Merkel Cell Polyomavirus Large T Antigen Unique Domain Regulates Its Own Protein Stability and Cell Growth

Merkel cell polyomavirus (MCV) is the only known human oncogenic virus in the polyomaviridae family and the etiological agent of most Merkel cell carcinomas (MCC). MCC is an aggressive and highly metastatic skin cancer with a propensity for recurrence and poor prognosis. Large tumor antigen (LT), is an essential oncoprotein for MCV transcription, viral replication, and cancer cell proliferation. MCV LT is a short-lived protein that encodes a unique domain: MCV LT unique regions (MURs). These domains consist of phosphorylation sites that interact with multiple E3 ligases, thus limiting LT expression and consequently, viral replication. In this study, we show that MURs are necessary for regulating LT stability via multiple E3 ligase interactions, resulting in cell growth arrest. While expression of wild-type MCV LT induced a decrease in cellular proliferation, deletion of the MUR domains resulted in increased LT stability and cell proliferation. Conversely, addition of MURs to SV40 LT propagated E3 ligase interactions, which in turn, reduced SV40 LT stability and decreased cell growth activity. Our results demonstrate that compared to other human polyomaviruses (HPyVs), MCV LT has evolved to acquire the MUR domains that are essential for MCV LT autoregulation, potentially leading to viral latency and MCC.

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Faculty Opinions recommendation of Merkel cell polyomavirus small T antigen controls viral replication and oncoprotein expression by targeting the cellular ubiquitin ligase SCFFbw7.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718077664.793485277 ◽

2013 ◽

Author(s):

Blossom Damania

Keyword(s):

Viral Replication ◽

Ubiquitin Ligase ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

Small T Antigen

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Merkel Cell Polyomavirus Downregulates N-myc Downstream-Regulated Gene 1, Leading to Cellular Proliferation and Migration

Journal of Virology ◽

10.1128/jvi.00899-19 ◽

2019 ◽

Vol 94 (3) ◽

Cited By ~ 1

Author(s):

Purnima Gupta ◽

Naveed Shahzad ◽

Alexis Harold ◽

Masahiro Shuda ◽

Assunta Venuti ◽

...

Keyword(s):

Gene Expression ◽

Cell Carcinoma ◽

Cellular Proliferation ◽

Expression Profiles ◽

Gene Expression Profiles ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Early Gene ◽

Merkel Cell ◽

Gene Expressing

ABSTRACT Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation. IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.

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Merkel Cell Polyomavirus Encodes Circular RNAs (circRNAs) Enabling a Dynamic circRNA/microRNA/mRNA Regulatory Network

mBio ◽

10.1128/mbio.03059-20 ◽

2020 ◽

Vol 11 (6) ◽

pp. e03059-20

Author(s):

Bizunesh Abere ◽

Hongzhao Zhou ◽

Jinghui Li ◽

Simon Cao ◽

Tuna Toptan ◽

...

Keyword(s):

Viral Replication ◽

Rna Sequencing ◽

Regulatory Network ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Circular Rnas ◽

Merkel Cell ◽

Rnase R ◽

Early Region ◽

Human Dna

ABSTRACTViral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.

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Merkel Cell Polyomavirus–Positive Merkel Cell Carcinoma Requires Viral Small T-Antigen for Cell Proliferation

Journal of Investigative Dermatology ◽

10.1038/jid.2013.483 ◽

2014 ◽

Vol 134 (5) ◽

pp. 1479-1481 ◽

Cited By ~ 38

Author(s):

Masahiro Shuda ◽

Yuan Chang ◽

Patrick S. Moore

Keyword(s):

Cell Proliferation ◽

Cell Carcinoma ◽

Merkel Cell Carcinoma ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

Small T Antigen

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Merkel Cell Polyomavirus Small T Antigen Controls Viral Replication and Oncoprotein Expression by Targeting the Cellular Ubiquitin Ligase SCFFbw7

Cell Host & Microbe ◽

10.1016/j.chom.2013.06.008 ◽

2013 ◽

Vol 14 (2) ◽

pp. 125-135 ◽

Cited By ~ 94

Author(s):

Hyun Jin Kwun ◽

Masahiro Shuda ◽

Huichen Feng ◽

Carlos J. Camacho ◽

Patrick S. Moore ◽

...

