Purification of Dengue and Zika Virus Non-structural Protein 5 for Crystallization and Screening of Antivirals

Abstract

Dengue Virus (DENV) and ZIKA Virus (ZIKV) are two important human pathogens that belong to the Flavivirus genus of positive strand RNA viruses. Symptoms of DENV infections range from asymptomatic or mild fever to life-threatening forms, while ZIKV can lead to teratogenic effects such as microcephaly in newborns and neurological disease like the Guillain–Barré syndrome.

Non-Structural Protein 5 (NS5) is the largest and most conserved enzyme across flaviviruses and hence constitutes a prime target for developing pan-flavivirus antiviral inhibitors. NS5 results from the gene fusion between a methyltransferase at the N-terminus of the protein and an RNA-dependent RNA polymerase (RdRp) at the C-terminal end. The NS5 protein plays key roles in replication and modification of viral RNA and its inhibition by potent antiviral drugs could prevent severe symptoms associated with infections.

We have optimized purification and crystallization protocols to obtain active recombinant proteins suitable for structure-based drug discovery for both the full-length NS5 protein and the polymerase domain of NS5 from DENV and ZIKV .

1 Introduction

Dengue virus (DENV) and Zika virus (ZIKV) are closely related mosquito-borne flaviviruses that belong to the Flaviviridae family. Four distinct DENV serotypes exist and are named DENV1–4. Among the seven non-structural proteins that are expressed solely during the intracellular phase of infection, NS5 plays a crucial role both for viral RNA synthesis and viral mRNA capping [1]. NS5 is well characterized and represents the most evolutionary conserved protein across various flaviviruses. NS5 comprises an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. The absence of a similar activity in the host cell and the major role that NS5 plays during viral replication makes NS5 an ideal target for drug development and antivirals discovery.

The NS5 protein from DENV3 and ZIKV share 66% of sequence identity and a similar 3D structure as revealed by X-ray crystallography. Several crystal structures of DENV and ZIKV have been reported in the recent years, for the catalytic domains [2,3,4,5] or for the NS5 full length [6,7,8,9]. The RdRp catalytic domain adopts a right-hand conformation consisting of three subdomains from N- to C-terminus: finger, palm, and thumb. The RdRp protein architecture is endowed with high flexibility in some functionally important segments of the protein, that appear disordered in the preinitiation structure that has been captured in the available crystal structures [4]. Following a tedious optimization process, the procedure to grow well-diffracting crystals of DENV3 RdRp was optimized allowing the determination of high-resolution crystal structures in the presence of inhibitors. However, several regions of NS5 RdRp remain poorly ordered in the available structures. These regions are likely to become ordered in the presence of incoming nucleotides and an RNA template.

There are currently no specific antiviral drugs available to treat DENV or ZIKV infections. Two different classes of RdRp inhibitors may be developed: nucleoside inhibitors (NIs) and non-nucleoside inhibitors (NNIs). The latter bind the polymerase allosteric pockets. Major efforts were made towards the discovery of NNIs targeting DENV or ZIKV RdRps leading recently to the characterization of two RNA tunnel inhibitors for DENV [3] and a series of derivatives targeting the same allosteric pocket on DENV and ZIKV [2, 10].

As a reliable structure is essential for structure-based drug design of potent new inhibitors, we developed a robust purification protocol leading to reproducible crystallization of successive batches of DENV and ZIKV NS5 proteins.

Herein, we describe a multi-step approach to obtain well-diffracting crystals of the full-length NS5 protein (or the RdRp ) from DENV2 and DENV3 as well as the RdRp domain from Zika virus. From a screening perspective these protocols are suitable for soaking of antivirals (Fig. 1).

Fig. 1

From gene to protein crystal structure (Created with BioRender.com)

2 Materials

2.1 Reagents

• 1. E. coli BL21 T1R.

• 2. LB (Luria-Bertani) medium: Tryptone (10 g/L), NaCl (10 g/L), Yeast extract (5 g/L).

• 3. LB agar medium: LB broth containing 1% agar powder and the desired selection antibiotic for LB agar plates preparation.

• 4. Protease inhibitor cocktail.

• 5. TEV (tobacco etch virus)/Thrombin protease (depending on the cleavage site).

• 6. SDS-PAGE gels, electrophoresis apparatus, and running buffer.

