Human Norovirus NS3 Has RNA Helicase and Chaperoning Activities

Abstract

RNA-remodeling proteins, including RNA helicases and chaperones, act to remodel RNA structures and/or protein-RNA interactions and are required for all processes involving RNAs. Although many viruses encode RNA helicases and chaperones, their in vitro activities and their roles in infected cells largely remain elusive. Noroviruses are a diverse group of positive-strand RNA viruses in the family Caliciviridae and constitute a significant and potentially fatal threat to human health. Here, we report that the protein NS3 encoded by human norovirus has both ATP-dependent RNA helicase activity that unwinds RNA helices and ATP-independent RNA-chaperoning activity that can remodel structured RNAs and facilitate strand annealing. Moreover, NS3 can facilitate viral RNA synthesis in vitro by norovirus polymerase. NS3 may therefore play an important role in norovirus RNA replication. Lastly, we demonstrate that the RNA-remodeling activity of NS3 is inhibited by guanidine hydrochloride, an FDA-approved compound, and, more importantly, that it reduces the replication of the norovirus replicon in cultured human cells. Altogether, these findings are the first to demonstrate the presence of RNA-remodeling activities encoded by Caliciviridae and highlight the functional significance of NS3 in the noroviral life cycle.IMPORTANCE Noroviruses are a diverse group of positive-strand RNA viruses, which annually cause hundreds of millions of human infections and over 200,000 deaths worldwide. For RNA viruses, cellular or virus-encoded RNA helicases and/or chaperones have long been considered to play pivotal roles in viral life cycles. However, neither RNA helicase nor chaperoning activity has been demonstrated to be associated with any norovirus-encoded proteins, and it is also unknown whether norovirus replication requires the participation of any viral or cellular RNA helicases/chaperones. We found that a norovirus protein, NS3, not only has ATP-dependent helicase activity, but also acts as an ATP-independent RNA chaperone. Also, NS3 can facilitate in vitro viral RNA synthesis, suggesting the important role of NS3 in norovirus replication. Moreover, NS3 activities can be inhibited by an FDA-approved compound, which also suppresses norovirus replicon replication in human cells, raising the possibility that NS3 could be a target for antinoroviral drug development.

Keywords: RNA chaperone; RNA helicase; norovirus.

FIG 1

Norovirus NS3 is similar to other virus-encoded SF3 helicases in consensus motifs and protein structures. (A) The genomic RNA of norovirus contains three ORFs (ORF1, -2, and -3). The 5′-proximal ORF1 encodes a polyprotein that is subsequently self-cleaved into six nonstructural proteins (NS1/2, NS3, NS4, NS5/VPg, NS6^Pro, and NS7/RdRP) by viral protease NS6^Pro. ORF2 and ORF3 encode structural proteins VP1 and VP2, respectively. (B) Sequence alignment of the helicase core domains of NV NS3 and other SF3 viral helicases, EV71 2C^ATPase, SV40 LTag, HPV18 E1, and AAV2 Rep40. The SF3 consensus motifs A, B, and C are indicated. Completely conserved residues (100% identity) are highlighted in black, and highly conserved residues (≥50% identity) are highlighted in gray. Dashes indicate no amino acid and are included for alignment purposes. (C) The structure of NV NS3 without its N-terminal domain (NS3ΔN) was predicted by the HMMSTR/Rosetta server. NS3ΔN containing the middle HC domain and the CTD is shown in green. SF3 motifs A, B, and C are indicated and highlighted in red. (D to F) Structural alignments of NV NS3ΔN (green) and SV40 LTag, HPV18E1, or AAV2 Rep40 (cyan). The N-terminal domain of E2 binding to HPV18 E1 is shown in brown. SF3 motifs A, B, and C of these viral helicases are highlighted in blue.

