Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

NOTE: The JFH1 strain (WT) used here was a kind gift from Takaji Wakita, National Institute of Infectious Diseases. The human hepatoma cell line, Huh7.5, was a kind gift from Charles Rice, The Rockefeller University. A schematic of the method is shown in Figure 1.

1. Genome-wide substitution mutagenesis of JFH1 using error-prone PCR

1. To perform ep-PCR, prepare the master mix for four sets of experiments with primers J-For 5'-GTTTTCCCAGTCAGCACGTTGTAAAACGACGGC-3' and J-Rev 5'-CATGATCTGCAGAGAGACCAGTTACGGCACTCTC -3' (Figure 1A), along with the reaction components described in Table 1 without the pJFH1 template (plasmid isolated from JFH1 strain).
2. Aliquot the master mix into four tubes, add 100 ng, 50 ng, 25 ng, and 10 ng of template into the individual tubes, and adjust the total volume of the reaction to 50 µL. Apply the cycling conditions described in Table 2 to amplify a 9736 base pair (bp) fragment comprising the T7 promoter and full-length HCV genome (Figure 2).
   NOTE: The ep-PCR product must contain the T7 promoter sequence to facilitate in vitro transcription of the viral genome. Therefore, the forward primer must be located upstream of the T7 promoter of the viral cDNA clone, and the reverse primer sequence should end at the 3'-end of the virus genome to facilitate transcription run-off. Optimize each component of the ep-PCR reaction within the range recommended by the manufacturer of Taq DNA polymerase to achieve high-yield amplification. In addition to varied template amounts, unbalanced dNTPs (especially low dATP and/or dGTP concentration) can also be used to increase diversity in the length of viral genomes from 6-8 kb long³.
3. Estimate the ep-PCR product amplified by loading equal volumes of the PCR products (5 µL) and comparing to known amounts of 1 kilobase (kb) DNA ladder by running a 0.8% Tris-acetate-EDTA (TAE)-based agarose gel electrophoresis¹¹. Purify the product using a column purification kit as directed by the manufacturer.
4. Estimate the concentration of the purified product by measuring the absorbance at 260 nm using a microvolume spectrophotometer¹². Vacuum concentrate the ep-PCR product if needed to obtain a product concentration ≥100 ng/µL.
   ​NOTE: More accurate estimation can be made by loading two-fold serial dilutions of ep-PCR product and comparing their intensities to the known amounts of 1 kb DNA ladder.

2. Viral RNA synthesis

1. Set up a 40 µL reaction to digest 5 µg of clonal-pJFH1 with 4 U of XbaI enzyme at 37 °C for 2 h, followed by column purification and measuring the absorbance at 260 nm in a microvolume spectrophotometer¹². Set up a 20 µL in vitro transcription reaction of the column-purified ep-PCR product or clonal-pJFH1 (WT) using a commercially available large-scale RNA production system according to the manufacturer's instructions.
2. In a 200 µL microcentrifuge tube, mix approximately 1 µg of the purified ep-PCR product (mutant libraries) or XbaI digested clonal-pJFH1, 10 µL of 2x buffer containing ribonucleotides, and 2 µL of T7 enzyme mix. Mix all the components and briefly spin down the components. Incubate the reaction mixture at 37 °C for 45 min, followed by the addition of 1 U RNase-free DNase enzyme and incubation at 37 °C for 30 min (Figure 1C).
3. Purify in vitro synthesized RNA using an RNA cleanup kit as per the manufacturer's instructions, elute in 40 µL of RNase- and DNase-free water, and estimate the concentration of the purified RNA by measuring the absorbance at 260 nm using a microvolume spectrophotometer¹². Make aliquots as needed (5 µg or 2 µg) to avoid freeze-thaw and store them at −80 °C. Verify the integrity and size of the viral transcripts by using 2 µg of RNA for a MOPS formaldehyde 0.8% agarose gel electrophoresis¹³ (Figure 3).

3. Estimation of the proportion of mutations in ep-PCR products (mutant libraries)

NOTE: In this step, the proportion of nucleotides mutated by subcloning the product obtained in step 2. was estimated to demonstrate the advantage of employing ep-PCR to create genetic heterogeneity using two full genome mutant libraries (ML50 and ML25) and clonal pJFH1-derived viral RNAs. The proportion of mutations was estimated in the HCV NS5A gene, which was also the phenotypic readout gene (drug resistance) in this study.