2.2 Plasmids

• 1. ZIKV-NS5 RdRp (French Polynesia strain, GenBank accession number ARU07075.1) cDNA cloned into a pNIC-Bsa4 vector with a hexa-histidine tag and a TEV (tobacco etch virus) protease cleavage site, fused at the N-terminus.

• 2. D2-NGC-NS5 FL (GenBank Accession Number AF038403.1) cDNA cloned into a pET28a vector with a hexa-histidine tag and a TEV protease cleavage site.

• 3. DENV-3 RdRp (amino acid residues 272 to 900 of DENV3 NS5) (GenBank accession number AY662691) cDNA cloned into a pNET15b vector.

• 4. DENV-3 FL (amino acid residues 6 to 895 of DENV3 NS5) (GenBank accession number AY662691) cDNA is cloned into a pNIC28-Bsa4 vector.

2.3 Buffers

Buffers are summarized in Table 1. Here are the buffers used in the following protocols.

• 1. Buffer A: 20 mM Tris–HCl pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM DTT, 10% glycerol.

• 2. Buffer B: 20 mM Tris–HCl pH 7.5, 500 mM NaCl, 400 mM imidazole, 1 mM DTT.

• 3. Buffer C: 20 mM Tris–HCl pH 7.5, 200 mM NaCl, 1 mM DTT, and 10% glycerol.

• 4. Buffer D: 20 mM Tris–HCl pH 7.5, 1 mM DTT, 10% glycerol.

• 5. Buffer E: 20 mM Tris–HCl pH 7.5, 1 M NaCl, 1 mM DTT, 10% glycerol.

• 6. Buffer F: 20 mM Na HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP, 10% glycerol.

• 7. Buffer G: 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 10 mM β-mercaptoethanol, and 10% glycerol.

• 8. Buffer H: 50 mM morpholineethanesulfonic acid (MES), pH 6.5, 0.3 M NaCl, 1 mM EDTA, and 5 mM β-mercaptoethanol.

• 9. Buffer I: 50 mM MES, pH 6.2, 50 mM NaCl, 5 mM β-mercaptoethanol, 1 mM EDTA.

• 10. Buffer J: 20 mM Tris–HCl at pH 6.8, 0.25 M NaCl, 1 mM EDTA, 2 mM β-mercaptoethanol, and 0.1% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS).

• 11. Buffer K: 20 mM Na HEPES at pH 7.0, 300 mM NaCl, and 5 mM TCEP.

• 12. Buffer L: 20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, and 10% glycerol.

• 13. Buffer M: 20 mM Na HEPES pH 7.5, 300 mM NaCl, 5 mM DTT, 10% glycerol.

• 14. Crystallization condition A: 20% (w/v) PEG 6000 and 0.2 M Na HEPES pH 8 at 20 °C.

• 15. Crystallization condition B: 11–15% (wt/vol) polyethylene glycol 4000, 22% (vol/vol) glycerol, 0.1 M Tris–HCl at pH 8, and 0.1 M bicine at pH 9 at 4 °C.

• 16. Crystallization condition C: 20–25% PEG 550 monomethyl ether and 0.1 M Tris–HCl, pH 8.0.

Table 1 DENV and ZIKV proteins purification conditions

2.4 Instruments

• 1. Water bath with adjustable temperature.

• 2. Benchtop centrifuge.

• 3. Shaking incubator with adjustable temperature.

• 4. Sonicator.

• 5. Cellulose acetate filters, 0.2 μm.

• 6. HisTrap column.

• 7. Ni²⁺-NTA agarose beads.

• 8. Heparin-affinity chromatography column.

• 9. Hi Load S200 16/60 size exclusion chromatography (SEC ) column.

• 10. Biorad NGC chromatography system.

• 11. SnakeSkin Dialysis Tubing, 7 K MWCO.

• 12. Concentrator (Vivaspin turbo).

• 13. Amicon® Ultra Centrifugal Filters.

3 Methods

3.1 ZIKV NS5 RdRp and DENV2 NS5 FL

3.1.1 Expression and Protein Purification

• 1. Transform the desired plasmid into E. coli strain BL21λ(DE3) cells via heat shock. 100 μL of cells are incubated for 10 min on ice before addition to the plasmid followed with 1 pulse of 45 s at 42 °C in the water bath. Keep 10 min on ice before adding 300 μL of 1× LB media for the recovery step 1 h at 37 °C.

