A New Subclass of Exoribonuclease-Resistant RNA Found in Multiple Genera of Flaviviridae

ABSTRACT Viruses have developed innovative strategies to exploit the cellular machinery and overcome the antiviral defenses of the host, often using specifically structured RNA elements. Examples are found in the Flavivirus genus (in the family Flaviviridae), where during flaviviral infection, pathogenic subgenomic flaviviral RNAs (sfRNAs) accumulate in the cell. These sfRNAs are formed when a host cell 5′ to 3′ exoribonuclease degrades the viral genomic RNA but is blocked by an exoribonuclease-resistant RNA structure (xrRNA) located in the viral genome’s 3′ untranslated region (UTR). Although known to exist in several Flaviviridae genera, the full distribution and diversity of xrRNAs in this family were unknown. Using the recently solved high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNAs in the remaining three genera of Flaviviridae: Pegivirus, Pestivirus, and Hepacivirus. We biochemically and structurally characterized several examples, determining that they are genuine xrRNAs with a conserved fold. These new xrRNAs look superficially similar to the previously described xrRNAs but possess structural differences making them distinct from previous classes of xrRNAs. Overall, we have identified the presence of xrRNA in all four genera of Flaviviridae, but not in all species. Our findings thus require adjustments of previous xrRNA classification schemes and expand the previously known distribution of xrRNA in Flaviviridae. IMPORTANCE The members of the Flaviviridae comprise one of the largest families of positive-sense single-stranded RNA (+ssRNA) and are divided into the Flavivirus, Pestivirus, Pegivirus, and Hepacivirus genera. The genus Flavivirus contains many medically relevant viruses such as Zika virus, dengue virus, and Powassan virus. In these, a part of the RNA of the virus twists up into a distinct three-dimensional shape called an exoribonuclease-resistant RNA (xrRNA) that blocks the ability of the cell to “chew up” the viral RNA. Hence, part of the RNA of the virus remains intact, and this protected part is important for viral infection. These xrRNAs were known to occur in flaviviruses, but whether they existed in the other members of the family was not known. In this study, we identified a new subclass of xrRNA found not only in flaviviruses but also in the remaining three genera. The fact that these structured viral RNAs exist throughout the Flaviviridae family suggests they are important parts of the infection strategy of diverse pathogens, which could lead to new avenues of research.