Project C02.2

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C2.2

Project Summary

Exploring the interactions and tethering between Merkel cell polyomavirus (MCPyV) genome and host cellular chromatin

Merkel Cell Polyomavirus (MCPyV) is a human tumor polyomavirus that establishes lifelong persistence in ~80% of the human population, yet causes a rare but highly aggressive skin cancer, Merkel Cell Carcinoma (MCC) [1]. Prior studies have shown that MCPyV can persist as extrachromosomal episomes in specific cell types in the absence of re-infection [2,3]. However, the molecular mechanism that enables long-term maintenance of MCPyV episomes remains unclear.

In other DNA viruses establishing lifelong persistence, viral genomes are maintained through tethering of episomes to host mitotic chromosomes, examples are herpesviruses EBV and KSHV as well as polyomaviruses. This process is mediated by episomal maintenance proteins (EMPs), which multimerize, bind viral DNA and simultaneously interact with specific regions of host chromatin, thereby ensuring faithful segregation of viral genomes to daughter cells during cell division [4]. 

Recent work has demonstrated that the MCPyV large T (LT) antigen selectively binds a specific pentameric DNA motif within cellular promoter regions, that is identical to the LT binding motif present in the viral origin of replication [5]. Genome-wide ChIP-seq analyses further revealed that this core set of LT binding sites is conserved across different cellular contexts. Importantly, transcriptomic profiling showed that LT binding to host DNA does not substantially alter gene expression, a feature reminiscent of established EMPs such as LANA from KSHV [6] and EBNA1 from EBV [7].

Based on these observations, we hypothesize that MCPyV LT functions as an episomal maintenance protein, tethering viral genomes to host chromatin to enable episome persistence through cell division. To test this hypothesis, we will employ chromosome conformation capture–based assays (3C) to determine whether and where MCPyV genomes physically associate with host chromatin in LT-expressing cells [8-10]. These experiments will be performed in cells transfected with MCPyV genomes and ectopically expressing LT. In parallel, we will directly visualize potential tethering interactions using DNAscope and super-resolution microscopy, providing spatial and molecular insight into MCPyV genome maintenance mechanisms.   

C2.2

References

1. Grundhoff A, Fischer N. Merkel cell polyomavirus, a highly prevalent virus with tumorigenic potential. Curr Opin Virol. 2015 Oct;14:129-37. doi: 10.1016/j.coviro.2015.08.010. 
2. Abere B, Zhou H, Li J, Cao S, Toptan T, Grundhoff A, Fischer N, Moore PS, Chang Y. Merkel Cell Polyomavirus Encodes Circular RNAs (circRNAs) Enabling a Dynamic circRNA/microRNA/mRNA Regulatory Network. mBio. 2020 Dec 15;11(6):e03059-20. doi: 10.1128/mBio.03059-20.
3. Riethdorf S, Hildebrandt L, Heinzerling L, Heitzer E, Fischer N, Bergmann S, Mauermann O, Waldispühl-Geigl J, Coith C, Schön G, Peine S, Schuler G, Speicher MR, Moll I, Pantel K. Detection and Characterization of Circulating Tumor Cells in Patients with Merkel Cell Carcinoma. Clin Chem. 2019 Mar;65(3):462-472. doi: 10.1373/clinchem.2018.297028.
4. Aydin I, Schelhaas M. Viral Genome Tethering to Host Cell Chromatin: Cause and Consequences. Traffic. 2016 Apr;17(4):327-40. doi: 10.1111/tra.12378.
5. Ohnezeit D, Huang J, Westerkamp U, Brinschwitz V, Schmidt C, Günther T, Czech-Sioli M, Weißelberg S, Schlemeyer T, Nakel J, Mai J, Schreiner S, Schneider C, Friedel CC, Schwanke H, Brinkmann MM, Grundhoff A, Fischer N. Merkel cell polyomavirus small tumor antigen contributes to immune evasion by interfering with type I interferon signaling. PLoS Pathog. 2024 Aug 7;20(8):e1012426. doi: 10.1371/journal.ppat.1012426.
6. Hu J, Yang Y, Turner PC, Jain V, McIntyre LM, Renne R. LANA binds to multiple active viral and cellular promoters and associates with the H3K4methyltransferase hSET1 complex. PLoS Pathog. 2014 Jul 17;10(7):e1004240. doi: 10.1371/journal.ppat.1004240.
7. Lu F, Wikramasinghe P, Norseen J, Tsai K, Wang P, Showe L, Davuluri RV, Lieberman PM. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1). Virol J. 2010 Oct 7;7:262. doi: 10.1186/1743-422X-7-262.
8. Wang P, Feng Y, Zhu K, Chai H, Chang YT, Yang X, Liu X, Shen C, Gega E, Lee B, Kim M, Ruan X, Ruan Y. In situ Chromatin Interaction Analysis Using Paired-End Tag Sequencing. Curr Protoc. 2021 Aug;1(8):e174. doi: 10.1002/cpz1.174.