Abstract
Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, although how they assemble remains unknown. During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue. The role of this posttranslational modification in the formation of viroplasms and its impact on virus replication remain obscure. Here, we investigated the role of NSP5 during RV infection by taking advantage of a modified fully tractable reverse-genetics system. A trans-complementing cell line stably producing NSP5 was used to generate and characterize several recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that an rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication. A number of mutants with impaired NSP5 phosphorylation were generated to further interrogate the function of this posttranslational modification in the assembly of replication-competent viroplasms. We showed that the rRV mutant strains exhibited impaired viral replication and the ability to assemble round-shaped viroplasms in MA104 cells. Furthermore, we investigated the mechanism of NSP5 hyperphosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, as well as MA104-derived stable transfectant cell lines expressing either wild-type NSP5 or selected NSP5 deletion mutants. Our results indicate that NSP5 hyperphosphorylation is a crucial step for the assembly of round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 in the formation of replication-competent viral factories. Such a complex NSP5 phosphorylation cascade may serve as a paradigm for the assembly of functional viral factories in other RNA viruses.IMPORTANCE 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.
Highlights
Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, how they assemble remains unknown
Rotavirus NSP5 Phosphorylation and Viroplasm Assembly additional modifications were introduced to trans-complement the potential loss of NSP5 function in the recombinant rotaviruses (rRVs) mutants: (i) an additional plasmid, pcDNA3-NSP5 (T7 and cytomegalovirus [CMV] promoter driven), encoding the open reading frame (ORF) of wild-type NSP5, was included in the transfection step of BHK-T7 cells, and (ii) each rescued rRV was amplified in a stable transfectant cell line, MA104-NSP5 (MA-NSP5), supplying the wt NSP5 in trans
Of the two nonstructural proteins involved in viroplasm assembly, NSP5 appears to play a crucial role by potentially providing a scaffold that allows for recruitment of additional viral proteins
Summary
Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, how they assemble remains unknown. During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue The role of this posttranslational modification in the formation of viroplasms and its impact on virus replication remain obscure. Our results indicate that NSP5 hyperphosphorylation is a crucial step for the assembly of round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 in the formation of replicationcompetent viral factories. 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. Biochemical evidence suggests that viroplasms are essential for RV replication, since virus production is highly impaired upon silencing of either NSP2 or NSP5 [12,13,14,15]
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