High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms

• Published in: PLoS pathogens 2020-08, Vol.16 (8), p.e1008562
• Main Authors: Czech-Sioli, Manja, Günther, Thomas, Therre, Marlin, Spohn, Michael, Indenbirken, Daniela, Theiss, Juliane, Riethdorf, Sabine, Qi, Minyue, Alawi, Malik, Wülbeck, Corinna, Fernandez-Cuesta, Irene, Esmek, Franziska, Becker, Jürgen C, Grundhoff, Adam, Fischer, Nicole
• Format: Article
• Language: English
• Subjects: Bioinformatics; Biology and Life Sciences; Breakpoints; Carcinoma; Causes of; Chromatin; Consortia; Deoxyribonucleic acid; DNA; DNA damage; DNA viruses; Epigenetics; Etiology; Gene sequencing; Gene therapy; Genetic aspects; Genomes; Health aspects; High resolution; Homology; Hygiene; Integration; Loci; Medical research; Medicine and Health Sciences; Merkel cell carcinoma; Metastasis; Molecular modelling; Mutation; Next-generation sequencing; Non-homologous end joining; Nucleotide sequence; Oncogenes; Papillomaviridae; Polyomavirus; Porosity; Proteins; Replication; Research and Analysis Methods; Ribonucleic acid; RNA; Sequence analysis; Skin cancer; Software; Solid state physics; Transcription; Tumors; Virology; Viruses
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1008562

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.