Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell cultures

Satoshi Komoto, Yuta Kanai, Saori Fukuda, Masanori Kugita, Takahiro Kawagishi, Naoto Ito, Makoto Sugiyama, Yoshiharu Matsuura, Takeshi Kobayashi, Koki Taniguchi

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6- deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmidonly- based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

Original languageEnglish
Article numbere00695-17
JournalJournal of Virology
Volume91
Issue number21
DOIs
Publication statusPublished - 01-11-2017

Fingerprint

Reverse Genetics
Rotavirus
virus replication
cell culture
Cell Culture Techniques
viruses
Viruses
Proteins
proteins
open reading frames
Open Reading Frames
Genome
genome
viral proteins
structural proteins
double-stranded RNA
Double-Stranded RNA
Viral Proteins
Growth
diarrhea

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Komoto, Satoshi ; Kanai, Yuta ; Fukuda, Saori ; Kugita, Masanori ; Kawagishi, Takahiro ; Ito, Naoto ; Sugiyama, Makoto ; Matsuura, Yoshiharu ; Kobayashi, Takeshi ; Taniguchi, Koki. / Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell cultures. In: Journal of Virology. 2017 ; Vol. 91, No. 21.
@article{b918b10558614e4bba92285d58142a9e,
title = "Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell cultures",
abstract = "The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6- deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmidonly- based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.",
author = "Satoshi Komoto and Yuta Kanai and Saori Fukuda and Masanori Kugita and Takahiro Kawagishi and Naoto Ito and Makoto Sugiyama and Yoshiharu Matsuura and Takeshi Kobayashi and Koki Taniguchi",
year = "2017",
month = "11",
day = "1",
doi = "10.1128/JVI.00695-17",
language = "English",
volume = "91",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "21",

}

Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell cultures. / Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki.

In: Journal of Virology, Vol. 91, No. 21, e00695-17, 01.11.2017.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Reverse genetics system demonstrates that rotavirus nonstructural protein NSP6 is not essential for viral replication in cell cultures

AU - Komoto, Satoshi

AU - Kanai, Yuta

AU - Fukuda, Saori

AU - Kugita, Masanori

AU - Kawagishi, Takahiro

AU - Ito, Naoto

AU - Sugiyama, Makoto

AU - Matsuura, Yoshiharu

AU - Kobayashi, Takeshi

AU - Taniguchi, Koki

PY - 2017/11/1

Y1 - 2017/11/1

N2 - The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6- deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmidonly- based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

AB - The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6- deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmidonly- based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches.

UR - http://www.scopus.com/inward/record.url?scp=85031101285&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85031101285&partnerID=8YFLogxK

U2 - 10.1128/JVI.00695-17

DO - 10.1128/JVI.00695-17

M3 - Article

C2 - 28794037

AN - SCOPUS:85031101285

VL - 91

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 21

M1 - e00695-17

ER -