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

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.

Fig 1. EV71 2C^ATPase is similar to other viral SF3 helicases in both motifs and structures.

(A) Schematic representation of enterovirus genome and the functional motifs in 2C^ATPase, including Zinc binding, oligomerization, membrane binding, and RNA binding motifs. Amino acid positions of each motif are numbered. (B) Domain alignments of EV71 2C^ATPase and SF3 viral helicases, including SV40 LTag, HPV18 E1, and AAV2 Rep40. SF3 helicase motifs A, B, and C are highlighted in orange. (C) The structure of EV71 2C^ATPase was predicted using the HMMSTR/Rosetta server. The N-terminal domain (NTD), the middle helicase core (HC) domain, and the C-terminal domain (CTD) are linked by flexible loops as indicated. (D) Structural alignment of EV71 2C^ATPase (slate) and AAV2 Rep40 (yellow). The ATP/ADP binding site is highlighted in red. (E) MBP-fusion EV71 2C^ATPase was expressed using a prokaryotic (E. coli) or eukaryotic (baculovirus) system. The purified recombinant proteins were subjected to 10% SDS-PAGE followed by Coomassie brilliant blue R250 staining. (F) RNA/RNA hybrid helix (R*/R) substrate (0.1 pmol) as illustrated in the upper panel was reacted with 20 pmol prokaryotically (lane 5) or eukaryotically expressed MBP-2C^ATPase (lane 6). The helix unwinding activity was detected via gel electrophoresis and scanning on a Typhoon 9200 imager. Native (lane 1) or boiled (lane 2) reaction mixture was used as negative or positive control. Asterisks indicate the HEX-labeled strands.

Fig 2. EV71 2C^ATPase unwinds both RNA and DNA helices.

(A) The standard RNA/RNA hybrid helix (R*/R) substrate (0.1 pmol) as illustrated in the upper panel was reacted with each indicated protein (20 pmol). The helix unwinding activity was detected via gel electrophoresis and scanning on a Typhoon 9200 imager. Lane 1, reaction mixture without 2C^ATPase; lane 2, boiled reaction mixture without 2C^ATPase; lane 3, complete reaction mixture with MBP alone; lane 4, complete reaction mixture with MBP-fusion EV71 2C^ATPase; lane 5, complete reaction mixture with MBP-fusion EoV 2C^ATPase; lane 6, complete reaction mixture with MBP-fusion HCV NS3. (B) to (D) As illustrated in each upper panel, 0.1 pmol DNA/DNA (D*/D) (B), RNA/DNA (R*/D) (C), or DNA/RNA (D*/R) (D) substrate was incubated in the presence (lane 3) or absence (lane 1) of MBP-fusion EV71 2C^ATPase. Asterisks indicate the HEX-labeled strands.

Fig 3. 2C^ATPase 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-2C^ATPase (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-2C^ATPase (20 pmol).

Fig 4. EV71 2C^ATPase possesses both ATP-dependent and ATP-independent helix unwinding activities.

(A) Schematic illustration of the standard RNA helix substrate that contains both 3′- and 5′-protrusions. (B) and (C) The standard RNA helix (0.1 pmol) was reacted with MBP-2C^ATPase in the absence or presence of the indicated NTPs (5 mM) (B) or in the presence of increasing concentrations (0–6 mM) of ATP as indicated (C). (D) The unwinding activities under different ATP concentrations were plotted as the percentage of the released RNA from the total RNA helix substrate (Y-axis) at each ATP concentration (X-axis). Error bars represent standard deviation (SD) values from three separate experiments. (E) Unwinding assays of 2C^ATPase using the standard helix substrate were performed in the absence or presence of ATP or ATP analog (AMP-PNP) as indicated. (F) The ATPase activity of MBP-2C^ATPase was measured as nanomoles of released inorganic phosphate at the indicated Mg²⁺ concentrations. Error bars represent SD values from three separate experiments. (G) and (H) Unwinding assays of 2C^ATPase were performed in the presence of 0–2.5 mM (G) or 3–20 mM (H) Mg²⁺ as indicated. (I) The ATPase activity of MBP-2C^ATPase was measured as nanomoles of released inorganic phosphate at the indicated GnHCl concentrations. Error bars represent SD values from three separate experiments. (J) Unwinding assays of 2C^ATPase were performed in the presence of 0–50 mM GnHCl as indicated.

