Abstract A34: The discovery of novel noncoding circular RNAs generated by high-risk human papillomavirus type 16

Recently, two independent research teams discovered thousands of conserved non-coding human circular RNAs (circRNAs), however, there are no studies suggesting the existence of circRNAs produced by human viruses. The objective of this study is to explore our hypothesis that human papillomavirus (HPV)-16 creates viral circRNAs that may alter host activity in order to promote viral replication and induction of carcinogenesis. CircRNAs are non-coding RNAs generated by the alternative splicing of a 59 acceptor and 29 or 39 donor sites, creating a “head” to “tail” covalent junction of exonic and/or intronic regions. Although the role of circRNAs is largely unknown, studies have demonstrated several functions for circRNAs including sequestering microRNAs (miRNAs) and regulation of transcription. Recently, circRNAs have also been associated with several cancers such as colorectal, breast, and gastric cancers. Infections with high-risk HPVs, such as types 16 and 18, are responsible for the majority of cervical cancers and a subset of head and neck squamous cell carcinomas (HNSCC). HPV is a small double-stranded circular DNA encoding six early proteins (E-1, -2, -4, -5, -6, and -7) and two late proteins (L-1, -2). HPV DNA is tightly regulated and undergoes extensive and complex splicing to achieve the expression of these proteins. Our preliminary data suggest that HPV-16 extensive splicing generates at least two viral circRNAs (hpv-circRNAs) that could potentially regulate host cellular processes promoting tumorigenesis. We demonstrated the existence of the “head” to “tail” junctions of hpv-circRNAs in HPV-16 viral RNA with reverse transcription polymerase chain reaction (RT-PCR) amplification using divergent primers in RNA extracted from cervical tumor cell lines. Quantitative RT-PCR (qRT-PCR) analysis showed the retention of the hpv-circRNAs in RNase R treated samples suggesting the absence of free RNA ends in these hpv-circRNAs. Moreover, these viral circRNAs persisted even after the cervical tumor cells were exposed to Actinomycin D for 48 hrs (to inhibit RNA synthesis), confirming the stability of these hpv-circRNAs in comparison to the short-lived lariat structures that also can occur during splicing. Together, our data suggest the existence of viral circRNAs produced in HPV-16. To the best of our knowledge, these are the first described human viral generated circRNAs. Currently, we are characterizing these hpv-circRNAs by conducting knockdown and rescue experiments followed by behavior assays (e.g. invasion, angiogenesis, and proliferation). We believe this project will be significant in that it will identify and show the importance of viral circRNAs in HPV-related cancers as well as give us new information about a novel mechanism by which high-risk HPV infection contributes to cervical and HNSCC carcinogenesis. Citation Format: Karen E. Hayes, Jamie A. Barr, Jeremy E. Wilusz, Ivan Martinez. The discovery of novel noncoding circular RNAs generated by high-risk human papillomavirus type 16. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A34.