Protein-mediated RNA folding governs sequence-specific interactions between rotavirus genome segments

Essential revisions:

The data in Figure 4 would be considerably strengthened by showing that interactions between S5:S11mut can be recovered by compensatory mutations in S5 and, vice versa, that S5mut:S11 interactions can be recovered by compensatory mutations in S11. Similar interaction 'rescue' experiments could be carried out for the S3:S11 and S6:S11 pairs presented in Figure 4—figure supplement 1.

One major concern of this paper is the lack of in vivo validation for the observations made, and a lack of a well-reasoned model for how the formation of the higher-order ssRNA-oligomer allows itself for replication, duplex formation, and packaging. As noted above, we understand that in vivo validation is not practical at this point. Still, there are several questions (some of which could be addressed by in vitro experiments as well) such as for example 1) given that NSP2 strongly interacts with NSP5 in the viroplasm (and perhaps with other proteins including VP1 and VP2), how do these interactions affect NSP2-RNA interactions and RNA remodeling? Can the experiments be done in the presence of both NSP2 and NSP5? Do NTPase/RTPase activities of NSP2 play a role in RNA-RNA interactions or remodeling? This can be tested perhaps by using a mutant of NSP2 that lacks NTPase/RTPase activities? Addition of NSP5 and/or elucidation of potential roles of NTPase/RTPase activities would greatly strengthen the manuscript by making the measurement and analysis more physiologically relevant.

Here we have tried our best to respond to the wider concerns expressed by the referees.

In particular, we have addressed three general issues raised. These are:

1) Can the disrupted interactions between the mutated RNA sequences be ‘rescued’ by introducing compensatory, ‘trans-complementary’ mutations?

2) How does the viroplasm-forming RNA-binding protein NSP5 affect inter-segment RNA-RNA interactions alone or in the presence of NSP2?

3) Does the enzymatic activity (NTPase/RTPase) of NSP2 have an effect on the detected RNA-RNA interactions?

Regarding point 1, we have included results obtained with mutated RNAs S5 (S5mut) and a mutant of S11 RNA (comp_mutS11) showing that stable base-pairing between S5mut and the interacting S11 RNA mutant is restored. The mutated RNA sequences strongly interact with each other, consistent with the predicted stability of the ‘rescued’ RNA-RNA contacts between the two RNAs. These results, together with extensive characterisation of the stability of the formed duplex (Tm analysis presented in Figure 4B) and mutagenesis analysis of both identified sites in S5 and S11 RNAs) further confirm that the identified RNA contacts are important for detected RNA-RNA interactions. We plan to ultimately test these sites in vivo, using the recently developed RG system by Kanai et al., 2017. S11 RNA was chosen for mutagenesis analysis due to its small size and the availability of secondary structure probing data that could be used for introducing nucleotide substitutions whilst minimizing their impact on the secondary structure of the RNA.

Regarding point 2, we have expanded our studies to include RNA-binding protein NSP5 into RNA-RNA interaction assays. While both recombinantly expressed NSP2 and NSP5 bind ssRNAs, only NSP2 was capable of promoting the formation of RNA-RNA contacts between ssRNAs. Furthermore, addition of submicromolar amounts of NSP5 to the reaction involving ssRNAs S5 & S11, co-incubated with NSP2, resulted in less efficient formation of RNA-RNA contacts. Together these results strongly suggest that formation of RNA-RNA contacts requires NSP2, while NSP5 appears to inhibit this process while having other, not fully explored functions. In any case these observations are consistent with the previously reported NSP5 binding to the RNA-binding grooves of NSP2 octamers that competes with RNA binding by NSP2. We have also noted severe aggregation of the full-length NSP5 in the presence of NSP2, previously reported by Jiang et al., 2006 which precluded further analysis of the RNA-RNA contact formation in the presence of both viroplasm-forming proteins NSP2 and NSP5.

Regarding point 3, we have included new results obtained with NSP2 in the presence of ATP. The novel data indicate that addition of ATP does not affect the ssRNA-binding activity of NSP2, nor does it affect the formation of the S5:S11 RNA-RNA complex. Furthermore, the S5:S11 RNA complex formation was significantly impaired when NSP2 was substituted with the DC-terminal NSP2 mutant that has reduced affinity for ssRNA, while still retaining its NTPase/RTPase catalytic residues. Furthermore, although not raised by the referees, we have investigated whether rotavirus group C NSP2 can facilitate the formation of RNA-RNA contacts between RNAs S5 and S11 of a group A rotavirus. Despite sharing similar tertiary and quaternary architectures and RTPase/NTPase enzymatic activities, rotavirus group C NSP2 was inefficient in stabilising the S5:S11 RNA-RNA contacts in rotavirus group A.

We feel that these additional results significantly enhance our initial conclusions and widen the appeal of the revised manuscript, and we hope that the Editor and the referees who felt the study was well designed, yet lacking in vivo validation for the observations made, are now more reassured.

Addressing these concerns and accommodating the new data generated have required us to rewrite sections throughout the manuscript (highlighted in yellow). We have also added a new figure (Figure 5) presenting a proposed model of NSP2-facilitated RNA-RNA assortment process, described in the Discussion.