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.

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32 Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas 33 (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express 34 viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns 35 exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. 36We here combined short and long-read (nanopore) next-generation sequencing and present the first 37 high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. 38We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that 39 map closely together, and complex integration loci that exhibit local amplification of genomic sequences 40 flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral 41 replication by integration of defective/linear genomes into host DNA double strand breaks via non-42 homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns 43 are mediated by microhomology-mediated break-induced replication, MMBIR.44 Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open 45 chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, 46 rather than transcription from juxtaposed host promoters. Taken together, our data explain the 47 characteristics of MCPyV integration and may also provide a model for integration of other oncogenic 48 DNA viruses such as papillomaviruses. 49 50 Author summary 51 Integration of viral DNA into the host genome is a key event in the pathogenesis of many virus-induced 52 cancers. One such cancer is Merkel cell carcinoma (MCC), a highly malignant tumor that harbors 53 monoclonally integrated and replication-defective Merkel cell polyomavirus (MCPyV) genomes. 54Although MCPyV integration sites have been analyzed before, there is very little knowledge of the 55 mechanisms that lead to mutagenesis and integration of viral genomes. We used multiple sequencing 56 technologies and interrogation of chromatin states to perform a comprehensive characterization of 3 57 MCPyV integration loci. This analysis allowed us to deduce the events that likely precede viral 58 integration. We provide evidence that the mutations which result in the replication defective phenotype 59 are acquired prior to integration and propose that the cellular DNA repair pathways non-homologous 60 end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) produce two 61 principal MCPyV integration patterns (simple and complex, respectively). We show that, although 62 MCPyV integrates predominantly in open chromatin regions, viral oncogene expression is independent 63 of host promoters and driven by the viral promotor region. Our findings are important since they can 64 explain the mechanisms of MCPyV integration. Furthermore, our model...