• 2. Spin the cells 2 min at 4000 × g. Resuspend the pellet in 100 μL of media and plate the transformed BL21λ(DE3) cells onto LB agar plate with 50 μg/mL of Kanamycin antibiotic. Incubate the plate overnight at 37 °C.

• 3. Pick one colony after incubation and resuspend in 10 mL of 1× LB media. Culture the cells at 37 °C under a 180 rpm shaking overnight.

• 4. Use 1 mL of the pre-culture suspension to inoculate 1 L of 1×X LB media and culture the cells at 37 °C under a 180 rpm shaking.

• 5. When an OD_600nm of 1.0 is reached (see Note 1), lower the temperature to 16 °C and culture the cells for 1 h prior to addition of 0.1 mM of IPTG to induce protein expression.

• 6. Culture the cells for 16 h under a 180 rpm constant shaking at 37 °C.

3.1.2 Cell Lysis

• 1. Harvest the cells by centrifugation at 4000 × g for 20 min at 4 °C.

• 2. Resuspend the pellet in 15 mL of buffer A per gram of wet cell mass (see Notes 2, 3 and Table 1).

• 3. Sonicate for 10 min at an amplitude of 40% with 5 s on/5 s off (see Note 4).

• 4. Harvest the cells by centrifugation for 1 h at 20,000 × g at 4 °C.

3.1.3 Metal Affinity Chromatography

The supernatant is purified via metal affinity chromatography using a HisTrap 5 mL column. The prepacked column is plugged into a Fast Protein Liquid Chromatography system. The column is equilibrated in 5 column volume of buffer A prior to sample application.

• 1. Wash the column using 5 column volumes of buffer A supplemented with 40 mM imidazole to eliminate unbound protein.

• 2. Use a linear gradient of 50–400 mM imidazole to elute the protein using buffers A and B.

• 3. Prepare an SDS-page polyacrylamide gel of 12%, collect and load protein fractions corresponding to the peak to confirm protein presence.

• 4. Pool the fractions containing the target protein (according to the size) into a SnakeSkin Dialysis Tubing and dialyze against the buffer C in the presence of TEV protease (see Note 5) to cleave the hexa-histidine tag. Carry out the dialysis overnight at 4 °C with continuous stirring.

• 5. Purify the cleaved protein using reverse metal affinity chromatography in order to remove TEV, cleaved hexa-histidine tag, and un-cleaved protein. To do so, incubate the protein with Ni²⁺-NTA agarose beads under agitation for 1 h at 4 °C.

• 6. Transfer the mixture into a gravity column. Collect the flow-through containing the cleaved protein of interest.

• 7. Check the content and purity of the fractions by SDS-PAGE (Figs. 2a and 3a).

Fig. 2

Protein Purification and Crystal of ZIKV NS5 RdRp . (a) SDS-PAGE analysis of ZIKAV NS5 RdRp protein at different purification stages. (b) ZIKV NS5 RdRp protein crystal

3.1.4 Heparin-Affinity Chromatography

• 1. Dilute the flow-through obtained in the previous step with buffer D to lower the NaCl concentration to ~50 mM and inject onto a heparin-affinity chromatography column (see Note 6).

• 2. Elute the protein using a gradient from 0% to 100% using buffers D and E.

• 3. Collect the fractions corresponding to the peak.

• 4. Check the content and purity of the fractions by SDS-PAGE (see Note 7, Figs. 2a and 3a).

3.1.5 Size Exclusion Chromatography

• 1. Pool the fractions obtained in the previous step and concentrate them to reach a volume of 2.5 mL.

• 2. Inject the sample onto a Hi Load S200 16/60 size exclusion chromatography (SEC ) column that had been pre-equilibrated with buffer F (see Note 8). Check the content and purity of the fractions corresponding to the peak by SDS-PAGE (Figs. 2a and 3a).

• 3. Pool the fractions containing the target protein and concentrate to 5 mg/mL using a concentrator prior to flash freezing at −80 °C (see Note 9).

3.1.6 Preparation of Protein Crystals

We identified crystallization conditions respectively for DENV2 NS5 and ZIKV NS5 RdRp [2, 7]. JCSG+ and Morpheus commercial kits (Molecular Dimensions) were screened. We identified crystals suitable for diffraction (see Notes 10, 11 and Table 2) for ZIKV RdRp and DENV2 FL in the crystallization conditions A and B, respectively.

• 1. Concentrate the purified protein at 5 mg/mL in buffer F.

