In vitro evaluation of bidirectional transcription levels of five types of non-coding control regions of Merkel cell polyomavirus

Merkel cell polyomavirus (MCPyV) has been identified as the causative agent of Merkel cell carcinoma, and its non-coding control region (NCCR) has been demonstrated to play a critical role in regulating viral transcription. While NCCR variants exist, their comparative impact on bidirectional promoter activity remains poorly characterized. The present study conducted an in vitro evaluation of bidirectional transcription levels of five major MCPyV NCCR types (I, IIa-1, IIa-2, IIb, IIc). The NCCRs were subsequently cloned into a bidirectional reporter vector, which expresses green (EGFP, early) and red (RFP, late) fluorescent proteins. Subsequent to transfection into HEK293 cells, promoter activity was quantitatively analyzed via fluorescence imaging and flow cytometry. Bioinformatic analysis revealed high sequence similarity (> 94%) among the five NCCRs and predicted conserved transcription factor binding sites. The results indicated that all the variants exhibited stronger late promoter activity compared with the early promoter activity (p < 0.01). These observations are in alignment with the established biology of MCPyV. However, no statistically significant differences in the early/late transcription ratio or overall fluorescence intensity were observed between the different NCCR types under these conditions. These findings suggest that the core promoter function is conserved among these major NCCR variants in this model system. This study provides a foundational comparison of MCPyV NCCR activity, highlighting the need for further investigation in more physiologically relevant models to understand how NCCR diversity may influence viral pathogenesis in vivo. Moreover, incorporating models of viral genome integration is essential to understand mechanism of MCPyV carcinogenesis and viral-host interaction.