Methods for Studying Circular RNAs in Viral Oncogenesis

Circular RNAs (circRNAs) are single-stranded, covalently closed RNA molecules expressed across diverse organisms, including humans and viruses. Emerging evidence indicates that circRNAs are critical modulators of the interplay between viral persistence, immune evasion, and oncogenesis. Viral- and host-derived circRNAs can rewire cellular signaling pathways through diverse mechanisms including microRNA sponging, protein scaffolding, and, in some cases, acting as templates for translation. As a result, circRNAs have emerged as potential therapeutic targets and disease biomarkers for diagnosis and prognosis of various malignancies. Nevertheless, the study of these molecules presents various intrinsic technical challenges due to their lack of a poly(A) tail, low abundance, and high sequence similarity with their linear RNA counterparts. Moreover, circRNA research is a rapidly evolving field that lacks standardized methodologies, contributing to variability in detection, validation, and reproducibility across studies. This review provides a landscape of methodologies used to study viral and host circRNAs, especially in the context of viral oncogenesis. We compare techniques ranging from the computational identification and quantification of circRNAs in transcriptome datasets to their experimental validation and functional characterization. Showcasing current technological advances leading to biological insights, this article aims to offer a guide for investigating the roles of circRNAs in viral oncogenesis for better therapeutic opportunities.