Molecular Mechanisms of Merkel Cell Polyomavirus Transformation and Replication.
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ABSTRACT:
SUBMITTER: Liu W
PROVIDER: S-EPMC7811827 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, β-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.
| S-EPMC8950423 | biostudies-literature
Project description:Merkel cell polyomavirus (MCPyV) is associated with approximately 80% of cases of Merkel cell carcinoma (MCC), an aggressive type of skin cancer. The incidence of MCC has tripled over the past twenty years, but there are currently very few effective targeted treatments. A better understanding of the MCPyV life cycle and its oncogenic mechanisms is needed to unveil novel strategies for the prevention and treatment of MCC. MCPyV infection and oncogenesis are reliant on the expression of the early viral oncoproteins, which drive the viral life cycle and MCPyV+ MCC tumor cell growth. To date, the molecular mechanisms regulating the transcription of the MCPyV oncogenes remain largely uncharacterized. In this study, we investigated how MCPyV early transcription is regulated to support viral infection and MCC tumorigenesis. Our studies established the roles of multiple cellular factors in the control of MCPyV gene expression. Inhibitor screening experiments revealed that the histone acetyltransferases p300 and CBP positively regulate MCPyV transcription. Their regulation of viral gene expression occurs through coactivation of the transcription factor NF-κB, which binds to the viral genome to drive MCPyV oncogene expression in a manner that is tightly controlled through a negative feedback loop. Furthermore, we discovered that small molecule inhibitors specifically targeting p300/CBP histone acetyltransferase activity are effective at blocking MCPyV tumor antigen expression and MCPyV+ MCC cell proliferation. Together, our work establishes key cellular factors regulating MCPyV transcription, providing the basis for understanding the largely unknown mechanisms governing MCPyV transcription that defines its infectious host cell tropism, viral life cycle, and oncogenic potential. Our studies also identify a novel therapeutic strategy against MCPyV+ MCC through specific blockage of MCPyV oncogene expression and MCC tumor growth. Author summary MCPyV is a ubiquitous skin infection that can cause one of the most aggressive and highly fatal skin cancers, MCC, which has been increasing in incidence in the decades since its initial discovery. Despite the growing concern presented by this cancer, there are currently no effective targeted therapies to treat MCC. MCC metastasizes rapidly and resists currently available chemotherapy and immune checkpoint inhibitor treatment strategies in a significant portion of patients. Approximately 80% of MCCs are caused by MCPyV, which normally maintains asymptomatic infection within the skin but in rare cases drives MCPyV+ MCC oncogenesis through the expression of the viral oncogenes. Our characterization of the largely unknown molecular mechanisms controlling MCPyV gene expression furthers our understanding of the link between MCPyV infection and MCC oncogenesis. Our study also identifies druggable targets that are exploitable to specifically repress MCPyV oncogene expression and MCC tumor growth. This work demonstrates that hampering viral oncogene transcription is a novel and effective therapeutic strategy to obliterate MCPyV-induced MCC tumorigenesis.
| S-EPMC10468096 | biostudies-literature
Project description:Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.
| S-EPMC3142164 | biostudies-literature
Project description:Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT's ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells.
| S-EPMC4190550 | biostudies-literature
Project description:Viruses are the cause of approximately 15% of all human cancers. Both RNA and DNA human tumor viruses have been identified, with Merkel cell polyomavirus being the most recent one to be linked to cancer. This virus is associated with about 80% of Merkel cell carcinomas, a rare, but aggressive cutaneous malignancy. Despite its name, the cells of origin of this tumor may not be Merkel cells. This review provides an update on the structure and life cycle, cell tropism and epidemiology of the virus and its oncogenic properties. Putative strategies to prevent viral infection or treat virus-positive Merkel cell carcinoma patients are discussed.
| S-EPMC7407210 | biostudies-literature
Project description:Merkel cell polyomavirus (MCPyV) causes the highly aggressive and relatively rare skin cancer known as Merkel cell carcinoma (MCC). MCPyV also causes a lifelong yet relatively innocuous infection and is one of 14 distinct human polyomaviruses species. Although polyomaviruses typically do not cause illness in healthy individuals, several can cause catastrophic diseases in immunocompromised hosts. MCPyV is the only polyomavirus clearly associated with human cancer. How MCPyV causes MCC and what oncogenic events must transpire to enable this virus to cause MCC is the focus of this essay.This article is part of the themed issue 'Human oncogenic viruses'.
| S-EPMC5597743 | biostudies-literature
Project description: Not available
| S-EPMC2603124 | biostudies-literature
Project description:Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post-integration (~80% cases), or by UV-mediated DNA damage. Interestingly, overall survival of MCV-positive Merkel cell carcinoma patients is better, making this differential information of significant diagnostic and prognostic value. Also, MCV provides a direct target for therapy in MCC patients. Currently, the methods used for diagnosis of MCV in tumors are often discordant and unreliable. Here we used a guided molecular scissors based-DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV-positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme-gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that MCV DETECTR system can detect MCV integrated in Merkel tumor rapidly, specifically and with femto-molar sensitivity. Our study is a preliminary, proof-of-principle analysis showing the use of CRISPR for MCV diagnosis. Further validation in human tumor samples is needed for its clinical use in the near future. This new system is promising and we hope it can be coupled with immunohistochemical studies to diagnose the viral status of MCC in clinics soon.
| S-EPMC7011098 | biostudies-literature
Project description:Infection with Merkel cell polyomavirus (MCPyV) can lead to Merkel cell carcinoma (MCC), a lethal form of skin cancer. However, the skin cell type productively infected by MCPyV remains a central question. We combined cell culture and ex vivo approaches to identify human dermal fibroblasts as natural host cells that support productive MCPyV infection. Based on this, we established a cell culture model for MCPyV infection, which will facilitate investigation of the oncogenic mechanisms for this DNA virus. Using this model, we discovered that induction of matrix metalloproteinase (MMP) genes by the WNT/?-catenin signaling pathway and other growth factors stimulates MCPyV infection. This suggests that MCC risk factors such as UV radiation and aging, which are known to stimulate WNT signaling and MMP expression, may promote viral infection and thus drive MCC. Our study also introduces the FDA-approved MEK antagonist trametinib as an effective inhibitor for controlling MCPyV infection.
| S-EPMC4900903 | biostudies-literature
Project description:APOBEC3s mutate Merkel Cell Polyomavirusl genome
| PRJDB11225 | ENA