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
N1 - Funding Information:
We thank Chihiro Yamashiro, Naoko Nagasawa, and Misa Onishi for their technical assistance. This study was supported in part by the MEXT-Supported Program for the Research Program on Emerging and Re-emerging Infectious Diseases of the Japan Agency for Medical Research and Development, AMED (K.T. and S.K.), and JSPS KAKENHI grant number 15K08505 (S.K.). We have no conflicts of interest to declare.
Publisher Copyright:
© 2017 American Society for Microbiology.
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.
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U2 - 10.1128/JVI.00695-17
DO - 10.1128/JVI.00695-17
M3 - Article
C2 - 28794037
AN - SCOPUS:85031101285
SN - 0022-538X
VL - 91
JO - Journal of Virology
JF - Journal of Virology
IS - 21
M1 - e00695-17
ER -