulating viral replication, the innate immune response, apoptosis, and transformation for SV40, Merkel cell polyomavirus (MCPyV), murine polyomavirus (MuPyV), and JC polyomavirus (JCPyV). However, the function of BK polyomavirus (BKPyV) tAg has been much less studied. Here, we constructed mutant viruses that do not express tAg, and we showed that, in contrast with other polyomaviruses, BKPyV tAg inhibits large T antigen (TAg) gene expression and viral DNA replication. However, this occurs only in an archetype viral background. We also observed that the transduction of cells with a lentivirus-expressing BKPyV tAg kills the cells. We further discovered that BKPyV tAg interacts not only with PP2A A and C subunits, as has been demonstrated for other polyomavirus tAg proteins, but also with PP2A B''' subunit members. Knocking down either of two B''' subunits, namely STRN or STRN3, mimics the phenotype of the tAg mutant virus. However, a virus containing a point mutation in the PP2A binding domain of tAg only partially affected virus TAg expression and DNA replication. These results indicate that BKPyV tAg downregulates viral gene expression and DNA replication and that this occurs in part through interactions with PP2A. IMPORTANCE BK polyomavirus is a virus that establishes a lifelong infection of the majority of people. The infection usually does not cause any clinical symptoms, but, in transplant recipients whose immune systems have been suppressed, unchecked virus replication can cause severe disease. In this study, we show that a viral protein called small T antigen is one of the ways that the virus can persist without high levels of replication. Understanding which factors control viral replication enhances our knowledge of the virus life cycle and could lead to potential interventions for these patients.

Interaction of DBC1 with polyoma small T antigen promotes its degradation and negatively regulates tumorigenesis.

Deleted in Breast Cancer 1 (DBC1) is an important metabolic sensor. Previous studies have implicated DBC1 in various cellular functions, notably cell proliferation, apoptosis, histone modification, and adipogenesis. However, current reports about the role of DBC1 in tumorigenesis are controversial and designate DBC1 alternatively as a tumor suppressor or a tumor promoter. In the present study, we report that polyoma small T antigen (PyST) associates with DBC1 in mammalian cells, and this interaction leads to the posttranslational downregulation of DBC1 protein levels. When coexpressed, DBC1 overcomes PyST-induced mitotic arrest and promotes the exit of cells from mitosis. Using both transient and stable modes of PyST expression, we also show that cellular DBC1 is subjected to degradation by LKB1, a tumor suppressor and cellular energy sensor kinase, in an AMP kinase-independent manner. Moreover, LKB1 negatively regulates the phosphorylation as well as activity of the prosurvival kinase AKT1 through DBC1 and its downstream pseudokinase substrate, Tribbles 3 (TRB3). Using both transient transfection and stable cell line approaches as well as soft agar assay, we demonstrate that DBC1 has oncogenic potential. In conclusion, our study provides insight into a novel signaling axis that connects LKB1, DBC1, TRB3, and AKT1. We propose that the LKB1-DBC1-AKT1 signaling paradigm may have an important role in the regulation of cell cycle and apoptosis and consequently tumorigenesis.

Polyomavirus Small T Antigen Induces Apoptosis in Mammalian Cells through the UNC5B Pathway in a PP2A-Dependent Manner.

UNC5B is a dependence receptor that promotes survival in the presence of its ligand, netrin-1, while inducing cell death in its absence. The receptor has an important role in the development of the nervous and vascular systems. It is also involved in the normal turnover of intestinal epithelium. Netrin-1 and UNC5B are deregulated in multiple cancers, including colorectal, neuroblastoma, and breast tumors. However, the detailed mechanism of UNC5B function is not fully understood. We have utilized the murine polyomavirus small T antigen (PyST) as a tool to study UNC5B-mediated apoptosis. PyST is known to induce mitotic arrest followed by extensive cell death in mammalian cells. Our results show that the expression of PyST increases mRNA levels of UNC5B by approximately 3-fold in osteosarcoma cells (U2OS) and also stabilizes UNC5B at the posttranslational level. Furthermore, UNC5B is upregulated predominantly in those cells that undergo mitotic arrest upon PyST expression. Interestingly, although its expression was previously reported to be regulated by p53, our data show that the increase in UNC5B levels by PyST is p53 independent. The posttranslational stabilization of UNC5B by PyST is regulated by the interaction of PyST with PP2A. We also show that netrin-1 expression, which is known to inhibit UNC5B apoptotic activity, promotes survival of PyST-expressing cells. Our results thus suggest an important role of UNC5B in small-T antigen-induced mitotic catastrophe that also requires PP2A.IMPORTANCE UNC5B, PP2A, and netrin-1 are deregulated in a variety of cancers. UNC5B and PP2A are regarded as tumor suppressors, as they promote apoptosis and are deleted or mutated in many cancers. In contrast, netrin-1 promotes survival by inhibiting dependence receptors, including UNC5B, and is upregulated in many cancers. Here, we show that UNC5B-mediated apoptosis can occur independently of p53 but in a PP2A-dependent manner. A substantial percentage of cancers arise due to p53 mutations and are insensitive to chemotherapeutic treatments that activate p53. Unexpectedly, treatment of cancers having functional p53 with many conventional drugs leads to the upregulation of netrin-1 through activated p53, which is counterintuitive. Therefore, understanding the p53-independent mechanisms of the netrin-UNC5B axis, such as those involving PP2A, assumes greater clinical significance. Anticancer strategies utilizing anti-netrin-1 antibody treatment are already in clinical trials.

