Recombinant Rotaviruses Rescued by Reverse Genetics Reveal the Role of NSP5 Hyperphosphorylation in the Assembly of Viral Factories
| Autor: | Catherine Eichwald, Francesca Arnoldi, Elisabeth M. Schraner, Christiaan A. Potgieter, Alexander Borodavka, Luca Venditti, Guido Papa, Oscar R. Burrone |
|---|---|
| Přispěvatelé: | 10085637 - Potgieter, Abraham Christiaan, University of Zurich, García-Sastre, Adolfo, Burrone, Oscar R |
| Rok vydání: | 2019 |
| Předmět: |
Rotavirus
Cytoplasm 1109 Insect Science 10077 Institute of Veterinary Anatomy viruses Mutant Viral Nonstructural Proteins Virus Replication Recombinant viruses Phosphorylation cascade Gene Knockout Techniques viral factories Protein phosphorylation Phosphorylation Sequence Deletion 0303 health sciences 2404 Microbiology Genome Replication and Regulation of Viral Gene Expression 3. Good health Cell biology RNA Viral 10244 Institute of Virology Gene Expression Regulation Viral NSP5 Immunology Hyperphosphorylation Biology Transfection Microbiology Rotavirus Infections Virus Cell Line reverse genetics Viral Proteins 03 medical and health sciences Virology Animals Viroplasm Viral factories recombinant viruses 030304 developmental biology Organelles 2403 Immunology 030306 microbiology Protein Virus Assembly RNA RNA virus biology.organism_classification Reverse genetics protein phosphorylation viroplasms Viral replication Insect Science Mutation 2406 Virology 570 Life sciences biology Capsid Proteins Viroplasms |
| Zdroj: | Journal of Virology |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.01110-19 |
| Popis: | 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. 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|