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.

Information Encoded by the Flavivirus Genomes beyond the Nucleotide Sequence

“…Some authors have contested this division, arguing that the tertiary interactions of the class 2 xrRNAs structures can be used to predict those of class 1 [ 103 ]. However, the results of a recent study combining bioinformatics, biochemical and structural analysis suggest the further redefinition of class 1 into two subclasses: 1a and 1b [ 107 ]. Although full consensus is yet to be reached, most authors agree that the use of the two-class classification system aids for a more comprehensive understanding of xrRNAs and provides a tool for searching for new xrRNAs in different viral systems.…”

“…A clear example of how different primary and secondary structures can lead to the same 3D xrRNA has been reported in the divergent flavivirus Tamana bat virus (TABV) [ 30 , 107 , 109 , 110 ]. X-ray crystallization studies have shown that while the ring-like structure of the xrRNA is preserved across TABV and other flaviviruses, significant variations occur in the corresponding nucleotide sequences and secondary structural elements [ 107 , 110 ]. One major difference resides in the shortening of the stems that form the ring-like structure.…”

“…Since XRN1 can completely degrade the RNA of any viral RNA genome, the existence of xrRNAs and sfRNA-like subgenomic RNAs in viruses other than flaviviruses was thought to be unlikely [ 26 ]. However, the discovery of xrRNAs in TABV suggested that they might actually exist [ 30 , 107 , 115 , 116 , 117 , 118 , 119 , 120 ]. In fact, the structural divergences observed in the xrRNA of TBAV are also seen in members of other genera of Flaviviridae family.…”

“…In fact, the structural divergences observed in the xrRNA of TBAV are also seen in members of other genera of Flaviviridae family. Computational studies have identified putative xrRNAs in representative members of the genera Pestivirus , Pegivirus and Hepacivirus , expanding the presence of these structural elements to the entire Flaviviridae family [ 107 ] and supporting the definition of the structural subclass 1b [ 107 ].…”

“…For example, viruses with subclass 1b xrRNAs do not produce them (and interestingly they do not infect different hosts), unlike the viruses containing subclass 1a xrRNAs that alternate between two host. In many cases, these 1b xrRNAs do not locate to the 3′UTR of the viral genome [ 107 , 115 , 119 ]. It may be that they function as translation and/or replication switches, rather than being involved in the production of functional subgenomic RNAs, but more information is required to determine exactly what they do.…”