Limits...
Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

Xia H, Wang P, Wang GC, Yang J, Sun X, Wu W, Qiu Y, Shu T, Zhao X, Yin L, Qin CF, Hu Y, Zhou X - PLoS Pathog. (2015)

Bottom Line: Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus.Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication.These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.

ABSTRACT
RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

No MeSH data available.


Related in: MedlinePlus

2CATPase unwinds RNA helices in a bidirectional manner.(A) and (B) Schematic illustration of the RNA helix substrate with the 3′ single-stranded protrusion (6 bases) (A) or 5′ single-stranded protrusion (6 bases) (B). Asterisks indicate the HEX-labeled strands. (C) MBP-2CATPase (20 pmol) was reacted with 3′-protruded (lane 3) or 5′-protruded (lane 6) RNA helix substrate (0.1 pmol). Native (lanes 1 and 4) or boiled reaction mixture (lanes 2 and 5) was used as a negative or positive (lane 2 and 5) control, respectively. (D) Schematic illustration of the blunt-ended RNA helix substrate. (E) The blunt-ended RNA helix (0.1 pmol) was reacted with MBP-2CATPase (20 pmol).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4517893&req=5

ppat.1005067.g003: 2CATPase unwinds RNA helices in a bidirectional manner.(A) and (B) Schematic illustration of the RNA helix substrate with the 3′ single-stranded protrusion (6 bases) (A) or 5′ single-stranded protrusion (6 bases) (B). Asterisks indicate the HEX-labeled strands. (C) MBP-2CATPase (20 pmol) was reacted with 3′-protruded (lane 3) or 5′-protruded (lane 6) RNA helix substrate (0.1 pmol). Native (lanes 1 and 4) or boiled reaction mixture (lanes 2 and 5) was used as a negative or positive (lane 2 and 5) control, respectively. (D) Schematic illustration of the blunt-ended RNA helix substrate. (E) The blunt-ended RNA helix (0.1 pmol) was reacted with MBP-2CATPase (20 pmol).

Mentions: For helicases, the directionality of helix unwinding is a fundamental characteristic [35]. To assess the unwinding directionality of EV71 2CATPase, we constructed three different RNA helix substrates containing a 3′ single-stranded protrusion, a 5′ single-stranded protrusion, and blunt ends, respectively (Fig 3A, 3B and 3D). Each substrate was then incubated with 2CATPase for the helix unwinding assay. Our results showed that EV71 2CATPase could unwind either the 3′- or 5′-protruded RNA helix (Fig 3C), and its efficiency to unwind the 3′-protruded helix was apparently higher than its efficiency to unwind the 5′-protruded one (Fig 3C, lanes 3 vs. 6). Moreover, 2CATPase could not unwind the blunt-ended helix (Fig 3E). All these experiments were independently repeated several times. Based on these results, we conclude that EV71 2CATPase possesses a bidirectional unwinding activity to RNA helices, while the 3′→5′ unwinding is more preferred.


Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

Xia H, Wang P, Wang GC, Yang J, Sun X, Wu W, Qiu Y, Shu T, Zhao X, Yin L, Qin CF, Hu Y, Zhou X - PLoS Pathog. (2015)

2CATPase unwinds RNA helices in a bidirectional manner.(A) and (B) Schematic illustration of the RNA helix substrate with the 3′ single-stranded protrusion (6 bases) (A) or 5′ single-stranded protrusion (6 bases) (B). Asterisks indicate the HEX-labeled strands. (C) MBP-2CATPase (20 pmol) was reacted with 3′-protruded (lane 3) or 5′-protruded (lane 6) RNA helix substrate (0.1 pmol). Native (lanes 1 and 4) or boiled reaction mixture (lanes 2 and 5) was used as a negative or positive (lane 2 and 5) control, respectively. (D) Schematic illustration of the blunt-ended RNA helix substrate. (E) The blunt-ended RNA helix (0.1 pmol) was reacted with MBP-2CATPase (20 pmol).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4517893&req=5

ppat.1005067.g003: 2CATPase unwinds RNA helices in a bidirectional manner.(A) and (B) Schematic illustration of the RNA helix substrate with the 3′ single-stranded protrusion (6 bases) (A) or 5′ single-stranded protrusion (6 bases) (B). Asterisks indicate the HEX-labeled strands. (C) MBP-2CATPase (20 pmol) was reacted with 3′-protruded (lane 3) or 5′-protruded (lane 6) RNA helix substrate (0.1 pmol). Native (lanes 1 and 4) or boiled reaction mixture (lanes 2 and 5) was used as a negative or positive (lane 2 and 5) control, respectively. (D) Schematic illustration of the blunt-ended RNA helix substrate. (E) The blunt-ended RNA helix (0.1 pmol) was reacted with MBP-2CATPase (20 pmol).
Mentions: For helicases, the directionality of helix unwinding is a fundamental characteristic [35]. To assess the unwinding directionality of EV71 2CATPase, we constructed three different RNA helix substrates containing a 3′ single-stranded protrusion, a 5′ single-stranded protrusion, and blunt ends, respectively (Fig 3A, 3B and 3D). Each substrate was then incubated with 2CATPase for the helix unwinding assay. Our results showed that EV71 2CATPase could unwind either the 3′- or 5′-protruded RNA helix (Fig 3C), and its efficiency to unwind the 3′-protruded helix was apparently higher than its efficiency to unwind the 5′-protruded one (Fig 3C, lanes 3 vs. 6). Moreover, 2CATPase could not unwind the blunt-ended helix (Fig 3E). All these experiments were independently repeated several times. Based on these results, we conclude that EV71 2CATPase possesses a bidirectional unwinding activity to RNA helices, while the 3′→5′ unwinding is more preferred.

Bottom Line: Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus.Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication.These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, China.

ABSTRACT
RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

No MeSH data available.


Related in: MedlinePlus