Human polyomavirus modulation of the host DNA damage response.

The human DNA damage response (DDR) is a complex signaling network constituting many factors responsible for the preservation of genomic integrity. Human polyomaviruses (HPyVs) are able to harness the DDR machinery during their infectious cycle by expressing an array of tumor (T) antigens. These molecular interactions between human polyomavirus T antigens and the DDR create conditions that promote viral replication at the expense of host genomic stability to cause disease as well as carcinogenesis in the cases of the Merkel cell polyomavirus and BK polyomavirus. This review focuses on the six HPyVs with disease association, emphasizing strain-dependent differences in their selective manipulation of the DDR. Appreciation of the HPyV-DDR interface at a molecular scale is conducive to the development of novel therapeutic approaches.

Merkel Cell Polyomavirus Small T Antigen Induces DNA Damage Response.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin with high rates of metastasis and mortality. Besides well-established factors including genetic mutations and UV-induced DNA damage in Merkel cell carcinogenesis, the recent discovery of the Merkel cell polyomavirus (MCPyV) has shed light on the viral etiology of MCC. In the current study, we provide novel evidence that MCPyV small T (sT) antigen induces the DNA damage response (DDR) pathway. Our data show that in human MCC cells, the presence of MCPyV is associated with hyperphosphorylation of histone H2AX, a marker for DNA damage. We observed that overexpression of MCPyV sT antigen induced the phosphorylation of histone H2AX as well as the activation of ataxia telangiectasia mutant (ATM), an upstream kinase important for H2AX phosphorylation. Moreover, we observed that MCPyV sT expression also induced the hyperphosphorylation of other ATM downstream molecules (including 53BP1 and CHK2) as well as the hypermethylation of histone 3 and histone 4. These findings disclose a novel link between MCPyV sT and the DDR pathway in MCC. Given that measurement of DDR is clinically useful for evaluating treatment response to radio- and chemotherapy, our findings warrant further investigation to evaluate the potential implications of this pathway for MCC management.

MicroRNAs as Potential Biomarkers in Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare and aggressive type of skin cancer associated with a poor prognosis. This carcinoma was named after its presumed cell of origin, the Merkel cell, which is a mechanoreceptor cell located in the basal epidermal layer of the skin. Merkel cell polyomavirus seems to be the major causal factor for MCC because approximately 80% of all MCCs are positive for viral DNAs. UV exposure is the predominant etiological factor for virus-negative MCCs. Intracellular microRNA analysis between virus-positive and virus-negative MCC cell lines and tumor samples have identified differentially expressed microRNAs. Comparative microRNA profiling has also been performed between MCCs and other non-MCC tumors, but not between normal Merkel cells and malignant Merkel cells. Finally, Merkel cell polyomavirus encodes one microRNA, but its expression in virus-positive MCCs is low, or non-detectable or absent, jeopardizing its biological relevance in tumorigenesis. Here, we review the results of microRNA studies in MCCs and discuss the potential application of microRNAs as biomarkers for the diagnosis, progression and prognosis, and treatment of MCC.

Merkel cell polyomavirus in Merkel cell carcinogenesis: small T antigen-mediates c-Jun phosphorylation.

Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine skin cancer associated with the Merkel cell polyomavirus (MCPyV). The MCPyV genome, which is clonally integrated in the majority of MCCs, encodes the regulatory small T (sT) antigen. Previously, reports have established MCPyV sT antigen as a potent oncogene capable of inducing cell transformation. In the current study, we demonstrate a distinct role for c-Jun hyperactivation in MCPyV sT antigen pathogenesis. As MCPyV sT antigen's association with aggressive cancer growth has been previously established, this finding may represent a potential therapeutic target for the treatment of MCCs.

Genetic and structural analysis of polyomavirus BK T-antigens reveal a higher density of mutations at inter-domain and hexamerization regions, regardless the status of infection.