1. Set up a 20 µL cDNA synthesis reaction by adding approximately 1 µg of the viral RNA synthesized in step 2., 5 µM reverse primer 5'-GTGTACCTAGTGTGTGCCGCTCTA-3', and 200 U reverse transcriptase as per the manufacturer's recommendations.
2. Using the cDNA, amplify a 2571 bp fragment that comprises the complete NS5A gene. To do this, prepare a reaction mixture containing 0.5 µM for 5272F and 7848R primers (final concentration; Table 3), 1.5 mM MgCl₂, 1x PCR buffer, and 1 U high-fidelity Taq polymerase in a total volume of 50 µL and run an amplification cycle using the conditions described in Table 4.
3. Run the product on a 0.8% TAE-based agarose gel to confirm the product size of 2571 bp and then purify the product using a column purification kit as directed by the manufacturer. Elute the column-purified product in 40 µL of sterile water.
4. To add a 3' A overhang to the product, add 0.5 µM dATP and 1 U of low-fidelity Taq DNA polymerase and incubate the entire PCR product at 70 °C for 30 min along with 1x PCR buffer and 1.5 mM MgCl₂ (final concentration). Purify the mixture using a column purification kit as directed by the manufacturer
   NOTE: Alternatively, excise the ~9.7 kb long product from the gel from step 1.4. and perform cloning using a commercially available kit for cloning of long-PCR product according to the manufacturer's instructions or perform NGS of the ep-PCR product using an Illumina platform².
5. Set up a ligation reaction by adding 5 µL of 2x ligation buffer, 50 ng of the T-vector DNA, approximately 3-fold molar excess of the insert DNA synthesized in step 3.4., 3 Weiss units of T4 DNA ligase, and nuclease-free water to a total volume of 10 µL. Incubate this reaction at room temperature for 3 h and then add 100 µL of Escherichia coli DH5α with the ligated DNA; heat shock the cells at 42 °C for 35 s (Figure 1B).
6. Add 1 mL of Luria-Bertani (LB) medium (without antibiotic) and incubate with gentle shaking for 1 h at 37 °C for recovery. Centrifuge the cell suspension at 13,800 x g, discard the supernatant, and resuspend in 200 µL of fresh LB medium.
7. Plate 100 µL of the transformed E. coli DH5α cells on an LB plate containing 50 µg/mL ampicillin and incubate at 37 °C for 16 h. Set up minipreps of 25-30 colonies in 5 mL of LB medium + 50 µg/mL ampicillin and grow overnight at 37 °C.
8. The next day, extract the plasmids with a plasmid purification kit according to the manufacturer's instructions and perform restriction enzyme digestion in 10 µL volume with 200 ng of the isolated plasmids, 2 U of EcoR1, and 1x restriction buffer for all the colonies.
9. After incubating the digestion mixtures at 37 °C for 3 h, resolve the products on 0.8% TAE-based agarose gel to confirm plasmid DNA insertion.
10. Perform Sanger sequencing of the plasmids of 25 positive clones using M13 Forward, M13 reverse, 6208-SPF, and 6748-SPF primers (Table 3) to determine the proportion of mutations (expressed as the proportion of nucleotides mutated) in the viral RNAs derived from the libraries and clonal pJFH1 (Figure 1B and Figure 4).

4. Viral RNA transfection of the Huh7.5 cell line

NOTE: Use RNase/DNase-free tissue culture materials and work in a sterile class II biosafety cabinet. Work in the recommended containment facility as per the biosafety guidelines of the organization.

