Human Papillomavirus Type 16 Circular RNA Is Barely Detectable for the Claimed Biological Activity

Human papillomavirus type 16 (HPV16) E7 oncoprotein plays an essential role in cervical carcinogenesis and is encoded predominantly by an E6*I mRNA through alternative RNA splicing of a P97 promoter-transcribed bicistronic E6E7 pre-mRNA. Recently, an HPV16 circular RNA, circE7, was detected in two HPV16-positive cervical cancer cell lines, CaSki and SiHa. It was generated through back-splicing of the E6E7 pre-mRNA. The reported findings showed that, because viral E6*I RNA was nuclear, E7 was mainly translated from the cytoplasmic circE7, and knockdown of circE7 in CaSki cells led to reduction of E7 oncoprotein, cell proliferation, and xenograft tumor formation. We have reanalyzed the published data, conducted detailed experiments, and found that the circE7 in CaSki cells is only 0.4 copies per cell, which is ∼1,640-fold lower than E6*I RNA and also barely detectable from two W12 subclone cell lines, 20861 (integrated HPV16) and 20863 (extrachromosomal HPV16) cells derived from a low-grade cervical lesion. We also determined HPV16 E6*I and E6*II RNAs in CaSki cells are mainly cytoplasmic in cell fractionation analyses, as reported in other studies. We further demonstrated that the claimed circE7 functions in the published report have resulted from off-target effects on E6*I RNA by the circE7 small interfering RNAs used in the reported study. IMPORTANCE RNA back-splicing is a rare splicing event accounting for <1% of canonical RNA splicing and, thus, is thought to have little or no biological significance. Recently, circular RNAs (circRNAs) from RNA back-splicing have been found widely in cells and tissues and may have a role in modulating RNA transcription, splicing, and interference and antiviral innate immunity. A recent report claimed that the predominant HPV16 E6*I RNA was nuclear and unable to encode E7. Rather, a viral circE7 was responsible for translating the oncoprotein E7 in CaSki cells, a cervical cancer cell line. However, we found that both HPV16 E6*I and circE7 RNAs in CaSki cells are primarily cytoplasmic and that the copy number of viral E6*I RNA is 656 copies per cell, whereas the viral circE7 is only 0.4 copies per cell. Most importantly, we found that the claimed circE7 function resulted from off-target effect on viral E6*I RNA by the small interfering RNA (siRNA) si-circE7 designed to knock down the back-spliced circE7 RNA.