Project C02

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C2

Project Summary

Characterisation of the structural and molecular mechanism how Merkel cell polyomavirus (MCPyV) Large T-Antigen tumps hallmark mutations occur

Merkel Cell Polyomavirus (MCPyV) is a dsDNA tumorigenic virus that infect up to the 80% of the adult world wide population (1). In most of the cases the virus persists in the host as an episome without causing symptoms, but in immunocompromised patients the virus can reactivate and integrate in the host genome by a mechanism of Non-homologous end-joining (NHEJ) or by Microhomology-mediated Break-Induced Replication (MMBIR) (2). The virus causatively contributes to tumor formation, it is the etiological agent of a skin cancer, the Merkel cell carcinoma (3). Tumor cells are dependent on viral oncoporotein expression, small T-Antigen (sT) and a truncated Large T-Antigen (LT) (4).  Further, the viral DNA genome is monoclonal integrated into the host genome, in form of one viral copy or multiple copies, concatemers (2). Different to the episomal DNA persisting in healthy persons, the integrated genomes contain mutations, deletions or insertions within the LT gene that inevitably truncate the protein into a replication deficient version (truncated, tLT) (5). These truncating mutation always occur downstream of a retinoblastoma protein binding region and upstream of the Helicase/ATPase region of LT, thus conserving the function to activate cell proliferation while losing the capability to initiate DNA replication (6). Beside the fact that these truncating mutations are a hallmark of MCC, the question on how these mutations occur is still unknown. It is hypothesized that the mutations on the LT gene has to appear during the process of viral DNA replication by the induction of stresses that could induce double strand breaks or by accidental errors of the polymerase in presence of dysfunctional DNA repair mechanisms (2,7).

This project aims to elucidate the molecular mechanisms that generate the hallmark of truncating mutations on the LT gene of MCPyV. For achieving that, due to the absence of appropriate in vitro and in vivo models for studying the replication of this virus, it will be necessary to establish an in vitro replication assay for MCPyV (Figure). After the establishment of the MCPyV in vitro replication assay, the system will be challenged in different ways to elucidate the molecular mechanisms of pathogenesis (Figure).

C2

References

1. Viscidi et al. Clinical and Vaccine Immunology, 2011
2. Czech-Sioli et al. PLOS Pathogens, 2020
3. Feng et al. Science, 2008
4. DeCaprio Annual review of Pathology, 2021
5. Shuda et al. PNAS, 2008
6. Borchert et al. Journal of Virology, 2014
7. Sowd et al. PLOS Pathogens, 2013