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

Abstract

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.

Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining

“…MMEJ is a Ku-independent, Polθ-dependent, DNA DSB repair pathway [26,[62][63][64] that could be involved aberrant IN-independent HIV-1 integration [47][48][49] and adeno-associated virus, papillomavirus and MCV integration [7,13,28,29]. We showed that a majority of the virus-host junctions bear 2-10 nts of MHS between MmuPV1 and host DNA.…”

“…Microhomology sequences (MHS) are present at aberrant HIV-1 integration sites that were not mediated by integrase (IN) [47][48][49][50] and at the integration sites of adeno-associated virus, papillomavirus and MCV [13,28,29]. We analyzed the virus-host junction sequences by both RNA-seq and targeted DNA-seq by comparing the sequences of the MmuPV1 genome and the mouse genome.…”

“…Microhomology-mediated end-joining (MMEJ) is a double stand break (DSB) repair mechanism that uses short regions of homologous DNA sequences (> = 1 bp) for error-prone end-joining [25][26][27]. MMEJ has been found in the integration sites of several pathologic viruses, including adeno-associated virus, Merkel cell polyomavirus (MCV) and papillomavirus [7,13,28,29].…”

Viral Manipulation of the Host Epigenome as a Driver of Virus-Induced Oncogenesis

“…Integration of MCPyV into the host genome is not part of the viral life cycle, as the virus does not contain integrase, and occurs through DNA repair mechanisms, such as nonhomologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) [216].…”

From Merkel Cell Polyomavirus Infection to Merkel Cell Carcinoma Oncogenesis

“…Point mutations in other regions of the genome and truncations of the MCPyV LT C-terminal domain, however, are common in viral MCCs (Liu et al, 2016a ). Expression of the viral oncoproteins is largely driven by the preserved MCPyV promoter rather than endogenous promoters, though there are conflicting reports as to whether MCPyV is more likely to integrate into specific regions of chromatin (Doolittle-Hall et al, 2015 ; Czech-Sioli et al, 2020 ). Sequencing of integration sites in multiple MCC tumors reveal that initial recombination of a linearized MCPyV genome with the host genome could lead to transient circularization and amplification of the viral genome and neighboring host DNA (Starrett et al, 2017 ).…”