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Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal...

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Autores principales: Sweeney, Trevor R., Cisnetto, Valentina, Bose, Daniel, Bailey, Matthew, Wilson, Jon R., Zhang, Xiaodong, Belsham, Graham J., Curry, Stephen
Formato: Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2915670/
https://www.ncbi.nlm.nih.gov/pubmed/20507978
http://dx.doi.org/10.1074/jbc.M110.129940
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author Sweeney, Trevor R.
Cisnetto, Valentina
Bose, Daniel
Bailey, Matthew
Wilson, Jon R.
Zhang, Xiaodong
Belsham, Graham J.
Curry, Stephen
author_facet Sweeney, Trevor R.
Cisnetto, Valentina
Bose, Daniel
Bailey, Matthew
Wilson, Jon R.
Zhang, Xiaodong
Belsham, Graham J.
Curry, Stephen
author_sort Sweeney, Trevor R.
collection PubMed
description Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.
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spelling pubmed-29156702010-08-05 Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism Sweeney, Trevor R. Cisnetto, Valentina Bose, Daniel Bailey, Matthew Wilson, Jon R. Zhang, Xiaodong Belsham, Graham J. Curry, Stephen J Biol Chem Enzymology Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection. American Society for Biochemistry and Molecular Biology 2010-08-06 2010-05-27 /pmc/articles/PMC2915670/ /pubmed/20507978 http://dx.doi.org/10.1074/jbc.M110.129940 Text en © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version full access. Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) applies to Author Choice Articles
spellingShingle Enzymology
Sweeney, Trevor R.
Cisnetto, Valentina
Bose, Daniel
Bailey, Matthew
Wilson, Jon R.
Zhang, Xiaodong
Belsham, Graham J.
Curry, Stephen
Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
title Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
title_full Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
title_fullStr Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
title_full_unstemmed Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
title_short Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
title_sort foot-and-mouth disease virus 2c is a hexameric aaa+ protein with a coordinated atp hydrolysis mechanism
topic Enzymology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2915670/
https://www.ncbi.nlm.nih.gov/pubmed/20507978
http://dx.doi.org/10.1074/jbc.M110.129940
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