A new subclass of exoribonuclease resistant RNA found in multiple Flaviviridae genera

Viruses have developed innovative strategies to exploit the cellular machinery and overcome the host antiviral defenses, often using specifically structured RNA elements. Examples are found in flaviviruses; 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 genomes 3untranslated region (UTR). Although known to exist in several Flaviviridae genera the full distribution and diversity of xRNAs in this virus family was unknown. Using the recent high-resolution structure of an xrRNA from the divergent flavivirus Tamana bat virus (TABV) as a reference, we used bioinformatic searches to identify xrRNA in the Pegivirus, Pestivirus, and Hepacivirus genera. 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. Our findings thus require adjustments of previous xrRNA classification schemes and expand on the previously known distribution of the xrRNA in Flaviviridae, indicating their widespread distribution and illustrating their importance. IMPORTANCEThe Flaviviridae comprise one of the largest families of positive sense single stranded (+ssRNA) and it is 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 viruss RNA twists up into a very special three-dimensional shape called an xrRNA that blocks the ability of the cell to "chew up" the viral RNA. Hence, part of the virus RNA remains intact, and this protected part is important for viral infection. This was known to occur in Flaviviruses but whether it existed in the other members of the family was not known. In this study, we not only identified a new subclass of xrRNA found in Flavivirus but also in the remaining three genera. The fact that this process of viral RNA maturation exists throughout the entire Flaviviridae family makes it clear that this is an important but underappreciated part of the infection strategy of these diverse human pathogens.