Plasmid construction
To prepare the wild-type (WT) NS3 and its mutated proteins, two sets of NS3 constructs containing single or simultaneous substitution in the four basic residues (R50, K74 and K94 in protease domain and H24 in NS4APCS) in the positively charged surface patch to alanine or aspartic acid (Fig. 1A) were generated by using a NS3 expression plasmid (pET28a-NS3S163A/NS4APCS) as a template via QuickChange mutagenesis method [49, 50]. The variants generated by mutating the basic residue to alanine (A) or aspartic acid (D) were clustered into Set 1 or Set 2, respectively. When the four basic residues were simultaneously substituted with alanine or aspartic acid, the variant was correspondingly named as Quad_A (Quadruple_A) or Quad_D (Quadruple_D). A helicase-only construct (NS3Hel, residues 204 to 683) and the variant harboring K232A mutation in NS3 to abolish its ATPase and helicase activities were used as a negative control. We also introduced the mutations of two sets into the full-length CSFV infectious clone [20] as previously described [45] to investigate the effect of the basic residue substitution on infectious virus production. All variants were confirmed by sequencing.
Protein expression and purification
The expression and purification of NS3S163A/NS4APCS and its variants were performed as described previously [22, 49]. Briefly, the expression plasmids were transformed into E. coli BL21-CodonPlus (DE3)-RIL strain and then the bacteria were cultured at 37°C in terrific broth (TB) medium containing 50 µg/ml of kanamycin and 25 µg/ml of chloramphenicol. When the optical density at 600 nm (OD600) of the culture reached 0.8, Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM. After an additional incubation at 25°C for 4 h, the cells were harvested for subsequent experiments.
The harvested cells were resuspended in a lysis buffer (150 mM Na2SO4, 50 mM Tris [pH 8.0], 10 mM imidazole, 0.02% [wt/vol] NaN3, 20% [vol/vol] glycerol) and then lysed by an AH-2010 homogenizer (ATS Engineering Ltd.) at 14,500 lb/in2. After centrifugation, the clarified lysate was loaded onto a HisTrap HP column (GE Healthcare) and the target protein was eluted with an elution buffer (300 mM imidazole, 50 mM Tris [pH 8.0], 150 mM Na2SO4,20% [vol/vol] glycerol and 0.02% [wt/vol] NaN3). The protein fractions were pooled, concentrated and run over a ENrich SEC 650 (Bio-Rad) equilibrated with 150 mM NaCl, 5 mM Tris 7.5, 10% (vol/vol) glycerol and 0.02% (wt/vol) NaN3. The concentrated protein was flash frozen in liquid nitrogen and stored as aliquots at -80°C. The protein concentration was determined by the Bradford method [6, 35].
ATPase assay
The ATPase activity was measured using a malachite green-based method as previously described [39, 43, 49]. Briefly, the 90-μl reaction mixture except for the ATP substrate was incubated at 37°C for 5 min. The reaction was initiated by addition of a 10-μl ATP solution to yield a final reaction mixture containing 10 nM NS3, 50 mM Tris (pH 7.5), 2.5 mM MgCl2, 50 mM NaCl, 5 to 500 μM ATP. After an additional incubation at 37°C for 15 min, the malachite green mixture (water/0.081% [wt/vol] malachite green/5.7% [wt/vol] ammonium molybdate in 6 M HCl ratio =3:2:1 [vol: vol: vol]) was added and the absorbance was immediately measured at 630 nm on a Multiskan MK3 microplate reader (Thermo Fisher Scientific). Initial ATPase catalytic rates were determined based on the slope of the initial absorbance change and the reference standard curve of absorbance versus phosphate concentration determined independently. The observed ATP hydrolysis rates at various ATP concentrations were fitted to Michaelis-Menten kinetics to yield the ATPase parameters (KMapp and kcat).
Helicase assay
A helicase unwinding pssRNA substrate T40:R20 was prepared by annealing the template strand (T40, 5′-GGGCCAAUCAUGCAUACGAGAAUGAACUAACCU-CGUAUAC-3′) and the release strand (R20, 5′-UAUCUCGUAUGCAUGAUUGG-3′) labeled with a 3′-6-TAMRASE (6-carboxy-tetramethylrhodamine N-succinimidyl ester) [49]. A typical 20-μl unwinding reaction mixture contains 8 U of RNasin (RiboLock; Thermo Scientific), 50 mM morpholinepropanesulfonic acid (MOPS)-NaOH (pH 7.0), 5 mM ATP, 2.5 mM MgCl2, 1 mM dithiothreitol (DTT), 0.5% Tween 20, 0.1 mg/ml of bovine serum albumin, 10 nM T40:R20 (according to the concentration of the R20), 100 nM unlabeled release strand (competitive strand) and 25 nM NS3 (protein/pssRNA molar ratio = 2.5:1). The unwinding reaction proceeded at 37°C for 30 min and was then terminated by addition of 2.2 µl of a 10× loading buffer (50 mM Tris [pH 7.5], 50 mM EDTA, 1% [wt/vol] SDS, 50% [vol/vol] glycerol, 0.1% [wt/vol] xylene cyanol). RNAs in the quenched reaction mixtures were resolved by 12% nondenaturing polyacrylamide gel electrophoresis. The fluorescent signal of 6-TAMRASE-labeled R20 was detected using a Molecular Imager PharosFX™ Plus System (Bio-Rad) with an excitation wavelength of 532 nm and a 605 nm emission filter. The band intensities were quantified by ImageJ (http://imagej.nih.gov/ij) and the unwound percentages were calculated based on the intensity fraction of the released R20. Each unwinding reaction was independently performed four times.
