The rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The nonstructural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully tractable reverse-genetics (RG) system for rotaviruses. Here, we show a novel application of a recently developed RG system by establishing a stable trans-complementing NSP5-producing cell line required to rescue rotaviruses with mutations in NSP5. This approach allowed us to provide the first direct evidence of the pivotal role of this protein during RV replication. Furthermore, using recombinant RV mutants, we shed light on the molecular mechanism of NSP5 hyperphosphorylation during infection and its involvement in the assembly and maturation of replication-competent viroplasms.

36Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories although 37 how they assemble remains unknown. 38During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by 39 the phosphorylation of a single serine residue. The role of this post-translational 40 modification in the formation of viroplasms and its impact on the virus replication 41 remains obscure. Here we investigated the role of NSP5 during RV infection by 42 taking advantage of a modified fully tractable reverse genetics system. An NSP5 43 trans-complementing cell line was used to generate and characterise several 44 recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that a 45 rRV lacking NSP5, was completely unable to assemble viroplasms and to 46 replicate, confirming its pivotal role in rotavirus replication. 47A number of mutants with impaired NSP5 phosphorylation were generated to 48 further interrogate the function of this post-translational modification in the 49 assembly of replication-competent viroplasms. We showed that the rRV mutant 50 strains exhibit impaired viral replication and the ability to assemble round-shaped 51 viroplasms in MA104 cells. Furthermore, we have investigated the mechanism of 52 NSP5 hyper-phosphorylation during RV infection using NSP5 phosphorylation-53 negative rRV strains, as well as MA104-derived stable transfectant cell lines 54 expressing either wt NSP5 or selected NSP5 deletion mutants. Our results 55 indicate that NSP5 hyper-phosphorylation is a crucial step for the assembly of 56 round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 57 in the formation of replication-competent viral factories. Such a complex NSP5 58 phosphorylation cascade may serve as a paradigm for the assembly of functional 59 viral factories in other RNA viruses. 60 61 62 63 64 65 66 . CC-BY-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/660217 doi: bioRxiv preprint 3 IMPORTANCE 67 Rotavirus (RV) double-stranded RNA genome is replicated and packaged into 68 virus progeny in cytoplasmic structures termed viroplasms. The non-structural 69 protein NSP5, which undergoes a complex hyperphosphorylation process during 70 RV infection, is required for the formation of these virus-induced organelles. 71 However, its roles in viroplasm formation and RV replication have never been 72 directly assessed due to the lack of a fully tractable reverse genetics (RG) 73 system for rotaviruses. Here we show a novel application of a recently developed 74 RG system by establishing a stable trans-complementing NSP5-producing cell 75 line required to rescue rotaviruses with mutations in NSP5. This approach 76 allowed us to provide the first direct evidence of the pivotal role of this protein 77 during RV replication. Furthermore, using recombinant RV mutants we shed light 78 on the molecular mechanism of NSP5 hyperphosphorylation during infection and 79 its i...

CRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localize to rotavirus viral factories, we achieve the nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multisegmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 is edited through the formation of precise deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we label the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote an advanced level of understanding of viral replication and host-pathogen interactions, also offering opportunities to develop therapeutics.

Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA–RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo–electron microscopy reconstructions of an NSP2–RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.

We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an example. Small SARS-CoV-2 peptide insertions (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titer and replication, demonstrating a limited tolerance for peptide insertions at this site.