Polyomavirus BK (BKPyV) T-antigens (large and small tumor antigens, or Lt-ag and st-ag, respectively), control key aspects of viral replication and are able to regulate cell cycle, promoting cell proliferation. However, the structural effects of genetic mutations on T-antigens are poorly investigated. In this study, 214 sequences of T-antigens from individuals with different BKPyV infections (16 renal transplant with nephropathy; 78 asymptomatic renal transplant; 24 hematopoietic stem cell transplant with hemorrhagic cystitis; 96 healthy non-transplant), were analyzed from the genetic and structural standpoints. We found a high concentration of non-synonymous mutations at inter-domains and hexamerization regions of both proteins, being five of them under positive selection in the Lt-ag but none in the st-ag. The in silico analysis indicated that two mutations, located at positions 164 in the st-ag and 592 in the Lt-ag, would significantly affect the interaction with PP2A and p53 cell targets, respectively, although they were not associated to a specific clinical status. No mutations were detected on the J-domains or at the ATPase motif. In sum, the profile of the mutations found seem not to be associated to increased morbidity. This is the first work to analyze structural modifications on T-antigens in different BKPyV infections, and managed to map conserved and variable regions of the T-antigens, which will be helpful for the study of new antiviral drugs.

Phosphorylation of Human Polyomavirus Large and Small T Antigens: An Ignored Research Field.

Protein phosphorylation and dephosphorylation are the most common post-translational modifications mediated by protein kinases and protein phosphatases, respectively. These reversible processes can modulate the function of the target protein, such as its activity, subcellular localization, stability, and interaction with other proteins. Phosphorylation of viral proteins plays an important role in the life cycle of a virus. In this review, we highlight biological implications of the phosphorylation of the monkey polyomavirus SV40 large T and small t antigens, summarize our current knowledge of the phosphorylation of these proteins of human polyomaviruses, and conclude with gaps in the knowledge and a proposal for future research directions.

The Small t Antigen of JC Virus Antagonizes RIG-I-Mediated Innate Immunity by Inhibiting TRIM25's RNA Binding Ability.

JC polyomavirus (JCV), a DNA virus that leads to persistent infection in humans, is the causative agent of progressive multifocal leukoencephalopathy, a lethal brain disease that affects immunocompromised individuals. Almost nothing is currently known about how JCV infection is controlled by the innate immune response and, further, whether JCV has evolved mechanisms to antagonize antiviral immunity. Here, we show that the innate immune sensors retinoic acid-inducible gene I (RIG-I) and cGMP-AMP synthase (cGAS) control JCV replication in human astrocytes. We further identify that the small t antigen (tAg) of JCV functions as an interferon (IFN) antagonist by suppressing RIG-I-mediated signal transduction. JCV tAg interacts with the E3 ubiquitin ligase TRIM25, thereby preventing its ability to bind RNA and to induce the K63-linked ubiquitination of RIG-I, which is known to facilitate RIG-I-mediated cytokine responses. Antagonism of RIG-I K63-linked ubiquitination and antiviral signaling is also conserved in the tAg of the related polyomavirus BK virus (BKV). These findings highlight how JCV and BKV manipulate a key innate surveillance pathway, which may stimulate research into designing novel therapies.IMPORTANCE The innate immune response is the first line of defense against viral pathogens, and in turn, many viruses have evolved strategies to evade detection by the host's innate immune surveillance machinery. Investigation of the interplay between viruses and the innate immune response provides valuable insight into potential therapeutic targets against viral infectious diseases. JC polyomavirus (JCV) is associated with a lifelong, persistent infection that can cause a rare neurodegenerative disease, called progressive multifocal leukoencephalopathy, in individuals that are immunosuppressed. The molecular mechanisms of JCV infection and persistence are not well understood, and very little is currently known about the relevance of innate immunity for the control of JCV replication. Here, we define the intracellular innate immune sensors responsible for controlling JCV infection and also demonstrate a novel mechanism by which a JCV-encoded protein acts as an antagonist of the type I interferon-mediated innate immune response.

Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol.

The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model.

A Comprehensive Proteomics Analysis of the JC Virus (JCV) Large and Small Tumor Antigen Interacting Proteins: Large T Primarily Targets the Host Protein Complexes with V-ATPase and Ubiquitin Ligase Activities While Small t Mostly Associates with Those Having Phosphatase and Chromatin-Remodeling Functions.

