Analysis of viral circular RNA expression in Zika virus infected cells

Zika virus (ZIKV) is an arthropod-borne virus with an RNA genome and a member of the Flaviviridae family, mainly transmitted by the bite of infected mosquitoes of the Aedes genus. Other transmission routes are mother-to-fetus and sexual transmission, as well as the transfusion of infected blood. The virus has a strong tropism for the nervous system, and it is predominantly associated with microcephaly in babies born to infected mothers and with Guillain-Barré syndrome in adults. Circular RNAs (circRNAs) are a class of single-stranded non-coding RNAs in a covalently closed configuration. Since they lack 5’- and 3’-ends they are more stable than linear RNAs and are also resistant to exonuclease-mediated degradation. CircRNAs can exert different functions, they can act as microRNAs decoys, protein sponges and in some cases, they can even be translated into peptides. In mammals, circRNAs are expressed in many different types of tissues across the body, with the brain being the tissue in which they are predominantly expressed. In recent years, it was discovered that some DNA viruses, e.g., Epstein-Barr virus (EBV), Kaposi’s sarcoma virus (KSHV), human cytomegalovirus (HCMV) and human papillomavirus (HPV), are able to encode their own circRNA molecules, and in some cases their function has also been determined. Only recently, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and hepatitis C virus (HCV), which are RNA viruses, were also found to produce viral circRNAs. The aim of this thesis was to investigate the presence of Zika virus-derived circRNA (vcircRNA) molecules in infected neural stem cells (NSCs). For this purpose, NSCs were infected with Zika virus and harvested at different days post-infection. Total RNA was extracted from infected NSCs, and samples were sequenced by an external service using Illumina sequencing technology. Several viral back-splice junctions (BSJs) were recovered from sequencing analysis in infected cells. To experimentally validate these putative ZIKV circRNAs, some identified viral BSJs were selected for rolling-circle reverse transcription followed by PCR with divergent primers. Using this protocol, only few vcircRNAs were resolved, so a different strategy based on rolling-circle reverse transcription, linear PCR, and Nanopore sequencing was adopted, which confirmed the circular structure of all tested vcircRNAs through the detection of repeated back-splice junctions. These findings provide the first evidence of vcircRNAs production in Zika virus during infection of NSCs and indicate that circular RNA biogenesis may occur more broadly among RNA viruses than previously appreciated.