Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture

Komoto, Satoshi; Kanai, Yuta; Fukuda, Seisuke; Kugita, Masanori; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

doi:10.1128/jvi.00695-17

Cited by 44 publications

(55 citation statements)

References 43 publications

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“…The 12-kDa protein NSP6 is encoded by ORF3, which is nested within the gene for nonstructural protein 5 (47), an arrangement analogous to the placement of the 1s coding sequence within the 1 ORF. Like 1s, NSP6 is not absolutely essential for viral replication but does promote efficient viral growth in cultured cells (48). The function of NSP6 is not fully defined, but NSP6 has the capacity to bind both single-stranded RNA (ssRNA) and dsRNA (49).…”

Section: Discussionmentioning

confidence: 99%

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression

Phillips

Stuart

Simon

2018

J Virol

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Reovirus non-structural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of the σ1s protein during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in SV40-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in the reovirus infection, as no difference in infectivity between the wild type and σ1s-null viruses was observed. However, wild type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and induction of type-1 interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired, but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production, but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.Hematogenous dissemination is critical a step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by σ1s promotes reovirus dissemination is unknown. Here, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Owing to reduced levels of viral protein synthesized in the absence of σ1s, maturation of viral factories was impaired and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required hematogenous reovirus dissemination.

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Section: Discussionmentioning

confidence: 99%

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression

Phillips

Stuart

Simon

2018

J Virol

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“…5(f)}. Recent reverse genetics experiments demonstrated that NSP6 is not essential for rotavirus replication in the cell culture [140].…”

Section: Regions Serotypesmentioning

confidence: 99%

Understanding the penetrance of intrinsic protein disorder in rotavirus proteome

Kumar

Singh

Kumar

et al. 2020

International Journal of Biological Macromolecules

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a b s t r a c tRotavirus is a major cause of severe acute gastroenteritis in the infants and young children. The past decade has evidenced the role of intrinsically disordered proteins/regions (IDPs)/(IDPRs) in viral and other diseases. In general, (IDPs)/(IDPRs) are considered as dynamic conformational ensembles that devoid of a specific 3D structure, being associated with various important biological phenomena. Viruses utilize IDPs/IDPRs to survive in harsh environments, to evade the host immune system, and to highjack and manipulate host cellular proteins. The role of IDPs/IDPRs in Rotavirus biology and pathogenicity are not assessed so far, therefore, we have designed this study to deeply look at the penetrance of intrinsic disorder in rotavirus proteome consisting 12 proteins encoded by 11 segments of viral genome. Also, for all human rotaviral proteins, we have deciphered molecular recognition features (MoRFs), which are disorder based binding sites in proteins. Our study shows the wide spread of intrinsic disorder in several rotavirus proteins, primarily the nonstructural proteins NSP3, NSP4, and NSP5 that are involved in viral replication, translation, viroplasm formation and/or maturation. This study may serve as a primer for understanding the role of IDPs/MoRFs in rotavirus biology, design of alternative therapeutic strategies, and development of disorder-based drugs.

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“…protein (shaded area in Figure 3) might not be translated because of missing of the start codon based on the sequence analysis. As recent our study demonstrated that NSP6 protein was involved in vitro replication efficiency of RVA, 20 virulence of RVA may be affected by the gene rearrangement with an additional copy of the NSP6 gene. In contrast to an immunocompetent healthy infant, high levels of RVA vaccine replication may be involved in the high frequency of RVA gene rearrangement in SCID patients.…”

Section: Discussionmentioning

confidence: 60%

“…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. [19][20][21] Gene rearrangements have been detected in chronic RVA infection in immunodeficient children, 22,23 and in vitro during passages at a high multiplicity of infection in cell culture. 24,25 Since PAGE analysis suggested that the gene rearrangements occurred in several genes including NSP5 gene in RVAs collected from the patient (Figure 2), sequence analysis was carried out to confirm the gene rearrangements.…”

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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Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture

Cited by 44 publications

References 43 publications

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression

Understanding the penetrance of intrinsic protein disorder in rotavirus proteome

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

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