The oncogenic potential of both the polyomavirus large (LT-Ag) and small (Sm t-Ag) tumor antigens has been previously demonstrated in both tissue culture and animal models. Even the contribution of the MCPyV tumor antigens to the development of an aggressive human skin cancer, Merkel cell carcinoma, has been recently established. To date, the known primary targets of these tumor antigens include several tumor suppressors such as pRb, p53, and PP2A. However, a comprehensive list of the host proteins targeted by these proteins remains largely unknown. Here, we report the first interactome of JCV LT-Ag and Sm t-Ag by employing two independent "affinity purification/mass spectroscopy" (AP/MS) assays. The proteomics data identified novel targets for both tumor antigens while confirming some of the previously reported interactions. LT-Ag was found to primarily target the protein complexes with ATPase (v-ATPase and Smc5/6 complex), phosphatase (PP4 and PP1), and ligase (E3-ubiquitin) activities. In contrast, the major targets of Sm t-Ag were identified as Smarca1/6, AIFM1, SdhA/B, PP2A, and p53. The interactions between "LT-Ag and SdhB", "Sm t-Ag and Smarca5", and "Sm t-Ag and SDH" were further validated by biochemical assays. Interestingly, perturbations in some of the LT-Ag and Sm t-Ag targets identified in this study were previously shown to be associated with oncogenesis, suggesting new roles for both tumor antigens in novel oncogenic pathways. This comprehensive data establishes new foundations to further unravel the new roles for JCV tumor antigens in oncogenesis and the viral life cycle.

Human Polyomaviruses: The Battle of Large and Small Tumor Antigens.

About 40 years ago, the large and small tumor antigens (LT-Ag and sT-Ag) of the polyomavirus (PyVs) simian vacuolating virus 40 have been identified and characterized. To date, it is well known that all the discovered human PyVs (HPyVs) encode these 2 multifunctional and tumorigenic proteins, expressed at viral replication early stage. The 2 T-Ags are able to transform cells both in vitro and in vivo and seem to play a distinct role in the pathogenesis of some tumors in humans. In addition, they are involved in viral DNA replication, transcription, and virion assembly. This short review focuses on the structural and functional features of the HPyVs' LT-Ag and sT-Ag, with special attention to their transforming properties.

Emerging differential roles of the pRb tumor suppressor in trichodysplasia spinulosa-associated polyomavirus and Merkel cell polyomavirus pathogeneses.

BACKGROUND: Merkel cell carcinoma (MCC) and trichodysplasia spinulosa (TS) are two proliferative cutaneous diseases caused by the Merkel cell polyomavirus (MCPyV) and trichodysplasia spinulosa-associated polyomavirus (TSPyV) respectively. Recently, studies have elucidated a key role of the small tumor (sT) antigen in the proliferative pathogenic mechanisms of MCPyV and likely TSPyV. While both sT antigens have demonstrated a capacity in regulating cellular pathways, it remains unknown whether MCPyV and TSPyV sT antigens contribute similarly or differentially to cell proliferation. OBJECTIVES: The present study aims to explore the proliferative potential of MCPyV and TSPyV sT antigens by investigating their regulatory effects on the retinoblastoma protein (pRb) tumor suppressor. STUDY DESIGN: Inducible cell lines expressing MCPyV sT or TSPyV sT were created using a lentiviral packaging system. Cellular proteins were extracted and subjected to SDS-PAGE followed by Western blot detection and densitometric analysis. RESULTS: Expression of TSPyV sT markedly enhanced the phosphorylation of pRb in Western blot experiments. In contrast, expression of MCPyV sT did not alter pRb phosphorylation under the same experimental conditions. Densitometric analysis revealed that TSPyV sT antigen expression nearly doubled the ratio of phosphorylated to total pRb (P<0.001, Student's T-test), while MCPyV sT antigen expression did not cause significant change in pRb phosphorylation status. CONCLUSION: Given that hyperphosphorylation of pRb is associated with dysregulation of the cell cycle, S-phase induction, and increased cell proliferation, our findings support an important role of TSPyV-mediated pRb deactivation in the development of TS. The observation that the pRb tumor suppressor is inactivated by TSPyV sT but not MCPyV sT provides further insights into the distinct pathobiological mechanisms of MCC and TS.

Limited variation during circulation of a polyomavirus in the human population involves the COCO-VA toggling site of Middle and Alternative T-antigen(s).

We have recently shown that the trichodysplasia spinulosa-associated polyomavirus (TSPyV) belongs to a large monophyletic group of mammalian polyomaviruses that experienced accelerated codon-constrained Val-Ala (COCO-VA) toggling at a protein site common to both Middle and Alternative T-antigens (MT/ALTO). Here we analyzed thirteen, mostly newly sequenced TSPyV genomes, representing ~40% of reported TS disease cases world-wide. We found two deletions and 30 variable sites (≤0.6%) that included only four sites with non-synonymous substitutions (NSS). One NSS site was under positive selection in the exon shared by Small and Middle T antigens, while three others were segregated in MT/ALTO. Two MT/ALTO sites covaried with five sites elsewhere in the genome and determined separation of twelve TSPyVs into two most populous phylogenetic lineages. The other, most distant TSPyV was distinguished by NSS at the COCO-VA site, observed for the first time during intra-species evolution. Our findings reveal a connection between micro- and macro-evolution of polyomaviruses.