• 2. Choose the suitable crystallization condition (see Table 2).

• 3. Use the sitting-drop vapor diffusion method to grow crystals. Mix 1 μL of protein solution with 1 μL of the reservoir solution.

• 4. Following crystal appearance (see Note 10, Figs. 2b and 3b), mount crystals with suitable size on a cryo-loop and flash freeze in liquid nitrogen.

Table 2 Growing crystal from protein conditions

3.1.7 Soaking of Compounds

• 1. For protein-compound complex structure determination, transfer protein crystals using a cryo-loop into a drop containing the crystallization solution supplemented with 1 mM of the desired compound (see Note 12).

• 2. Soak for 6 h prior to flash freezing in liquid nitrogen.

3.2 DENV3 NS5 RdRp

3.2.1 Expression and Protein Purification

• 1. Transform the plasmid into E. coli strain BL21 cells (RIL).

• 2. Grow the cells at 37 °C in LB medium containing 100 μg/mL ampicillin and 50 μg/mL chloramphenicol until the OD_600nm reaches 0.6 to 0.8.

• 3. Lower the temperature to 16 °C and add IPTG to a final concentration of 0.4 mM.

• 4. After overnight growth, harvest the cells by centrifugation at 8000 × g for 10 min at 4 °C.

3.2.2 Cell Lysis

• 1. Resuspend the cell pellet in buffer G supplemented with a tablet of EDTA-free protease inhibitor .

• 2. Sonicate for 10 min at an amplitude of 40% with 5 s on/5 s off (see Note 4).

• 3. Harvest the cells by centrifugation for 30 min at 30,000 × g at 4 °C.

3.2.3 Metal Affinity Chromatography

The supernatant is purified via metal affinity chromatography using a HisTrap HP column. The prepacked column is plugged into a fast protein liquid chromatography system. The column is equilibrated in 5 column volume of buffer G prior to sample application.

• 1. Wash the column using 5 column volume of buffer G supplemented with 25 mM and 125 mM imidazole.

• 2. Use a linear gradient of 125–500 mM imidazole to elute the protein.

• 3. Pool the fractions containing the protein and dialyze overnight against buffer H in the presence of thrombin to remove the hexa-histidine tag.

3.2.4 Cation-Exchange Chromatography

The cation-exchange column (15S) is plugged into a fast protein liquid chromatography system. The equilibration, sample application, and elution steps are done at a flow of 1 mL/min. The column is pre-equilibrated in buffer I for 5 column volume.

• 1. Use a cation-exchange chromatography (15S) column to further purify the protein.

• 2. Use a linear gradient of 0.05 to 1.5 M NaCl in buffer I for 20 column volumes.

• 3. Concentrate the pooled fractions by ultrafiltration using a 30 kDa cutoff (Amicon).

3.2.5 Size Exclusion Chromatography

• 1. Inject the sample onto a HiPrep 16/26, Superdex 200 size exclusion chromatography (SEC ) column that had been pre-equilibrated with buffer J for 1 column volume at 1 ml/min. The elution is run for 1 column volume at 1 mL/min. Protein is detected using UV reading at 280 nm. Purity is assessed by SDS-PAGE.

• 2. Concentrate the protein to ∼11 mg/mL in buffer K by ultrafiltration using a 30 kDa cutoff (Amicon).

3.2.6 Preparation of Protein Crystals

• 1. Use the hanging-drop vapor diffusion method at 18 °C to grow crystals. Mix 1 μL of protein solution with 1 μL of the crystallization condition C reservoir solution.

• 2. For cryoprotection, transfer the crystals to the crystallization solution supplemented with 10% glycerol.

3.2.7 Co-crystallization

• 1. Mix at 4 °C the DENV-3 RdRp in buffer K with the compound (see Table 2, from a stock solution; 10 mM in 100% DMSO) to reach final concentrations of ∼130 μM (RdRp ) and 1 mM of the compound, respectively.

• 2. The mixture is then used for co-crystallization using the optimized conditions described above.

3.3 DENV3 NS5 FL

3.3.1 Protein Expression

• 1. Transform the plasmid into E. coli strain BL21-CodonPluscells.

• 2. Grow the cells at 37 °C in LB medium containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol until the OD_600nm reaches 0.6 to 0.8.

• 3. Lower the temperature to 18 °C and add IPTG to a final concentration of 0.5 mM.

