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. 2013 May;87(9):5205-18.
doi: 10.1128/JVI.00245-13. Epub 2013 Feb 28.

The nonstructural protein 2C of a Picorna-like virus displays nucleic acid helix destabilizing activity that can be functionally separated from its ATPase activity

Zhenyun Cheng  1 , Jie Yang, Hongjie Xia, Yang Qiu, Zhaowei Wang, Yajuan Han, Xiaoling Xia, Cheng-Feng Qin, Yuanyang Hu, Xi Zhou
Affiliations

The nonstructural protein 2C of a Picorna-like virus displays nucleic acid helix destabilizing activity that can be functionally separated from its ATPase activity

Zhenyun Cheng et al. J Virol. 2013 May.

Abstract

Picorna-like viruses in the Picornavirales order are a large group of positive-strand RNA viruses that include numerous important pathogens for plants, insects, and humans. In these viruses, nonstructural protein 2C is one of the most conserved proteins and contains ATPase activity and putative RNA helicase activity. Here we expressed 2C protein of Ectropis obliqua picorna-like virus (EoV; genus Iflavirus, family Iflaviridae, order Picornavirales) in a eukaryotic expression system and determined that EoV 2C displays ATP-independent nucleic acid helix destabilizing and strand annealing acceleration activity in a concentration-dependent manner, indicating that this picornaviral 2C is more like an RNA chaperone than like the previously predicted RNA helicase. Our further characterization of EoV 2C revealed that divalent metal ions, such as Mg(2+) and Zn(2+), inhibit 2C-mediated helix destabilization to different extents. Moreover, we determined that EoV 2C also contains ATPase activity like that of other picornaviral 2C proteins and further assessed the functional relevance between its RNA chaperone-like and ATPase activities using mutational analysis as well as their responses to Mg(2+). Our data show that, when one of the two 2C activities was dramatically inhibited or almost abolished, the other activity could remain intact, showing that the RNA chaperone-like and ATPase activities of EoV 2C can be functionally separated. This report reveals that a picorna-like virus 2C protein displays RNA helix destabilizing and strand annealing acceleration activity, which may be critical for picornaviral replication and pathogenesis, and should foster our understanding of picorna-like viruses and viral RNA chaperones.