Fluorescence polarization (FP) -based RNA binding assay
To determine the RNA-binding ability of NS3 and its variants, we designed the pssRNA-2 by annealing the T40 (mentioned above) and a 33-mer release strand (R33, 5′-UCCACCAAUCAAGUA-UCUCGUAUGCAUGAUUGG-3′) labeled with a 5′-FAM for RNA binding assay. A FP-based RNA binding assay was used to estimate the affinity of each protein for RNA as previously described [28, 31]. Reactions were performed in a 96-well plate (Corning; Flat Bottom, Non-Binding Surface, Black Polystyrene). For each reaction, a total 72-μl reaction buffer mixture was incubated at 37°C for 5 min and the reaction was initiated by adding NS3 to make the final reaction mixture containing 10 nM 5′-FAM labeled pssRNA-2, 50 mM MOPS-NaOH (pH 7.0), 2.5 mM MgCl2, 50 mM NaCl, 1 to 1000 nM NS3 (the molar ratio of NS3:pssRNA-2 is 0.1:1 to 100:1). After incubation at 37°C for 30 min, polarization was monitored using a Cytation 3 Cell Imaging Multi-Mode Reader (Bio Tek) by exciting at 485 nm (20 nm bandwidth) and measuring total fluorescence intensity, parallel and perpendicular polarized light at 528 nm (20 nm bandwidth). G-factor (the instrument calibration factor) was calculated from wells with 10 nM pssRNA-2 alone. The data at different NS3 concentrations ([S]) were fitted to the quadratic equation: f = A´{(Kd+[S]+10)/2-sqrt[(Kd+[S]+10)2/4-[S]´10]}, where the “A” represents the amplitude of the FP value change and 10 is the concentration of pssRNA-2 (10 nM) and Kd is the dissociation constant of the NS3-RNA binding complex.
Virus rescue and titration
The virus was rescued as previously described [20, 45, 46]. Briefly, 2 μg of WT CSFV cDNA clone and its variants containing two sets of mutations were transfected into PK-15 cells using Lipofectamine 3000 (Invitrogen), respectively. After incubation at 37°C for 72 h, the virus production was monitored by indirect immunofluorescence assay (IFA) using anti-NS3 rabbit polyclonal antibody as primary antibody [20] and an Alexa Fluor 488-conjugated secondary antibody (goat anti-rabbit IgG, Invitrogen). The cultured supernatant was harvested and clarified by centrifugation for virus titration. The virus titers were calculated by IF staining [20] with anti-NS3 antibody in 96-well plates using the Reed-Muench method [32] and expressed as tissue culture infectious doses (50% endpoint, TCID50) per milliliter.
RT-qPCR
Viral RNA copy numbers were determined using a reverse transcription-quantitative PCR (RT-qPCR) [19, 30]. PK-15 cell monolayers in 24-well plates were infected with the virus at an MOI of 0.001. Total RNA was extracted from the infected cells at 6, 12 and 24 hpi using a TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa) and 500 ng of total RNA was reverse-transcribed using a ReverTra Ace qPCR RT Kit (TaKaRa) with a specific primer (5′-TAGCCTAATAGTGGGCCTCTG -3′). Then the cDNA transcribed from 50 ng of total RNA was analyzed by qPCR using a THUNDERBIRD Probe qPCR Mix kit (TaKaRa) with a 5′-FAM labeled probe (5'-TCAGGTCGTACTCCCATCACGTGGTGTGA-3') and the primers CSF-F174 (5′-ACAGGACAGTCGTCAGTAGTTC-3′)/CSF-R345 (5′-TAGCCTAATAGTGGGCCTCTG-3′). The quantitative cDNA containing the 5'UTR of CSFV genome was used as a standard for qPCR. The cycling parameters consisted of denaturation at 95°C for 60 s, followed by 40 amplification cycles (95°C for 15 s and 60°C for 60 s) and the fluorescent signals were detected by a BioRad CFX connect Real-time PCR detection System. The RNA copy numbers (log10 copies/μg) were calculated from 3 independent experiments.
Statistical analysis
Statistical analysis of the data was performed using the Student’s t test. A p-value of less than 0.05 was considered significant.