scholarly journals Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

2021 ◽  
Vol 12 ◽  
Author(s):  
Tatjana Sauerer ◽  
Christopher Lischer ◽  
Adrian Weich ◽  
Carola Berking ◽  
Julio Vera ◽  
...  
Keyword(s):  
Differential Expression ◽  
Cell Lines ◽  
Single Molecule ◽  
Immune Escape ◽  
Immune Surveillance ◽  
T Antigen ◽  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
Significant Differential Expression ◽  
Single Molecule Sequencing

Merkel cell carcinoma (MCC) is a rare and highly aggressive cancer, which is mainly caused by genomic integration of the Merkel cell polyomavirus and subsequent expression of a truncated form of its large T antigen. The resulting primary tumor is known to be immunogenic and under constant pressure to escape immune surveillance. Because interferon gamma (IFNγ), a key player of immune response, is secreted by many immune effector cells and has been shown to exert both anti-tumoral and pro-tumoral effects, we studied the transcriptomic response of MCC cells to IFNγ. In particular, immune modulatory effects that may help the tumor evade immune surveillance were of high interest to our investigation. The effect of IFNγ treatment on the transcriptomic program of three MCC cell lines (WaGa, MKL-1, and MKL-2) was analyzed using single-molecule sequencing via the Oxford Nanopore platform. A significant differential expression of several genes was detected across all three cell lines. Subsequent pathway analysis and manual annotation showed a clear upregulation of genes involved in the immune escape of tumor due to IFNγ treatment. The analysis of selected genes on protein level underlined our sequencing results. These findings contribute to a better understanding of immune escape of MCC and may help in clinical treatment of MCC patients. Furthermore, we demonstrate that single-molecule sequencing can be used to assess characteristics of large eukaryotic transcriptomes and thus contribute to a broader access to sequencing data in the community due to its low cost of entry.

scholarly journals Merkel Cell Polyomavirus-Infected Merkel Cell Carcinoma Cells Require Expression of Viral T Antigens

2010 ◽  
Vol 84 (14) ◽  
pp. 7064-7072 ◽  
Author(s):  
Roland Houben ◽  
Masahiro Shuda ◽  
Rita Weinkam ◽  
David Schrama ◽  
Huichen Feng ◽  
...  
Keyword(s):  
Cell Carcinoma ◽  
Cell Lines ◽  
Merkel Cell Carcinoma ◽  
Annexin V ◽  
T Antigen ◽  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
T Antigens ◽  
Negative Cell ◽  
Exon 1

ABSTRACT Merkel cell carcinoma (MCC) is the most aggressive skin cancer. Recently, it was demonstrated that human Merkel cell polyomavirus (MCV) is clonally integrated in ∼80% of MCC tumors. However, direct evidence for whether oncogenic viral proteins are needed for the maintenance of MCC cells is still missing. To address this question, we knocked down MCV T-antigen (TA) expression in MCV-positive MCC cell lines using three different short hairpin RNA (shRNA)-expressing vectors targeting exon 1 of the TAs. The MCC cell lines used include three newly generated MCV-infected cell lines and one MCV-negative cell line from MCC tumors. Notably, all MCV-positive MCC cell lines underwent growth arrest and/or cell death upon TA knockdown, whereas the proliferation of MCV-negative cell lines remained unaffected. Despite an increase in the number of annexin V-positive, 7-amino-actinomycin D (7-AAD)-negative cells upon TA knockdown, activation of caspases or changes in the expression and phosphorylation of Bcl-2 family members were not consistently detected after TA suppression. Our study provides the first direct experimental evidence that TA expression is necessary for the maintenance of MCV-positive MCC and that MCV is the infectious cause of MCV-positive MCC.

scholarly journals Merkel Cell Polyomavirus Large T Antigen Disrupts Lysosome Clustering by Translocating Human Vam6p from the Cytoplasm to the Nucleus

2011 ◽  
Vol 286 (19) ◽  
pp. 17079-17090 ◽  
Author(s):  
Xi Liu ◽  
Jennifer Hein ◽  
Simon C. W. Richardson ◽  
Per H. Basse ◽  
Tuna Toptan ◽  
...  
Keyword(s):  
T Antigen ◽  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
Large T Antigen

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

2019 ◽  
Vol 94 (3) ◽  
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.

scholarly journals Merkel Cell Polyomavirus Small T Antigen Activates Noncanonical NF-κB Signaling to Promote Tumorigenesis

2020 ◽  
Vol 18 (11) ◽  
pp. 1623-1637
Author(s):  
Jiawei Zhao ◽  
Yuemeng Jia ◽  
Shunli Shen ◽  
Jiwoong Kim ◽  
Xun Wang ◽  
...  
Keyword(s):  
T Antigen ◽  
Merkel Cell Polyomavirus ◽  
Merkel Cell ◽  
Small T Antigen

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

mBio ◽  
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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