• 4. After 20 h culture, harvest the cells by centrifugation at 8000 × g for 10 min at 4 °C.

3.3.2 Cell Lysis for Protein Purification

• 1. Resuspend the cell pellet in 15 mL of buffer L per gram of wet cell mass.

• 2. Lyse the cells by sonication (same conditions than in Subheading 3.2.3).

• 3. Harvest the cells by centrifugation for 60 min at 20,000 × g at 4 °C.

3.3.3 Metal Affinity Chromatography

The supernatant is purified via metal affinity chromatography using a HisTrap HP column. The prepacked column is plugged into a fast protein liquid chromatography system. The column is equilibrated in 5 column volume of buffer L prior to sample application.

• 1. Wash the column using 5 column volume of buffer L supplemented with 40 mM imidazole.

• 2. Use a linear gradient of 40–500 mM imidazole to elute the protein for 20 column volumes.

• 3. Pool the fractions containing the protein and dialyze overnight at 4 °C against buffer M using a SnakeSkin Dialysis Tubing (7 kDa) in the presence of TEV (see Note 5) to remove the hexa-histidine tag.

3.3.4 Size Exclusion Chromatography

• 1. Inject the sample onto a HiPrep 16/26, Superdex 200 size exclusion chromatography (SEC ) column that had been pre-equilibrated with buffer M for 1 column volume at 1 ml/min. The elution is run for 1 column volume at 1 mL/min. Protein is detected using UV reading at 280 nm. Purity is assessed by SDS-PAGE.

• 2. Concentrate the protein to 10 mg/mL by ultrafiltration using a 30 kDa cutoff (Amicon) (Fig. 3).

Fig. 3

Protein Purification and Crystal of DENV2 NS5 FL. (a) SDS-PAGE analysis of DENV2 NS5 FL protein at different purification stages. (b) DENV2 NS5 FL protein crystal

3.3.5 Preparation of Protein Crystals

• 1. Use the sitting-drop vapor diffusion method at 20 °C to grow crystals. Mix 1 μL of protein solution at 4–6 mg/mL with 1 μL of the reservoir solution (0.2 M calcium acetate or magnesium acetate, 0.1 M sodium cacodylate, pH 6.4, 10–20% (w/v) PEG 8000).

• 2. For cryoprotection, soak the crystals in a cryoprotecting solution containing 20% glycerol.

4 Notes

• 1. We have noted that an OD_600nm of 0.6 might not be sufficient for an optimal expression. We observed that a higher OD_600nm of 1.0 is necessary to reach a sufficient level of protein expression. An OD_600nm of 1.0 is typically reached in 3–4 h.

• 2. Use cellulose 0.2 μm acetate filters to filter all the buffers before starting the purification procedure.

• 3. Use 15 mL of lysis buffer per 1 g of cell pellet to limit protein aggregation if so.

• 4. Sonication: The Becker containing the lysate is kept at 4 °C during the sonication. To do so, one can use a sonicator in a cold room or keep the lysate in an icebox during the process. The sonication can be paused and the ice surrounding the Becker topped-up in order to prevent the lysate to heat.

• 5. TEV protease: Use a substrate-to-enzyme ratio of 40:1, w/w.

• 6. The flow-through needs to be diluted before injection in Heparin column to reduce the salt concentration to 50 mM NaCl.

• 7. In order to obtain an extra pure recombinant protein suitable for crystallization, we advise to carry a 3-steps purification protocol. However, if the protein is aimed to be used in biochemical activity assays, we suggest skipping the heparin-affinity chromatography step to prevent loss of protein activity.

• 8. We noticed that replacing DTT with TCEP in the buffers of the last steps of purification (SEC buffer) prevent protein oxidation and minimize protein precipitation [5].

• 9. Flash freezing of the purified protein is important to prevent X-ray induced radiation damage to the protein crystal during data processing.

• 10. Crystals grow to their final dimension in 2–4 weeks at 4 °C for DENV2, and 20 °C for ZIKV .

• 11. Soaking: Several conditions should be tested for optimal soaking, time of soaking (from 6 to 24 h) as well as different concentrations of the compound (from 1 to 10 mM if the DMSO percentage allows it).

• 12. Using those conditions, it is possible to achieve highly reproducible crystals with high-resolution diffraction, ideal for structure-based drug design. In order to obtain structures in complex with active compounds against DENV or ZIKV respectively, apo crystals need to be soaked in the crystallization solution supplemented with the desired compound.