New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: Conservation, folding, and host adaptation

Two tertiary interactions form an unexpected structure and suggest a folding pathway

The MVE xrRNA structure reveals two long-range tertiary interactions important for stabilizing the active conformation,³⁶ which may be defining characteristics of an xrRNA. The first is a pseudoknot interaction between the L3 loop and the single-stranded S4 segment (Fig. 2, yellow). This interaction was predicted based on phylogenetic co-variation, functional studies, and chemical probing.^19,33-35 Interestingly, in the crystal structure the sequences predicted to form this pseudoknot are not base-paired, rather they are located near each other and appear “poised” to pair. In the crystal, these nucleotides are involved in crystal contacts and thus it is not clear whether this unpaired conformation is due to crystallization or might reflect a functionally important state. As discussed later, the possible labile nature of this pseudoknot may have implications for the xrRNA folding pathway and for modulating function. The second long-range tertiary interaction was unexpected, comprising a base triple and base pairs formed between the 5′ end of the xrRNA (S1) and nucleotides within the RNA 3-way junction (S3) (Fig. 2A, red, cyan). This functionally important interaction is what positions the 5′ end within the ring-like structure; the base pairs in the S1-S3 interaction can be considered to comprise a second pseudoknot.

The xrRNA structure presents an interesting folding problem: in the context of the full viral genomic RNA, how does the 5′ end thread through the ring-like element? Consider a thought experiment in which the L3-S4 pseudoknot formed and closed the ring before the S1-S3 interaction between the 5′ end and the 3-way junction formed. In such a scenario, the entire viral genomic RNA, starting at the 5′ end, would have to thread through the closed ring (a seemingly impossible task). Fortuitously, the fact that within the crystal structure the L3-S4 pseudoknot was unpaired, yet the ring is largely formed and the 5′ end is docked, provides a hypothesis for how this scenario is avoided (Fig. 3A & B). Briefly, until the 5′ nucleotides interact with those in the 3-way junction, the junction is unstructured and the ring is open. Once these interactions form, the junction folds, causing helices P3 and P1 to swing into position and form the ring around the 5′ end. This positions the L3 and S4 bases to form the pseudoknot, “latching” the ring shut and fully stabilizing the Xrn1-resistant conformation.

Is there evidence for this model? In addition to the crystal structure, chemical probing experiments with an xrRNA from Dengue Virus 2 (DENV2) suggest the L3-S4 pseudoknot is conformationally dynamic or transiently formed.³⁵ The importance of this pseudoknot for Xrn1 resistance also appears to vary between xrRNAs, suggesting that the specific characteristics of this pseudoknot may modulate the robustness of Xrn1 resistance for different xrRNAs.^35,36 These ideas, suggested by the structure, remain largely untested, but application of modern biophysical methods linked with virology promises to give additional insight.

How conserved is the xrRNA structure and ability to robustly resist Xrn1?

Our previously published analysis of Xrn1 resistance by elements of the DENV2 3′ UTR showed that 2 xrRNAs in this UTR resist Xrn1 degradation in a reconstituted resistance assay and also share similar secondary structures.³⁵ To extend these findings we probed three other xrRNAs. This included the first xrRNA from MVE (MVExrRNA1) (the crystal structure was of the second MVE xrRNA, MVExrRNA2), an upstream xrRNA from West Nile Virus (WNVxrRNA1) and the single identified xrRNA from Yellow Fever Virus (YFxrRNA). Chemical probing was conducted as previously described.³⁵ All displayed similar probing patterns suggesting that despite variation in primary sequence they form similar secondary structures (Fig. 4A), consistent with previous predictions.^19,33 To determine if these xrRNAs are all capable of quantitatively resisting Xrn1, we employed a fluorescence-monitored time-resolved assay that was previously described.³⁵ All of these xrRNAs resisted Xrn1 over more than an hour (Fig. 4B). Although it was previously shown qualitatively that these RNAs could resist Xrn1³⁶, this result reveals the ability to do so quantitatively when challenged over long time periods in vitro. This is interesting, because examination of the pattern of sfRNAs formed during infection suggests that xrRNAs are not completely quantitative in cells and may have different Xrn1-halting efficiencies; clearly, this requires further examination. Together, these data add strong credence to the idea that the 3-dimensional folded structures of these RNAs are similar and thus the structural basis of Xrn1 resistance is conserved across diverse FVs. This invites further exploration of differences in the function of these elements in vivo vs. in vitro.

