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. 2001 May;75(10):4519-27.
doi: 10.1128/JVI.75.10.4519-4527.2001.

Identification and characterization of the helix-destabilizing activity of rotavirus nonstructural protein NSP2

Z F Taraporewala  1 , J T Patton
Affiliations

Identification and characterization of the helix-destabilizing activity of rotavirus nonstructural protein NSP2

Z F Taraporewala et al. J Virol. 2001 May.

Abstract

The rotavirus nonstructural protein NSP2 self-assembles into homomultimers, binds single-stranded RNA nonspecifically, possesses a Mg2+-dependent nucleoside triphosphatase (NTPase) activity, and is a component of replication intermediates. Because these properties are characteristics of known viral helicases, we examined the possibility that this was also an activity of NSP2 by using a strand displacement assay and purified bacterially expressed protein. The results revealed that, under saturating concentrations, NSP2 disrupted both DNA-RNA and RNA-RNA duplexes; hence, the protein possesses helix-destabilizing activity. However, unlike typical helicases, NSP2 required neither a divalent cation nor a nucleotide energy source for helix destabilization. Further characterization showed that NSP2 displayed no polarity in destabilizing a partial duplex. In addition, helix destabilization by NSP2 was found to proceed cooperatively and rapidly. The presence of Mg2+ and other divalent cations inhibited by approximately one-half the activity of NSP2, probably due to the increased stability of the duplex substrate brought on by the cations. In contrast, under conditions where NSP2 functions as an NTPase, its helix-destabilizing activity was less sensitive to the presence of Mg2+, suggesting that in the cellular environment the two activities associated with the protein, helix destabilization and NTPase, may function together. Although distinct from typical helicases, the helix-destabilizing activity of NSP2 is quite similar to that of the sigmaNS protein of reovirus and to the single-stranded DNA-binding proteins (SSBs) involved in double-stranded DNA replication. The presence of SSB-like nonstructural proteins in two members of the family Reoviridae suggests a common mechanism of unwinding viral mRNA prior to packaging and subsequent minus-strand RNA synthesis.

