In the “Circ du RNA” episode of the podcast This Week in Virology, Racaniello and colleagues interview Patrick Moore on his investigation into circular RNAs (circRNA) made by Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV). Specifically, they delineate functions of the viral circRNAs, how they are formed, and some potential applications in EBV- and KSHV-related cancer diagnostics.
One focus of this episode is the logic behind a virus making circRNA. Moore describes how his team characterized circRNAs generated from two viral genetic loci in either EBV or KSHV. They used polysome fractionation to show that these viral circRNAs do not associate with translation machinery, and thus are likely noncoding RNAs (1,2). These noncoding circRNAs are hypothesized to downregulate a cellular antiviral response (1,2). CircRNAs can act as sponges that ‘soak up’ microRNA (miRNA) and RNA-binding proteins (RBPs) that are involved in antiviral responses (1,2,3). For example, circRNAs can be complementary to specific miRNAs, which allows them to sequester miRNA through base pairing (1). Similarly, circRNAs can bind to specific RBPs and compete with the normal target RNAs (1). One can speculate that since cellular circRNAs are known to exist, it’s possible that viral circRNA can avoid being sensed as viral RNA by cellular antiviral systems by adopting a similar structure.
The EBV and KSHV circRNAs are formed through an interesting mechanism called back-splicing whereby a downstream 5’ splice donor site joins to an upstream 3’ splice acceptor site. The specific circRNAs described can be formed either with or without an intron, the presence or absence of which determines the subcellular localization of the circRNA; circRNAs that contain an intron are retained in the nucleus whereas circRNAs that exclude an intron primarily localize to the cytoplasm (2). CircRNAs have been found across domains of life, including in prokaryotes. Since splicing in prokaryotes is relatively rare, this could suggest that there are alternative methods of circRNA formation besides splicing. Historically it was thought that formation of circRNAs was accidental; however, the discovery of functional viral circRNAs seems to point to a more intentional formation.
Another focus of this episode involves the use of viral circRNAs as diagnostic tools for EBV-positive cancers. Toptan et al. found that every EBV-associated tumor that they examined contained a specific circRNA (2). This could suggest that the viral circRNA plays an essential role in tumor cell reproductive fitness for this specific viral-induced cancer. Since circRNAs are relatively stable, this may offer a robust method to diagnose this cancer type. Increased stability of circRNAs can be attributed to their lack of free 5’ and 3’ ends which renders them resistant to exonuclease degradation. Moore emphasizes this point, “In one case we measured circRNA that had been retained in a tumor sample for decades at room temperature” (1). If one could induce the degradation or downregulation of these circRNAs, it’s plausible that this could be a therapeutic option. For instance, an endonuclease specific to a sequence within the circRNA could be used if its delivery to the tumor were facilitated. In order for this to be feasible, it first would have to be demonstrated that the circRNAs are indeed required for tumor formation and not just a side effect.
Viral circRNAs are an exciting subject of research and are relatively unexplored with respect to tumor-causing viruses including EBV and KSHV. A more complete understanding of the formation and function of viral circRNAs will likely lead to advances in diagnostics and therapeutic approaches for a range virus-induced diseases.
References
- Moore, Patrick, Guest. Racaniello, Vincent, Host. Despommier, Dickson, Host. Dove, Alan, Host. Spindler, Kathy, Host. “Circ du RNA.” This Week in Virology, January 20th 2019, http://www.microbe.tv/twiv/twiv-531/.
- T. Toptan, B. Abere, M.A. Nalesnik, S.H. Swerdlow, S. Ranganathan, N. Lee, K.H. Shair, P.S. Moore, Y. Chang. Circular DNA tumor viruses make circular RNAs Proc. Natl. Acad. Sci. USA, 115 (2018), pp. E8737-E8745
- Barrett, S. P. & Salzman, J. Circular RNAs: analysis, expression and potential functions. Development 143, 1838–1847 (2016).