Fig 5. EV71 2C^ATPase contains both RNA helicase and chaperone activities.

(A) Schematic illustration of the RNA helix substrate with the 3′ single-stranded (upper panel) or 5′ single-stranded (lower panel) protrusion. (B) The 3′-protruded RNA helix (0.1 pmol) was reacted with MBP-2C^ATPase (20 pmol) in the presence of increasing concentrations of ATP as indicated (lanes 3–7). (C) and (D) The 5′-protruded RNA helix (0.1 pmol) was reacted with MBP-fusion Wt (C) or GK134AA mutant 2C^ATPase (D) in the presence of increasing concentrations of ATP as indicated (lanes 3–7). (E) For each indicated RNA helix substrate, the unwinding activities of Wt or GK134AA mutant 2C^ATPase at different ATP concentrations were plotted as the percentage of the released RNA from the total RNA helix substrate (Y-axis) at each ATP concentration (X-axis). Error bars represent SD values from three separate experiments.

Fig 6. 2C^ATPase destabilizes structured RNA strands and stimulates annealing.

(A) Schematic illustration of the stem-loop structures of the two complementary 42-nt RNA substrates. Asterisk indicates the HEX-labeled strand. (B) The two strands were 1:1 mixed (0.1 pmol each) and reacted with 5 pmol each indicated protein. (C) The two strands were mixed (0.1 pmol each) and reacted with increasing amounts (0–20 pmol) of MBP-fusion EV71 2C^ATPase as indicated. (D) The hybridization assay as in (B and C) was performed in the absence (lanes 3–6) or presence (lanes 7–10) of 5 pmol MBP-fusion EV71 2C^ATPase for different reaction times (5–20 min) as indicated. For (B-D), the mix of two strands was preannealed (lane 1) or boiled (lane 2) as a positive or negative control, respectively. The hybridized and free strands are indicated.

Fig 7. 2C^ATPase enhances hammerhead ribozyme activity.

(A) Schematic illustrations of the sequences and secondary structures of hammerhead ribozyme (purple) and its substrate RNA (red and blue). The ribozyme is a 58-nt unlabeled RNA synthesized by T7 RNA polymerase, and the 28-nt RNA substrate was synthesized with its 5′ end being HEX labeled. Under the correct structure of the hammerhead ribozyme, the cleavage of the substrate takes place between the 12^th and 13^th nucleotides from the 3′ end (indicated by arrow). (B) The RNA substrate was incubated with the ribozyme in the absence (lane 3) or presence (lanes 4–8) of increasing amounts (0.1–2 pmol) of MBP-2C^ATPase as indicated. The HEX-labeled 16-nt cleavage product (blue) was detected via denaturing gel electrophoresis and scanning. Non-ribozyme supplementation in the absence (lane 1) or presence (lane 2) of 2C^ATPase was used as a negative control. The non-cleaved substrate and cleaved product strands are indicated.

Fig 8. The CTD of 2C^ATPase is required for its RNA chaperone activity.

(A) The standard RNA helix (0.1 pmol) was reacted with MBP-fusion 2C^ATPase Wt (lane 3), GK134AA mutant (lane 4), or a CTD fragment (lane 5). Native (lane 1) or boiled reaction mixture (lane 2) was used as a negative or positive control, respectively. (B) The ATPase activity of the indicated proteins was measured as nanomoles of released inorganic phosphate as indicated. MBP alone was used as the negative control. Error bars represent SD values from three separate experiments. (C) and (D) The standard RNA helix (0.1 pmol) was reacted with MBP-2C^ATPase Wt (lane 3), GK134AA mutant (lane 5), or ΔCTD (lane 4) in the absence (C) or presence (D) of 5 mM ATP as indicated. (E) The unwinding activities of Wt or indicated mutant 2C^ATPase in the absence or presence of 5 mM ATP were plotted as the percentage of the released RNA from the total RNA helix substrate. Error bars represent SD values from three separate experiments.