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Figures

FIG. 1
FIG. 1
Expression and purification of recombinant NSP2. NSP2 expressed in E. coli with a C-terminal His tag was purified by NTA affinity chromatography, and the final eluate was dialyzed against LSB. The eluate (lane 2) and protein dialyzed in LSB (lane 1) were resolved by SDS-polyacrylamide gel electrophoresis and stained with Coomassie blue. M, molecular size marker.
FIG. 2
FIG. 2
NSP2 possesses helix-destabilizing activity. (A) A schematic representation of the 32P-labeled DNA-RNA partial duplex A11-StyI–18AD. (B) From 1 to 200 pmol of NSP2 (lanes 4 to 9) was incubated with 0.1 pmol of A11-StyI–18AD for 30 min at 37°C. Afterwards, the reaction mixtures were analyzed by nondenaturing gel electrophoresis and autoradiography. Reaction mixtures containing 0.1 pmol of the 32P-labeled 18AD DNA oligonucleotide instead of the duplex (lane 1), 0.1 pmol of the duplex and no NSP2 (lane 2), and 0.1 pmol of the duplex denatured by heating at 95°C for 2 min and containing no NSP2 (lane 3) were also analyzed.
FIG. 3
FIG. 3
Directionality of the unwinding activity of NSP2. (A) A schematic representation of the possible duplexes formed by annealing the 32P-labeled DNA oligonucleotides 5′18AD and 3′22AD to the A11-SacII RNA. (B) A standard strand displacement assay was performed by incubating 0.1 pmol of the duplex mixture as shown in panel A with 1 to 100 pmol of recombinant NSP2 (rNSP2) (lanes 4 to 7). The reaction mixtures were analyzed by nondenaturing gel electrophoresis and autoradiography. Reaction mixtures containing 32P-labeled 5′18AD (lane 1) or 5′22AD (lane 2) and no duplexes were also analyzed.
FIG. 4
FIG. 4
Helix destabilization by NSP2 is cooperative. (A) A schematic representation of the 32P-labeled DNA-RNA duplex A11-SacII–5′18AD, formed by incubating the DNA oligonucleotide, 5′18AD, with the A11-SacII RNA. (B) A set of standard strand displacement assays was performed in parallel by incubating 0.1 pmol of the duplex with 1 to 200 pmol of NSP2. The reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The percent helix-destabilizing activity of NSP2 was plotted as a function of the amount of NSP2 in the reaction mixtures.
FIG. 5
FIG. 5
Kinetics of helix destabilization by NSP2. The 32P-labeled DNA-RNA duplex A11-StyI–5′18AD (0.1 pmol) was incubated with 50 (●), 100 (▴), or 200 (■) pmol of NSP2 for 2, 10, 15, or 30 min at 37°C. Afterwards, the reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The percent helix-destabilizing activity of NSP2 was plotted as a function of reaction time.
FIG. 6
FIG. 6
Effect of cations on helix destabilization by NSP2. The DNA-RNA duplex, A11-StyI–18AD (0.1 pmol), was incubated with NSP2 (200 pmol) in the presence of 0 to 10 mM MgCl2 (○), CaCl2 (□), or MnCl2 (◊) (panel A) or 0 to 250 mM NaCl (●) (panel B). The reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The percent helix-destabilizing activity was plotted as a function of salt concentration, with the value obtained for the reaction mixture lacking salt normalized to 100%.
FIG. 7
FIG. 7
Combined effect of Mg2+ and NTP on helix destabilization by NSP2. (A) NSP2 (200 pmol) was incubated with 0.1 pmol of the DNA-RNA duplex, A11-StyI–18AD, in the absence or presence of 5 mM ATP, GTP, CTP, or UTP for 30 min at 37°C. (B) Reaction mixtures containing the same components as those of the reaction mixtures in panel A, except that these contained 5 mM MgCl2, were also incubated. (C) NSP2 (100 pmol) was incubated with 0.1 pmol of the DNA-RNA duplex, A11-StyI–18AD, in the absence or presence of 1 mM MgCl2 or in the presence of 1 mM MgCl2 and 1 mM ATP, ADP, or ATP-γ-S. The reaction mixtures were analyzed by nondenaturing gel electrophoresis, and the percent helix-destabilizing activity was determined using a PhosphorImager. The values were normalized to that of 100% for the assays performed in the absence of MgCl2.
FIG. 8
FIG. 8
Destabilization of an RNA-RNA duplex by NPS2. (A) A schematic representation of the 32P-labeled RNA-RNA duplex A11-StyI–14AR produced by annealing the RNAs A11-StyI and 14AR. (B) Strand displacement assays were performed by incubating 0.1 pmol of the RNA-RNA duplex with 1 to 200 pmol of NSP2 in the presence (lanes 4 to 9) or absence (lanes 10 to 15) of 5 mM MgCl2. As controls, reaction mixtures were also prepared that contained 0.1 pmol of the 14AR RNA instead of the duplex (lane 1), 0.1 pmol of A11-StyI–14AR duplex and no NSP2 (lane 2), or 0.1 pmol of A11-StyI–14AR and no NSP2, denatured by heating at 95°C for 2 min (lane 3). (C) Strand displacement assays were performed by incubating 5 to 20 fmol of the RNA-RNA duplex in the presence or absence of recombinant NSP2 (rNSP2). The reaction mixtures were analyzed by nondenaturing gel electrophoresis and autoradiography. A PhosphorImager was used to determine the percent helix-destabilizing activity for each reaction. The values were normalized to that of 100% for the assay reaction wherein the substrate duplex was denatured by heating.
FIG. 9
FIG. 9
NSP2 destabilizes a short RNA-RNA duplex with greater efficiency. (A) A schematic representation of the 32P-labeled RNA-RNA duplex A11-StyI–10AR produced by annealing the RNAs A11-StyI and 10AR. (B) Strand displacement assays were performed by incubating 0.1 pmol of the RNA-RNA duplex with 1 to 200 pmol of NSP2 (lanes 5 to 10). As controls, reaction mixtures were also prepared that contained 0.1 pmol of the 10AR RNA instead of the duplex (lane 1), 0.1 pmol of the A11-StyI–14AR duplex and no NSP2 (lane 2 and 4), or 0.1 pmol of A11-StyI–10AR and no NSP2, denatured by heating at 95°C for 2 min (lane 3). A PhosphorImager was used to determine the percent helix-destabilizing activity for each reaction. The values were normalized to that of 100% for the assay reaction wherein the substrate duplex was denatured by heating.
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