Keyword(s):

Viral Replication ◽

Ubiquitin Ligase ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

Small T Antigen

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Merkel cell polyomavirus T antigens promote cell proliferation and inflammatory cytokine gene expression

Journal of General Virology ◽

10.1099/jgv.0.000287 ◽

2015 ◽

Vol 96 (12) ◽

pp. 3532-3544 ◽

Cited By ~ 19

Author(s):

Kathleen F. Richards ◽

Anna Guastafierro ◽

Masahiro Shuda ◽

Tuna Toptan ◽

Patrick S. Moore ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cellular Proliferation ◽

Human Fibroblasts ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Cytokine Gene Expression ◽

Merkel Cell ◽

T Antigens ◽

Gene Expression Alterations

Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T-antigen locus that result in deletions of C-terminal replicative functions of the LT antigen. Despite these truncations, the LT LXCXE retinoblastoma (Rb) pocket protein family binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication and genes involved in cellular movement. The LXCXE mutant LT largely reversed gene expression alterations detected with the WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.

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Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model

PLoS ONE ◽

10.1371/journal.pone.0142329 ◽

2015 ◽

Vol 10 (11) ◽

pp. e0142329 ◽

Cited By ~ 44

Author(s):

Masahiro Shuda ◽

Anna Guastafierro ◽

Xuehui Geng ◽

Yoko Shuda ◽

Stephen M. Ostrowski ◽

...

Keyword(s):

Cell Proliferation ◽

Mouse Model ◽

Transgenic Mouse ◽

Transgenic Mouse Model ◽

T Antigen ◽

Merkel Cell Polyomavirus ◽

Merkel Cell ◽

Small T Antigen

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Protein-mediated viral latency is a novel mechanism for Merkel cell polyomavirus persistence

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1703879114 ◽

2017 ◽

Vol 114 (20) ◽

pp. E4040-E4047 ◽

Cited By ~ 13

Author(s):

Hyun Jin Kwun ◽

Yuan Chang ◽

Patrick S. Moore

Keyword(s):

Viral Latency ◽

E3 Ligase ◽

Virus Genome ◽

Merkel Cell Polyomavirus ◽

Nutrient Loss ◽

Viral Gene ◽

Genome Replication ◽

Merkel Cell ◽

E3 Ligases ◽

Dna And Rna

Viral latency, in which a virus genome does not replicate independently of the host cell genome and produces no infectious particles, is required for long-term virus persistence. There is no known latency mechanism for chronic small DNA virus infections. Merkel cell polyomavirus (MCV) causes an aggressive skin cancer after prolonged infection and requires an active large T (LT) phosphoprotein helicase to replicate. We show that evolutionarily conserved MCV LT phosphorylation sites are constitutively recognized by cellular Fbw7, βTrCP, and Skp2 Skp-F-box-cullin (SCF) E3 ubiquitin ligases, which degrade and suppress steady-state LT protein levels. Knockdown of each of these E3 ligases enhances LT stability and promotes MCV genome replication. Mutations at two of these phosphoreceptor sites [serine (S)220 and S239] in the full viral genome increase LT levels and promote MCV virion production and transmission, which can be neutralized with anti-capsid antibody. Virus activation is not mediated by viral gene transactivation, given that these mutations do not increase late gene transcription in the absence of genome replication. Mechanistic target of rapamycin inhibition by either nutrient starvation or use of an active site inhibitor reduces Skp2 levels and stabilizes LT, leading to enhanced MCV replication and transmission. MCV can sense stresses in its intracellular environment, such as nutrient loss, through SCF E3 ligase activities, and responds by initiating active viral transmission. Protein-mediated viral latency through cellular SCF E3 ligase targeting of viral replication proteins is a unique form of latency that may promote chronic viral persistence for some small DNA and RNA viruses.

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