Evidence for conserved tertiary structures and unexpected patterns in diverse FV 3′ UTRs

The three-dimensional xrRNA structure revealed two critical tertiary interactions; if the hypothesis that this structural architecture is used by a wide range of xrRNAs from diverse FVs is correct, we should be able to predict these tertiary interactions in a wide range of FV 3′ UTRs. To this end, we examined a large set of mosquito-borne FV 3′ UTRs to determine if they contained sequence patterns consistent with conservation of 3-dimensional tertiary structure. We found patterns of putative xrRNA elements in a variety of viral 3′ UTRs, all with the potential tertiary interactions observed in the 3-dimensional structure of the MVE xrRNA (Fig. 5). This includes contacts which provide the ability to form the long-range L3-S4 pseudoknot and the S1-S3 interaction (base pairs and base triples) between the 5′ end and the 3-way junction. Although the majority of these elements have not been directly tested for Xrn1 resistance, we predict that all are capable of this function and hence all should produce sfRNAs during infection by blocking Xrn1, using a similar 3-dimensional structure. Note that our analysis did not include sequences from more divergent arthropod-borne FVs that do not have obvious sequence similarity^27,38 and whose Xrn1 resistance properties have not yet been extensively examined biochemically. We also did not include the recently described elements from the 5′ ends of Hepatitis C Virus and Bovine Viral Diarrhea Virus (BVDV) as these appear to be structurally unrelated.³⁷

The analysis presented in Figure 5 shows that many of these FVs have two likely SL-type xrRNAs in their 3′ UTR, while some have only one. This was first predicted by a survey of a smaller set of FVs, and more recently for a larger group.^19,38,39 As previously mentioned, the presence of multiple Xrn1 resistant structures in a FV 3′ UTR appears to give rise to multiple sfRNA species in various cell types (Fig. 1A & B), although this has only been examined in a few FVs.^19,22,33-36 Multiple studies indicate that during WNV infection (which has 2 SL-type xrRNAs) the most abundant sfRNA is the largest (sfRNA1), with smaller species produced from a second SL-type xrRNA or dumbbell (DB) structures near the 3′ end (Fig. 1C).^19,21,33-36 Preventing the production of sfRNA1 and sfRNA2 together during WNV infection leads to serious defects in replication, infection-induced pathogenicity and cytopathicity.¹⁹ This appears to be due in part to disruption of the type I interferon response of the infected cells,²¹ although other sfRNA-induced effects are also likely important.^27,28 Interestingly, although WNV consistently produces multiple sfRNAs, sfRNA1 appears to be most important as mutations to the second xrRNA (WNVxrRNA2) to prevent sfRNA2 production have much more modest effects on viral growth and cytopathicity.^19,33 In addition, when WNVxrRNA1 is mutated and sfRNA 1 production is lost, the amount of sfRNA2 does not increase to sfRNA1 levels^19,33,35,36, suggesting there are programmed levels of Xrn1 resistance in these tandem SL-type xrRNAs. It is not clear if these patterns are conserved across the diverse FVs, but these observations from WNV raise interesting new questions: Why do some FVs apparently have 2 xrRNAs, and some only one? What is the purpose of the second xrRNA (and smaller sfRNAs)? Are there patterns to this tandem xrRNA organization?

Although we cannot yet answer these questions, as a first step we compared characteristics of the sequences in Figure 5. We measured the length of each xrRNA element in two ways: (1) from the first nucleotide predicted to form base pairs with the 3-way junction (the 5′ end) to the last nucleotide of the predicted L3-S4 pseudoknot, and (2) from the 5′ end to the last nucleotide in the predicted P4 stem (which we predict to be dispensable for halting Xrn1 in vitro with an xrRNA from DENV2).³⁵ Interestingly, an unexpected pattern emerged (Fig. 5C & D):

1. In the FV 3′UTRs that contain 2 putative xrRNAs, there is a clear trend; within each individual 3′ UTR the length of the upstream (#1) xrRNA is longer than the downstream (#2) xrRNA. This was true for all FVs except DENV2. On average, the #2 xrRNAs are shorter overall. This is true whether or not the P4 stem-loop is included in the length measurement.

2. The average length of putative xrRNAs in FV 3′ UTRs that are predicted to have just one copy is roughly the same as the length of the first xrRNA (#1) in those predicted to contain two copies if the P4 stem-loop is not included.