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Figures

Fig 1
Fig 1
Amino acid sequence alignment of 2C proteins of EoV and other picorna-like viruses. (A) Schematic representation of the EoV genome. (B) The three conserved motifs (A, B, and C) for SF3 helicases are indicated. The left numbers indicate the starting amino acid positions of the aligned sequences. The middle numbers indicate the numbers of amino acids between the conserved motif regions. PnV, Perina nuda virus; IFV, flacherie virus of silkworm; DWV, deformed wing virus; VDV-1, Varroa destructor virus-1; PV, poliovirus; EV71, enterovirus 71; EWCV, encephalomyocarditis virus; HAV, hepatitis A virus.
Fig 2
Fig 2
EoV 2C destabilizes both RNA and DNA helices. Purified MBP-2C was incubated with standard RNA helix (R*/R substrate), DNA helix (D*/D substrate), or RNA/DNA hybrid helix (R*/D substrate) as illustrated in the left panels. Asterisks indicate the HEX-labeled strand. The preparations of destabilizing substrates are indicated in Materials and Methods. Substrate (0.1 pmol) was incubated in standard reaction mixtures in the presence or absence of 10 pmol MBP-2C as indicated, and the destabilizing activity was assessed via gel electrophoresis and scanning on a Typhoon 9200 imager. (A) R*/R substrate (left panel). Lane 1, reaction mixture without 2C addition; lane 2, complete reaction mixture with negative-control MBP alone; lane 3, complete reaction mixture with MBP-2C; lane 4, boiled reaction mixture without 2C addition. (B) D*/D substrate (left panel). Lanes 1 and 2, boiled (lane 1) or native (lane 2) reaction mixtures without 2C addition; lane 3, complete reaction mixture with MBP-2C. (C) R*/D substrate (left panel). Lanes 1 and 2, boiled (lane 1) or native (lane 2) reaction mixtures without 2C addition; lane 3, complete reaction mixture with MBP-2C.
Fig 3
Fig 3
EoV 2C destabilizes RNA helices in a bidirectional manner. (A) MBP-2C (10 pmol) was reacted with 3′-tailed (lane 2) or 5′-tailed (lane 4) RNA helix substrate (0.1 pmol) as illustrated in the upper panel, respectively, under standard reaction conditions. Asterisks indicate the HEX-labeled strand. Each substrate alone (lanes 1 and 3) or boiled 5′-tailed substrate (lane 5) was used as a control. (B) MBP-2C (10 pmol) was reacted with blunt-ended RNA helix (0.1 pmol) as illustrated in the upper panel in the absence (lanes 3) or presence (lane 4) of ATP.
Fig 4
Fig 4
The RNA helix destabilizing activity of EoV 2C is NTP independent. (A) Standard RNA helix substrate (3′ plus 5′ tailed) (0.1 pmol) as illustrated in the left panel was reacted with MBP-2C in the absence or presence of indicated NTP (2.5 mM). Native or boiled substrates without MBP-2C addition were used as controls. (B) Schematic illustration of the 5′-tailed RNA helix substrate that was used in the experiments represented by panels C to G. Asterisks indicate the HEX-labeled strand. (C) The 5′-tailed RNA helix (0.1 pmol) was reacted with MBP-2C (10 pmol) in the absence or presence of the indicated NTP (2.5 mM). Native or boiled substrates without 2C addition were used as controls. (D to F) The 5′-tailed RNA helix (0.1 pmol) was reacted with MBP-2C (10 pmol) in the presence of 0.5 to 10 mM ATP (D), 1 to 400 μM ATP (E), or 1 to 900 nM ATP (F). (G) The 5′-tailed RNA helix (0.1 pmol) was reacted with MBP-2C (1 pmol) in the absence or presence of 2.5 mM ATP.
Fig 5
Fig 5
The length of 3′ tail of RNA helices shows no obvious effect on the helix destabilizing activity of EoV 2C. (A) Schematic illustration of RNA helix substrates with the indicated lengths of 3′ single strands. The shorter strand is HEX labeled. (B) A 0.1-pmol volume of the indicated 3′-tailed RNA helices was reacted with 10 pmol of MBP-2C. Neither ATP or another NTP was supplemented in the reaction mixture.
Fig 6
Fig 6
Characterization of the RNA helix destabilizing activity of 2C. Schematic illustrations of RNA helix substrates with indicated lengths of 3′ and 5′ tails and matched base pairs are shown in the upper panels. A 0.1-pmol volume of RNA helix substrate was reacted with 5 pmol MBP-2C in the absence of ATP or other NTP. A 5-pmol volume of MBP-fusion HCV NS3 (plus 2.5 mM ATP) and MBP alone were used as controls for the RNA helix destabilizing assay. Asterisks indicate the HEX-labeled strand.
Fig 7
Fig 7
EoV 2C destabilizes structured RNA strands. (A) Schematic illustrations of the stem-loop structures of the two 42-nt RNA substrates predicted by mfold. The two RNA strands are complementary. One strand has a HEX-labeled 5′ end as indicated by asterisk (right), while the other strand was not labeled (left). (B) The two complementary strands were mixed (0.1 pmol each) and reacted in the absence (lanes 3 to 6) or presence (lanes 7 to 10) of MBP-2C (5 pmol) for the indicated times (5 to 20 min). (C) The two complementary strands were mixed (0.1 pmol each) and reacted with increasing amounts of MBP-2C (0 to 15 pmol) for 20 min. For panels B and C, the mixture of the two strands was boiled (to prevent spontaneous annealing) (lane 1) or treated with a thermal cycler (lane 2) as a negative or positive control, respectively. Samples were subjected to gel electrophoresis (see Materials and Methods).
Fig 8
Fig 8
EoV 2C accelerates the annealing of RNA strands. (A) Schematic illustration of the 46-nt RNA strand (upper) that is complementary to the 146-nt RNA strand. The 46-nt strand is HEX labeled as indicated by an asterisk. (B) The two strands were mixed (0.1 pmol for each strand) and reacted with increasing amounts of MBP-2C (2 to 10 pmol) for 20 min. The mix of the two strands was boiled (to prevent spontaneous annealing) (lane 1) or treated with a thermal cycler (lane 2) as a negative or positive control, respectively. Samples were subjected to gel electrophoresis (see Materials and Methods).
Fig 9
Fig 9
Optimal conditions for EoV 2C RNA helix destabilization. (A) Standard RNA helix substrate (3′ plus 5′ tailed) (0.1 pmol) as illustrated in the left panel was reacted with MBP-2C in the presence of the indicated divalent metal ions at 2.5 mM. Native and boiled substrates without 2C addition were used as controls (lanes 1 and 7). (B) Schematic illustration of 5′-tailed RNA helix substrate that was used in the experiments represented by panels C to E. Asterisks indicate the HEX-labeled strand. (C and D) 5′-tailed RNA helix substrate (0.1 pmol) was reacted with MBP-2C in the presence of indicated concentrations of MgCl2 (C) or ZnCl2 (D). (E) Helix destabilizing activity was determined at the indicated pH in the absence of divalent metal ions.
Fig 10
Fig 10
EoV 2C has NTPase activity. (A) MBP-2C was reacted with the indicated NTP or dNTP. The NTPase activity of MBP-2C was measured as nanomoles of released inorganic phosphate. (B to E) The NTPase activity of EoV 2C was determined at the indicated pH (B), at the indicated concentrations of NaCl (C) orMgCl2 (D), or with indicated divalent metal ions at a concentration of 2.5 mM (E). For panels A to E, the complete reaction mixture with MBP alone was used as the negative control, and error bars represent standard deviation values from three separate experiments.
Fig 11
Fig 11
The RNA helix destabilizing and ATPase activities of EoV 2C can be separated. (A) Sequence analysis of the EoV 2C ORF and the mutagenesis strategy, with sites of replacement with alanine indicated by a star. (B) Expressed and purified eukaryotic proteins were subjected to 10% SDS-PAGE followed by Western blotting with anti-MBP polyclonal antibody. (C) Equal amounts of MBP-2C wild-type and mutant strains were reacted with ATP, and ATPase activity was measured as nanomoles of released inorganic phosphate. Error bars represent standard deviation values from three separate experiments. (D) 5′-tailed RNA helix substrate (as illustrated in Fig. 9B) was reacted with equal amounts of MBP-2C wild-type and mutant strains in the absence of NTP.
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