FIG 2

NV NS3 has NTPase activity. (A) MBP-NS3 was reacted with the indicated NTPs. The NTPase activity was measured as nanomoles of released inorganic phosphate. (B to D) The NTPase activity of MBP-NS3 was determined at the indicated concentrations of ATP (B), at the indicated concentrations of Mg²⁺ (C), or at the indicated pH (D). (B and C) MBP alone was used as the negative control. (A to D) The error bars represent standard deviation (SD) values from the results of three separate experiments.

FIG 3

NV NS3 has nucleic acid helix-unwinding activity. (A) The standard RNA/RNA helix (R*/R substrate) is illustrated in the upper diagram. R*/R substrate (0.1 pmol) was reacted with 20 pmol of each indicated protein. Nonboiled reaction mixture (lane 1) and reaction mixture with MBP alone (lane 3) were used as negative controls, and boiled reaction mixture (lane 2) and reaction mixture with purified MBP fusion with EV71 2C^ATPase (lane 5) were used as positive controls. (B to D) DNA/DNA helix (D*/D substrate), DNA/RNA hybrid helix (D*/R), and RNA/DNA hybrid helix (R*/D) are illustrated in the upper diagrams; 0.1 pmol of each indicated substrate was reacted with 20 pmol of each indicted protein. Lanes 1, nonboiled reaction mixture; lanes 2, boiled reaction mixture; lanes 3, reaction mixture with purified MBP-NS3 fusion; lanes 4, reaction mixture with purified MBP-EV71 2C^ATPase fusion. Helix unwinding was detected via gel electrophoresis and scanning on a Typhoon 9500 imager. The asterisks indicate the HEX-labeled strands.

FIG 4

Biochemical characterization of RNA helix-unwinding activity of NV NS3. (A) Illustration of the standard RNA helix (R*/R) substrate. (B to D) R*/R substrate (0.1 pmol) was reacted with 20 pmol MBP-NS3 in the absence or presence of individual NTP (B), increasing concentrations of Mg²⁺ (C), or different pH values (D) as indicated. Nonboiled (lanes 1) or boiled (lanes 2) reaction mixture was used as a negative or positive control. The asterisks indicate the HEX-labeled strands. Helix-unwinding activity was detected by gel electrophoresis and scanning on a Typhoon 9500 imager.

FIG 5

NV NS3 unwinds the RNA helix in a bidirectional manner. (A and B) Schematic illustration of the RNA helix substrate with the 5′ single-strand protrusion (A) or 3′ single-strand protrusion (B). The asterisks indicate the HEX-labeled strands. (C) MBP-NS3 (20 pmol) was incubated with 5′ single-strand protruded (lane 3) or 3′ single-strand protruded (lane 6) RNA helix substrate (0.1 pmol). Nonboiled (lanes 1 and 4) or boiled (lanes 2 and 5) reaction mixtures were used as negative or positive controls. (D and E) MBP-NS3 (20 pmol) was reacted with 5′-protruded (D) or 3′-protruded (E) RNA helix substrate in the presence of the indicated concentrations of ATP. Helix unwinding was detected as described for Fig. 3.

FIG 6

SF3 motif A is critical for the helicase activity of NV NS3. (A) Amino acid sequence alignment of NV NS3, SHV NS3, and MNV NS3. The SF3 consensus motifs A, B, and C are indicated. Completely conserved residues (100% identity) are highlighted in black. (B) The standard RNA helix (R*/R) substrate (0.1 pmol) was reacted with 20 pmol WT (lane 4), GK168AA (lane 5), or GK168AA-DD212AA (lane 6) MBP-NS3. Nonboiled reaction mixture (lane 1) and reaction mixture with addition of MBP alone (lane 3) were used as negative controls; the boiled reaction mixture (lane 2) was used as a positive control. (C) The standard RNA helix (R*/R) substrate (0.1 pmol) was reacted with 20 pmol GK168AA mutant MBP-NS3 in the presence of the indicated concentrations of ATP. (B and C) The asterisks indicate the HEX-labeled strands.

