PO-021 Merkel cell polyomavirus-encoded miRNA subverts the host cell immune response

Abstract

IntroductionVirus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of the rare but deadly skin cancer, Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection, which is a prerequisite for oncovirus-mediated transformation.Material and methodsWe adopted an unbiased RNA-Seq-based approach to identify differentially-expressed (DE) targets of MCV-miR-M1 in cell expressing either the 5 p or 3 p arm of the miRNA. Bioinformatic analysis of DE transcripts was carried out using DAVID. DE targets identified by RNA-seq were confirmed via RT-qPCR in a variety of cell lines expressing either MCV-miR-M1 mimics or an MCPyV replication system based on synthetic MCPyV genomes. MCV-miR-M1 interaction with full length 3’UTR regions of DE genes was analysed via dual-luciferase assay to identify direct MCV-miR-M1 targets. IL-8 secretion was determined by ELISA and neutrophil chemotaxis assessed via transwell migration assays.Results and discussionsBioinformatic analysis of DE transcripts revealed numerous genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Intriguingly, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in IL-8 secretion in cells harbouring synthetic MCPyV genomes. Moreover, the observed MCV-miR-M1-mediated reduction in IL-8 secretion was significantly increased by ectopic expression of MCV-miR-M1-resistant SP100. The attenuation of IL-8 secretion resulted in a significant reduction in neutrophil chemotaxis towards cells harbouring replicative MCPyV.ConclusionThese data represent the fist global analysis of MCPyV miRNA targets. Based on our observations, we propose that MCV-miR-M1 targets key immune response regulators in the host cell to help facilitate persistent infection, in part by attenuating neutrophil chemotaxis towards infected cells.