Entirely plasmid-based reverse genetics system for rotaviruses

Kanai, Yuta; Komoto, Satoshi; Kawagishi, Takahiro; Nouda, Ryotaro; Nagasawa, Naoko; Onishi, Misa; Matsuura, Yoshiharu; Taniguchi, Koki; Kobayashi, Takeshi

doi:10.1073/pnas.1618424114

Cited by 187 publications

(295 citation statements)

References 48 publications

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“…Taken together, our data suggest a phosphorylation-dependent mechanism for the formation of RV viroplasms. With the development of a reverse genetic system for RVs, these and other observations can be systematically assessed in the RV system (50). Most intriguingly, however, our work suggests common mechanisms may exist for assembling the virus replication factories from other RNA viruses, one that utilizes a common cellular kinase, CK1α, for localizing key components to sites of virus factory assembly.…”

Section: Discussionmentioning

confidence: 88%

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Criglar

Anish

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.M any viral pathogens, including both DNA viruses (poxviruses) and RNA viruses [rotavirus (RV), orthoreovirus, hepatitis C virus, dengue virus, Marburg virus] replicate in cytoplasmic compartments that are physical scaffolds composed of both viral and cellular proteins (1-3). These compartments, excluded from the rest of the cytoplasm, are sites where the virus genome is replicated and progeny particles are assembled. Such replication compartments are alternately known as virus factories, virus replication centers or complexes, and viroplasms. Exactly how these virus factories form is largely unknown, so it is intriguing to consider that there may be common mechanisms that initiate their formation and regulate their assembly.RV-induced severe, dehydrating gastroenteritis remains a leading cause of morbidity and mortality in infants and children less than 5 y of age worldwide and accounts for greater than 200,000 deaths annually (4). To begin to understand the mechanism of replication factory assembly, we studied the assembly of RV replication complexes known as viroplasms. Viroplasm formation requires the interaction of two nonstructural proteins, NSP2 and NSP5, and expression of NSP2 with NSP5 in cells lacking all other viral proteins results in the formation of viroplasm-like structures (VLS) (5). Loss of either NSP2 or NSP5 abolishes viroplasm formation and rotavirus replication (6-9). While information about the individual characteristics of NSP2 and NSP5 and some knowledge of how they physically interact are available, exactly...

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Section: Discussionmentioning

confidence: 88%

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Criglar

Anish

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…After many efforts 182,183 , a fully tractable, plasmid only-based reverse genetics system for rotaviruses has been developed 184 . This system can produce rotaviruses with predetermined changes in the genome, which enables functional analysis of the rotavirus NSPs and can be used to rescue rotaviruses containing heterologous inserts in one of their genes 184 .…”

Section: Discussionmentioning

confidence: 99%

“…This system can produce rotaviruses with predetermined changes in the genome, which enables functional analysis of the rotavirus NSPs and can be used to rescue rotaviruses containing heterologous inserts in one of their genes 184 . The reverse genetics system is versatile and very exciting and will be developed further to permit experiments that could not be previously conducted (BOX 3) and to move the field forward.…”

Section: Discussionmentioning

confidence: 99%

Rotavirus infection

Crawford

Ramani

Tate

et al. 2017

Nat Rev Dis Primers

531

558

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Rotavirus infections are a leading cause of severe, dehydrating gastroenteritis in children <5 years of age. Despite the global introduction of vaccinations for rotavirus over a decade ago, rotavirus infections still result in >200,000 deaths annually, mostly in low-income countries. Rotavirus primarily infects enterocytes and induces diarrhoea through the destruction of absorptive enterocytes (leading to malabsorption), intestinal secretion stimulated by rotavirus non-structural protein 4 and activation of the enteric nervous system. In addition, rotavirus infections can lead to antigenaemia (which is associated with more severe manifestations of acute gastroenteritis) and viraemia, and rotavirus can replicate in systemic sites, although this is limited. Reinfections with rotavirus are common throughout life, although the disease severity is reduced with repeat infections. The immune correlates of protection against rotavirus reinfection and recovery from infection are poorly understood, although rotavirus-specific immunoglobulin A has a role in both aspects. The management of rotavirus infection focuses on the prevention and treatment of dehydration, although the use of antiviral and anti-emetic drugs can be indicated in some cases.

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“…As suggested by other investigators, present result together with the previous studies suggest that vaccine‐derived double reassortant G1P[8] can replicate efficiently in human intestinal tissue and may be more virulent. To determine whether this hypothesis is correct or not, it is necessary to evaluate the superiority of G1 type VP7 and P[8] type VP4 in RVA infection by using a reverse genetics system of RVA in future studies …”

Section: Discussionmentioning

confidence: 99%

Persistent systemic rotavirus vaccine infection in a child with X‐linked severe combined immunodeficiency

Yoshikawa

Ihira²,

Higashimoto

et al. 2019

Journal of Medical Virology

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Objective The main aims of the present study were to elucidate the systemic group A rotavirus (RVA) infection and to clarify the genetic changes of persistent virus in the X‐linked severe combined immunodeficiency (SCID) patient. Methods RotaTeq vaccine (RV5) genotype‐specific real‐time reverse transcription polymerase chain reaction was used to monitor viral RNA load in serially collected serum and stool samples. Next‐generation sequence analysis was used to determine the genotype of the virus by sequencing 11 gene segments. Polyacrylamide gel electrophoresis (PAGE) analysis was used to identify rearrangement of viral genes. The gene rearrangement was examined in NSP5 gene by using Sanger sequence. Results A 7‐month‐old boy demonstrated chronic diarrhea following the third administration of RV5 and failure to thrive. He was diagnosed with X‐linked SCID and successfully underwent cord blood transplantation. High copy numbers of RV5 genotype G1 RNA were detected in serially collected stool and serum samples and the kinetics of viral RNA loads were correlated with the degree of clinical disease. Next‐generation sequence analysis revealed genetic reassortment at least between the strains WI79‐9/G1P7[5] and WI79‐4/G6P1A[8] in the VP7 gene and the VP4 gene among the vaccine‐derived rotavirus strains. In addition, PAGE analysis suggested genetic rearrangements in several genes, and it was confirmed in the NSP5 gene by sequence analysis. Conclusions The kinetics of RVA RNA load in serum and stool samples was consistent with the clinical course of the patient. Among five genotypes of RV5 vaccine, G1 genotype replicated well in this patient. Reassortment and rearrangements were demonstrated in persistently infected G1 genotype of RV5.

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Entirely plasmid-based reverse genetics system for rotaviruses

Cited by 187 publications

References 48 publications

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

Rotavirus infection

Persistent systemic rotavirus vaccine infection in a child with X‐linked severe combined immunodeficiency

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