FIG 7

NV NS3 has RNA-chaperoning activity to destabilize structured RNA strands and promote annealing. (A) The standard RNA helix (R*/R) substrate (0.1 pmol) was reacted with 20 pmol MBP-NS3 in the absence or presence of 5 mM ATP or ATP analog (AMP-PNP) as indicated. Lane 1, nonboiled reaction mixture; lane 2, boiled reaction mixture; lane 3, reaction mixture with MBP-NS3 in the absence of ATP; lanes 4 and 5, reaction mixture with MBP-NS3 in the presence of AMP-PNP or ATP. (B) Schematic illustrations of the stem-loop structures of the two complementary 42-nt RNA strands. The asterisk indicates the HEX-labeled strand. (C) The two strands were mixed 1:1 (0.1 pmol each) and reacted with the indicated amounts of MBP-NS3. (D) The hybridization assay (as for panel C) was conducted in the absence (lanes 3 to 6) or presence (lanes 7 to 10) of 20 pmol MBP-NS3 for the indicated reaction times. (C and D) The preannealed (lanes 1) or boiled (lanes 2) strands were used as a positive or negative control, respectively. The hybridized and free strands are indicated.

FIG 8

NV NS3 facilitates noroviral RNA synthesis in vitro. (A) Schematic illustration of the experimental procedures. (B and D) The in vitro-transcribed NV 3′ end (nt 1 to 400) of (−)-vRNA template was incubated with 10 pmol recombinant NS7/RdRP and NTP mixture in the absence or presence of 10 pmol MBP-NS3 (B) or MBP-NS3_GK168AA (D) at 30°C for 30, 60, or 90 min as indicated. The reaction products were detected by Northern blotting. (C and E) The synthesized (+)-RNA products in panels B and D were quantified with Bio-Rad Quantity One software. The relative RNA production was determined by comparing the level of RNA products in the presence of MBP-NS3 at each indicated time point with that in the absence of NS3 at 30 min (lanes 2). The error bars represent SD values from the results of three separate experiments.

FIG 9

GuHCl inhibits the biochemical activities of NV NS3. (A) The NTPase activity of MBP-NS3 was measured as nanomoles of released inorganic phosphate in the presence of the indicated concentrations (0 to 10 mM) of GuHCl. MBP alone was used as the negative control. (B) The standard RNA helix (R*/R) substrate is illustrated in the upper diagram. R*/R substrate (0.1 pmol) was reacted with 20 pmol MBP-NS3 in the presence of the indicated concentrations (0 to 10 mM) of GuHCl. Native (lane 1) or boiled (lane 2) reaction mixture was used as a negative or positive control, respectively. Helix unwinding was detected by gel electrophoresis and scanning on a Typhoon 9500 imager. The asterisk indicates the HEX-labeled strand. (C) The unwinding activities under different GuHCl concentrations were plotted as percentages of the released RNA from the total RNA helix substrate (y axis) at each indicated GuHCl concentration (x axis) with Bio-Rad Quantity One software. The error bars represent standard deviation values from the results of three separate experiments. (D) The two strands shown in Fig. 7B were mixed 1:1 (0.1 pmol each) and reacted with 10 pmol MBP-NS3 for 30 min in the presence of different concentrations of GuHCl in the absence of NTP. The hybridization of the two strands was detected by gel electrophoresis and scanning on a Typhoon 9500 imager. The preannealed (lane 1) or boiled (lane 2) reaction mixture was used as a positive or negative control, respectively. The hybridized and free strands are indicated. (E) The effect of GuHCl or 2CMC on Norwalk virus RNA levels was determined by examining the impact on NV replicon RNA levels in HGT-NV cells following 3 days of treatment. HGT-NV cells were treated with the indicated levels of either GuHCl or 2CMC, and the levels of viral RNA were calculated as a percentage of the mock-treated control. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) HGT-NV cells were treated as for panel D, and the number of viable cells was determined. The cell viability was calculated as a percentage of that of the mock-treated control. The error bars represent SD (n = 3). Statistical significance was determined using an unpaired t test. The experiment was repeated at least two independent times, with one representative data set presented.