3. If the P4 stem-loop is included in the length measurement, the single xrRNAs were larger on average than either of the xrRNAs when two copies are present. Thus, in general, the P4/L4 stem-loop appears to have expanded in these single xrRNAs.

Is there any biological or virological significance to these patterns? The answer is unknown, in large part because the measure of length of xrRNA is not a value we can yet relate to a specific characteristic that relates to function. Does length correlate with thermodynamic stability of secondary structures or tertiary interactions, with robustness of Xrn1 resistance, or with some other undiscovered feature? Have the longer xrRNAs evolved additional functionality unrelated to Xrn1 resistance? We also note that the potential length of the L3-S4 pseudoknot varies dramatically between xrRNAs - could this correlate with stability or activity? In addition, we do not know the significance of having one xrRNA versus 2; this further complicates efforts to understand the patterns. Clearly, more experiments are needed to understand how individual xrRNA structures operate within a larger context and if, within the highly conserved xrRNAs, there are subtle variations in stability, structure, or conformational dynamics that modulate function.

A model linking xrRNA tertiary structure, sfRNA production, and viral host adaptation

We do not yet understand how an individual xrRNA tertiary structure relates to the full architecture of a FV 3′ UTR and to patterns of sfRNA production during infection, but recent observations from several papers lead to an interesting model. First, when we infected human cells with the Kunjin strain of WNV (WNV_KUN), the pattern of sfRNA production demonstrated that the first xrRNA (WNVxrRNA1) was very efficient in halting Xrn1, with most of the sfRNAs produced by this upstream xrRNA (Fig. 6A).^35,36 Consistent with previous studies, when this xrRNA was mutated to disrupt its tertiary structure, this sfRNA disappeared as expected, but levels of the second sfRNA that results from the action of the downstream xrRNA (WNVxrRNA2) increased only marginally.^19,33,35,36 Thus, the downstream xrRNA is less efficient in its ability to halt Xrn1. More surprising, when WNVxrRNA2 was mutated to disrupt its tertiary structure the amount of sfRNA produced from WNVxrRNA1 also decreased, an effect noted previously but not yet explained.^19,33,35,36 This suggests that the efficiency of sfRNA production from WNVxrRNA1 is coupled to the integrity of the tertiary structure of WNVxrRNA2 by some completely unknown mechanism (Fig. 6A). To date, this coupling has only been examined or detected in WNV, clearly more exploration is needed to assess its significance.

The second observation comes from the recent work of Villordo et al..³⁸ They showed that when Dengue infects mosquitos, the virus acquires mutations in the second of its 2 xrRNAs (DVxrRNA2) that revert when the mutant viruses are moved back to a mammalian host. We note that the mutations which accumulate during infection of mosquitos are specific to the parts of the xrRNA that form the two functionally critical tertiary interactions (Fig. 6B). In other words, when DENV virus moves back and forth between mosquitos and humans, it seems to be modulating the tertiary structure of the second (downstream) of its 2 xrRNAs while maintaining the overall secondary structure and leaving xrRNA1 intact. What is the result of this? If the xrRNA coupling that we observed with WNV is also present in DENV (not yet tested), we predict these mutations result in a complete loss of sfRNA2 production and a decrease in the production of sfRNA1 during mosquito infection (Fig. 6C). Upon movement to a human host, the virus can mutate to restore the tertiary interactions in xrRNA2, increasing sfRNA1 production. This model postulates that FVs with 2 xrRNAs maintain the potential for efficient sfRNA1 production by keeping xrRNA1 intact in both humans and mosquitos, but in mosquitos they “turn it down” by modulating specific tertiary structure interactions in xrRNA2 and the observed, as yet unexplained coupling effect. This would alter which sfRNAs are made and in what amount as the virus alternates between arthropod and mammalian cells (Fig. 6C), and thus could alter the aforementioned sfRNA-dependent effects during infection. This model is in agreement with the conclusions of Villordo et al.³⁸, who propose that mutation of one xrRNA and not the other helps confer robustness as the virus moves between hosts. Our model remains speculative, in part because we are merging observations from three viruses (MVE, WNV_KUN, DENV). However, it provides a starting point for new experiments that may link overall 3′ UTR architecture, RNA tertiary structure modulation, sfRNA production, coupling of tandem xrRNAs, and diverse sfRNA-dependent processes to the ability of these viruses to adapt to different hosts.