Identification and characterization of RNA duplex unwinding and ATPase activities of an alphatetravirus superfamily 1 helicase

Abstract

Dendrolimus punctatus tetravirus (DpTV) belongs to the genus omegatetravirus of the Alphatetraviridae family. Sequence analysis predicts that DpTV replicase contains a putative helicase domain (Hel). However, the helicase activity in alphatetraviruses has never been formally determined. In this study, we determined that DpTV Hel is a functional RNA helicase belonging to superfamily-1 helicase with 5'-3' dsRNA unwinding directionality. Further characterization determined the length requirement of the 5' single-stranded tail on the RNA template and the optimal reaction conditions for the unwinding activity of DpTV Hel. Moreover, DpTV Hel also contains NTPase activity. The ATPase activity of DpTV Hel could be significantly stimulated by dsRNA, and dsRNA could partially rescue the ATPase activity abolishment caused by mutations. Our study is the first to identify an alphatetravirus RNA helicase and further characterize its dsRNA unwinding and NTPase activities in detail and should foster our understanding of DpTV and other alphatetraviruses.

Fig. 1

(A) Amino acid sequence alignment of helicases from SARS coronavirus (SARS-CoV; GenBank: NC_004718), hepatitis E virus (HEV; GenBank: JX121233), Rubella virus (RV; GenBank: JN635296), HaSV (GenBank: NC_001981), NβV (GenBank: AF102884) and DpTV (GenBank: AY594352). Helicase motifs are labeled as motifs I to VI. (B) The mutagenesis strategy to generate point mutations in motifs I, II and VI. (C) Electrophoresis analysis of purified MBP-Hel and its mutants. Proteins were subjected to 12% SDS-PAGE, and protein bands were stained with Coomassie blue. Lane M, molecular weight markers; lane 1, MBP alone; lane 2, MBP-Hel; lane 3, MBP-Hel (I); lane 4, MBP-Hel (II); lane 5, MBP-Hel (VI). (I), (II) or (VI) indicates the mutation in motifs I, II or VI, respectively. (D) MBP-Hel and its mutants were subjected to Western blots with anti-MBP or anti-Hel polyclonal antibodies. The lanes are labeled as in (C).

Fig. 2

Duplex unwinding activities of DpTV Hel and its mutants. (A–D) Purified MBP-Hel was incubated with standard dsRNA, dsDNA or RNA/DNA hybrid substrates in the presence or absence of Mg²⁺ or ATP (RNA: green line; DNA: blue line; HEX: red asterisk). The preparations of unwinding substrates are presented in the “Materials and methods” section. Substrates were incubated in standard reaction mixtures (except as noted) and the unwinding activity was assessed by gel electrophoresis followed by scan with a Typhoon9200. (A) Standard dsRNA substrates. (B) D^⁎/R substrates. (C) R^⁎/D substrates. (D) D^⁎/D substrates. Lanes 1 and 2: reaction mixtures lacking enzyme that were either left native (lane 1) or boiled (lane 2); Lane 3, complete reaction mixture with negative control MBP alone; lanes 4 and 5, complete reaction mixtures with MgCl₂ omitted (lane 4) or with ATP omitted (lane 5); lane 6, complete reaction mixture. (E) dsRNA unwinding activities of DpTV Hel mutants. Lanes 1 and 2: reaction mixtures lacking enzyme that were either left native (lane 1) or boiled (lane 2); lanes 3, 4, 5 and 6, complete reaction mixture with MBP-Hel (lane 3), MBP-Hel (I) (lane 4), MBP-Hel (II) (lane 5) or MBP-Hel (VI) (lane 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

DpTV Hel unwinds dsRNA in the 5′–3′ direction, and its unwinding efficiency depends on the length of 5′ single stranded tail. (A–C) MBP-Hel was reacted with 5′-tailed (A), 3′-tailed (B) or blunt ended (C) dsRNA substrates in the presence or absence of ATP. The short RNA strand was also HEX-labeled as in Fig. 2. Substrates alone (lane 1), boiled substrates (lane 2) and MBP alone (lane 3) were used as controls. (D) Schematic of the unwinding substrates with different 5′-tail length. The length of 5′ tail was indicated above each dsRNA substrate. (E) Each substrate in (D) was incubated with MBP-Hel in standard reaction condition, and the unwinding activity was graphed as the percentage of the released RNA from the total dsRNA substrate at each time point. The 5′ tail length was indicated on the right. Error bars represent S.D. values from three separate experiments.

Fig. 4

Optimal conditions for DpTV Hel dsRNA unwinding. (A) Standard dsRNA substrate was reacted with MBP-Hel in the presence of 2.5 mM indicated divalent metal ions. Substrates alone or boil substrates were used as controls. (B–D) Standard dsRNA substrate was reacted with MBP-Hel in the presence of indicated concentrations of MgCl₂ (B), MnCl₂ (C) or KCl (D). The unwinding activities were determined and graphed as in Fig. 3E. (E) In the presence of 2 mM MgCl₂ and 50 mM KCl, the unwinding activities were determined at indicated pH, and then graphed as above. 5 mM ATP was used in all experiment, and error bars represent S.D. values from three separate experiments.

Fig. 5

Optimal conditions for DpTV Hel ATPase activity. (A–C) The ATPase activities were measured as nmol of released inorganic phosphate in 20 μl reaction volume at 37 °C for 20 min, in the presence of indicated concentrations of MgCl₂ (A) or KCl (B), or at indicated pH (C). Error bars represent S.D. values from three separate experiments.

Fig. 6

ATPase activities of DpTV Hel and its mutants are stimulated by dsRNA. The ATPase activity of MBP-Hel (A) or mutants of motifs I, II or VI (B) was determined as in Fig. 5 in the presence of dsRNA or dsDNA. The absence of any oligonucleotide was used as control. Error bars represent S.D. values from three separate experiments.