Fig 9. 2C^ATPase facilitates 3D^pol-mediated enteroviral RNA synthesis in vitro.

(A) Schematic of the experimental procedures. (B) The in vitro transcribed EV71 3′-end (-)RNA template (10 pmol) and an excessive amount of primers (50 pmol) were preannealed and reacted with recombinant His-tagged EV71 3D^pol and DIG RNA labeling mix in the absence or presence of 5 pmol (upper panel) or 10 pmol (lower panel) MBP-2C^ATPase at 22°C for 30, 60, and 90 min as indicated. The reaction products were analyzed via electrophoresis on a denaturing formaldehyde-agarose gel. (C) The synthesized (+)RNA (DIG-labeled) products from the experiments in (B) were measured via Bio-Rad Quantity One software, and the relative RNA production was determined by comparing the RNA product level in the presence of the indicated amount of MBP-2C^ATPase at each time point with the RNA product level in the absence of MBP-2C^ATPase at 30 min. (D) The in vitro transcribed (-)RNA template and primers were preannealed and reacted with recombinant 3D^pol and DIG RNA labeling mix in the absence or presence of increasing amounts (1–10 pmol) of MBP-2C^ATPase at 22°C for 60 min as indicated. Upper panel: the amount of template (50 pmol) exceeded that of primer (10 pmol); lower panel: the amount of primer (50 pmol) exceeded that of template (10 pmol). (E) The synthesized (+)RNA products from the experiments in (C) were measured as in (B). Under either template- or primer-excessive conditions, the relative RNA production was determined by comparing the RNA product level in the presence of the indicated amount of MBP-2C^ATPase with the RNA product level in the absence of MBP-2C^ATPase. (F) The RdRP reactions were conducted as in (B) under different conditions as indicated. (G) The synthesized (+)RNA products from the experiments in (F) were measured as in (B). Under each condition, the relative RNA production was determined by comparing the RNA product level in the presence of the indicated protein with the RNA product level in the absence of MBP-2C^ATPase (F, lane 5). For (C, E, and G), error bars represent SD values from three separate experiments.

Fig 10. Effect of 2C^ATPase helicase-defective GK134AA mutation on EV71 RNA synthesis and viability.

(A) Wt and GK134AA mutant EV71 RNA transcripts were transfected into human RD cells. The total RNA from differently transfected cells were exacted, and the levels of EV71 RNA were measured via qRT-PCR 24 and 48 hours post-transfection (h.p.t). For either Wt or mutant EV71 transcript, the relative EV71 RNA production 48 h.p.t. was determined by comparing the RNA product level at 48 h.p.t. with that at 24 h.p.t. Error bars represent SD, n = 3. (B) The virus production of either Wt (upper panel) or GK134AA mutant (lower panel) EV71 transcript in RD cells 24 h.p.t. was detected via immunofluorescent staining of EV71 VP1 with anti-VP1 antibody (green). The cell nuclei were stained with DAPI (blue). The merged image represents the digital superimposition of green and blue signals.

Fig 11. CAV16 2C^ATPase also contains both RNA helicase and chaperone activities.

(A) Schematic illustration of the RNA helix substrate with the 3′ single-stranded protrusion. (B) The 3′-protruded RNA helix (0.1 pmol) was reacted with MBP-fusion CAV16 2C^ATPase (20 pmol) in the absence (lane 3) or presence (lanes 4–7) of increasing concentrations of ATP as indicated. (C) Schematic illustration of the RNA helix substrate with the 5′ single-stranded protrusion. (D) The 5′-protruded RNA helix (0.1 pmol) was reacted with MBP-fusion CAV16 2C^ATPase (20 pmol) in the absence (lane 3) or presence (lanes 4–7) of increasing concentrations of ATP as indicated. Asterisks indicate the HEX-labeled strands.