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. 2016 Apr 14;8(4):100.
doi: 10.3390/v8040100.

Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

Nadya Urakova  1   2   3 , Natalie Netzler  4 , Andrew G Kelly  5 , Michael Frese  6   7   8   9 , Peter A White  10 , Tanja Strive  11   12   13
Affiliations

Purification and Biochemical Characterisation of Rabbit Calicivirus RNA-Dependent RNA Polymerases and Identification of Non-Nucleoside Inhibitors

Nadya Urakova et al. Viruses. .

Abstract

Rabbit haemorrhagic disease virus (RHDV) is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus). The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp) is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C) for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV.

Keywords: RCV-A1; RHDV; antiviral agents; non-nucleoside inhibitors; polymerase.

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Figures

Figure 1
Figure 1
Amino acid alignment of RHDV and RCV RdRps. The alignment compares RHDV Czech strain V351 (GenBank accession number KF594473.1) and RCV-A1 (GenBank accession number EU871528.1) and was conducted with the BioEdit software. Conserved motifs (A–E, F1–F3) attributed to RdRps of single-stranded positive-sense RNA viruses [30] are shown in black boxes.
Figure 2
Figure 2
Chemical structure of compounds tested as potential antiviral agents against rabbit caliciviruses (from [44]). (a) NIC02; (b) NIC10; (c) NIC12. Mw—molecular weight, g/mol.
Figure 3
Figure 3
Comparison of RHDV and RCV RdRp activities. Purified recombinant RdRps were used to generate dsRNA from a poly(C) RNA (20 ng/μL) template using rGTP (0.5 mM) as substrate. Following incubation at 30 °C, reactions were stopped with 5 mM EDTA and dsRNA was quantified using the fluorescent dye PicoGreen. RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point. (a) The effect of RHDV and RCV RdRp concentrations on dsRNA formation over 15 min; (b) The synthesis of dsRNA catalysed by 20 ng of RHDV and RCV RdRps over a 1-h period. RFU—relative fluorescence units.
Figure 4
Figure 4
Impact of MnCl2, pH and temperature on calicivirus RdRp activity. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA (20 ng/μL) as template and rGTP (0.5 mM) as substrate. Following a 15-min incubation in the presence of different (a) MnCl2 concentrations; (b) pH levels; or (c) temperatures, reactions were stopped with EDTA at a final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. Unless indicated otherwise, reactions were incubated at 30 °C. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. Averages of relative fluorescence levels were calculated and plotted with standard deviations. The results were generated from two (a) or three (b and c) independent experiments with triplicate reactions for each measurement point.
Figure 5
Figure 5
Kinetics of template and substrate utilisation by calicivirus RdRps. Purified recombinant RdRps (40 ng of RHDV and RCV RdRps and 200 ng of NoV RdRp) were used to generate dsRNA with poly(C) RNA as template and rGTP as substrate. (a) The kinetics of template utilisation was examined by titrating poly(C) from 0 to 40 ng/μL in the presence of 0.5 mM rGTP; (b,c) The kinetics of nucleotide incorporation was examined by titrating rGTP from 0 to 0.5 mM and 0 to 2.0 mM in the presence of 20 ng/μL of poly(C). Following a 15-min incubation at 30 °C, reactions were stopped with EDTA at final concentration 5 mM and dsRNA was quantified using the PicoGreen reagent. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares and NoV RdRp activity is shown in black as circles. The results from a representative experiment are shown with average values and standard deviations from triplicate reactions for each measurement point.
Figure 6
Figure 6
NIC02 inhibits de novo polymerase activity of rabbit calicivirus RdRps in vitro. The inhibitory effects of NIC02 on the de novo activity of (a) RHDV; and (b) RCV RdRps were analysed by monitoring the formation of dsRNA from a single-stranded poly(C) homopolymeric template. NIC02 concentrations were varied between 0.1–100 μM and compared to the relative activity in mock-treated samples containing the vehicle (0.5% v/v DMSO) only. Log (inhibitor concentration) vs. response curves were plotted. RHDV RdRp activity is shown in red as triangles, RCV RdRp activity is shown in blue as squares. Averages of relative fluorescence levels were calculated and plotted with standard deviations. Results from three independent experiments with triplicate reactions for each measurement point are shown.
Figure 7
Figure 7
NIC02 inhibits primed elongation activity of rabbit calicivirus RdRps in vitro. A gel-based RdRp activity assay was used to evaluate effects of NIC02 on the primed elongation activity of RHDV and RCV RdRps. (a) PE46-C template. (b) Products generated in the in the presence of 19.9 (IC50) and 99.5 µM (5× IC50) NIC02 for RHDV RdRp, 13.5 (IC50) and 67.5 µM (5× IC50) NIC02 for RCV RdRp or in the presence of the vehicle (0.5% v/v DMSO) alone (no inhibitor). Products were separated on a 15% denaturing polyacrylamide gel and stained with SYBR green II. Inhibition of the RdRp of Φ6 bacteriophage was carried out using 200 µM of the nucleoside analogue chain terminator 3′dGTP as an inhibition control.
Figure 8
Figure 8
Limited inhibitory effects of NIC10, NIC12 and NIC12 derivatives on de novo polymerase activity of rabbit calicivirus RdRps in vitro. The effects of (a) NIC10; (b) NIC12 and the NIC12 derivatives; (c) NIC12-2; (d) NIC12-3; (e) NIC12-4; and (f) NIC-12-5 on the de novo activity of RHDV and RCV RdRps were analysed by monitoring the formation of dsRNA. The chain terminator 3′dGTP (10 µM) was used as a positive control (a,b). RHDV RdRp activity is shown in red, RCV RdRp activity is shown in blue. The results are averages of relative fluorescence levels determined from three independent experiments with triplicate reactions for each measurement point plotted with standard deviations.
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