Nucleotide triphosphatase and RNA chaperone activities of murine norovirus NS3

Abstract

Modulation of RNA structure is essential in the life cycle of RNA viruses. Immediate replication upon infection requires RNA unwinding to ensure that RNA templates are not in intra- or intermolecular duplex forms. The calicivirus NS3, one of the highly conserved nonstructural (NS) proteins, has conserved motifs common to helicase superfamily 3 among six genogroups. However, its biological functions are not fully understood. In this study we report the oligomeric state and the nucleotide triphosphatase (NTPase) and RNA chaperone activities of the recombinant full-length NS3 derived from murine norovirus (MNV). The MNV NS3 has an Mg²⁺-dependent NTPase activity, and site-directed mutagenesis of the conserved NTPase motifs blocked enzyme activity and viral replication in cells. Further, the NS3 was found via fluorescence resonance energy transfer (FRET)-based assays to destabilize double-stranded RNA in the presence of Mg²⁺ or Mn²⁺ in an NTP-independent manner. However, the RNA destabilization activity was not affected by mutagenesis of the conserved motifs of NTPase. These results reveal that the MNV NS3 has an NTPase-independent RNA chaperone-like activity, and that a FRET-based RNA destabilization assay has the potential to identify new antiviral drugs targeting NS3.

Keywords: NS3; NTPase; RNA chaperone; RNA replication; helicase; norovirus.

Fig. 1.

Expression and purification of recombinant MBP-NS3 proteins. (a) Schematic diagram of recombinant MNV-1 NS3. The N-terminal 12× His tag and 10× Asn linker between MBP and NS3 are indicated in black and grey, respectively. The arrow head represents the TEV cleavage site. The mutation sites of K169A, D213A and N260A are indicated. Amino acid sequence alignment of putative SF3 helicases from viruses belonging to the families Caliciviridae and Picornaviridae (NCBI Entrez accession numbers: MNV, NC008311; MD145, AAK50354; NV, AAB50465; SV, Q6XDK8; FCV, AAA79323; RHDV, P27410; PV, ACS88256; EV71, KC954662; FMDV, P03305). Well-conserved motifs A, B (also known as Walker A and B) and C are indicated. The arrow heads indicate the positions where amino acids were substituted with alanine. (b) Purification of recombinant proteins after size-exclusion chromatography. Purified native and mutant MBP and MBP-NS3 proteins were analysed by 12 % SDS-PAGE. The molecular masses of standard proteins are shown in kDa on the left. (c) Native PAGE and dynamic light scattering (DLS) results for MBP-NS3 protein. Left panel: Purified native MBP-NS3 protein was analysed by 6 % PAGE. The molecular masses of standard proteins are shown in kDa, and the right inset shows oligomeric forms of MBP-NS3 (indicated by arrows). Right panel: DLS results for MBP-NS3 protein, showing oligomers with average molecular sizes of 35–40 nm and larger aggregates.

Fig. 2.

NTPase activity of MNV NS3. (a) MBP or MBP-NS3 was subjected to NTPase assay with 0, 10, or 100 µM ATP. The amount of released inorganic phosphate was measured. The effects of the amount of ATP (upper panel), MBP or MBP-NS3 (lower panel) on NTPase activity were tested. (b) The effects of temperature, pH and MgCl₂ concentration on NTPase activity were tested. The NTPase activities of MNV-NS3 with different nucleotides as a substrate were also tested (right lower panel).

Fig. 3.

RNA chaperone activity of MNV NS3. (a) Schematic diagram of FRET-based RNA destabilization assay. 5′ and 3′ protruded dsRNA substrates labelled with Cy3 and Cy5 dyes at the end of each strand were used for the assays. (b) Purified MBP or MBP-NS3 was subjected to RNA chaperone assays. Both Cy3 and Cy5 emission signals were monitored every 1 min. (c) The FRET efficiency was calculated by the equation F _Cy5/(F _Cy3+F _Cy5) and plotted for 2 h.

Fig. 4.

Optimum conditions for NS3-mediated RNA destabilization. The effects of temperature, pH, NaCl, divalent cations, MgCl₂, MnCl₂ and ATP on FRET efficiency were measured and plotted for 2 h (from the top left panel). RNA chaperone activity with different nucleotides was also measured (right bottom panel).

Fig. 5.

NTPase and RNA chaperone activities of MBP-NS3 mutants. (a) Wild-type and MBP-NS3 mutant proteins, K169A, D213A and N260A, were subjected to NTPase assay. The standard deviations of triplicate independent samples are shown as vertical error bars. Statistical analysis was performed by a one-way ANOVA (**, P<0.005; ***, P<0.001). (b) Growth analysis of recombinant MNVs in 293T-CD300LF cells transfected with plasmids producing MNVs with the indicated mutations. Data are expressed as mean±sem. The dashed line indicates the limit of detection. N=3. (c) The RNA chaperone activities of wild-type and mutant MBP-NS3 proteins were measured and plotted for 2 h.