1. Maintain Huh7.5 cells by incubating in a 5% CO₂ incubator at 37 °C in a T75 cm² tissue culture flask containing 15 mL of complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL; Figure 1D).
2. Split the cells in a 1:3 ratio when confluency reaches 90%. Wash the cells with 1x prewarmed phosphate-buffered saline (PBS; pH 7.4). Add 5 mL of 1x trypsin-EDTA solution to completely cover the cell layer, gently swirl the flask, and then incubate the flask at 37 °C for 5 min.
   NOTE: Incubation time may vary; incubate the cells until the cell layer is completely detached from the cell culture flask surface.
3. Add 10 mL of 1x prewarmed complete DMEM, disperse the cells by repeat pipetting, and collect the cell suspension in a 15 mL or 50 mL sterile centrifuge tube. Centrifuge the cell suspension at 252 x g for 5 min at room temperature. Discard the supernatant, resuspend the cell pellet in 6 mL of complete DMEM, and incubate the cells at 37 °C in a humidified incubator with 5% CO₂.
4. For continuous maintenance of naïve, transfected, or virus-infected Huh7.5 cells, repeat step 4.2. and step 4.3.
5. 1 day prior to transfection, split the cells as described in steps 4.1.-4.3., count the number of viable cells using a hemocytometer, seed the Huh7.5 cells at a density of 0.6 x 10⁶ in 35 mm dishes in 2 mL of complete DMEM, and incubate the cells at 37 °C in a humidified incubator with 5% CO₂.
6. On the following day, prepare the transcript-lipid complex. To do this, dilute 10 µL of transfection reagent in 50 µL of minimal essential medium, and separately dilute 5 µg of viral transcripts in 50 µL of minimal essential medium.
7. Incubate both mixtures at room temperature for 10 min, and then mix them in a single sterile microcentrifuge tube and further incubate the mixture at room temperature for 30 min to form the transcript-lipid complex.
8. After 16 h of cell incubation, remove the culture medium from the culture dish, wash the cells 2x with prewarmed 1x PBS, and add 1.5 mL of minimal essential medium. Slowly add the transcript-lipid complex to the dish and gently swirl with hands to ensure uniform distribution of the complexes.
9. Incubate the cells in a 5% CO₂ incubator at 37 °C for 10 h. Then, remove the medium, wash the transfected cells 2x with 1 mL of prewarmed 1x PBS, and add 2 mL of complete DMEM.

5. Virus production

1. Split the transfected Huh7.5 cells every 2 days or 3 days when they reach 90% confluency. Collect virus supernatants at every split and store them at −80 °C for further analysis.
2. At every split, monitor the spread of the virus using a focus forming assay (FFA) as described in step 6.2. Harvest the virus until the virus spread reaches >80% of transfected cells (Figure 1D).
   NOTE: Split the transfected cells at 1:1 ratio for the first few passages to rescue the maximum number of viral variants. Virus spread slows and viability decreases with increasing proportions of mutations in the full-genome MLs².

6. Quantification of virus titers

1. Quantification of viral RNA
   1. Isolate viral RNA from 140 µL of culture supernatants using a viral RNA isolation kit according to the manufacturer's instructions.
   2. Set up a 10 µL qRT-PCR reaction using a commercial qRT-PCR kit according to the manufacturer's instructions. Use the forward primer R6-130-S17, reverse primer R6-290-R19 (final concentration 0.2 µM each), and probe R9-148-S21FT (final concentration 0.3 µM) for HCV RNA quantification (Table 3). Set the run conditions as 48 °C for 20 min, 95 °C for 10 min, and then 45 cycles of 95 °C for 15 s and 60 °C for 1 min.
      NOTE: The probe should contain the fluorescent reporter dye 6-carboxyfluorescein (FAM) and the quencher dye 6-carboxy-tetramethyl-rhodamine (TAMRA) at the 5' and 3' ends, respectively.
   3. Run reactions in a real-time PCR machine. Setup negative controls, i.e., RNA extracted from the supernatants of mock-infected cells and nuclease-free water simultaneously to ensure the absence of cross-contamination.
   4. In parallel, generate a standard curve using a 10-fold serially diluted known copy number of HCV transcripts (1 x 10⁸ to 0) for the quantification of viral RNA. Perform the quantification in triplicate (Figure 1D).
2. Infectious virus titer quantification using 50% tissue culture infectious dose (TCID₅₀)
   1. Approximately 16 h prior to adding the virus, plate 6.5 x 10³ Huh7.5 cells/well in a 96-well plate and incubate the cells at 37 °C in a 5% CO₂ incubator.
   2. In a biosafety class II cabinet, perform 10-fold serial dilutions (1 mL each) covering 1 x 10⁻¹ to 1 x 10⁻⁶ dilutions (eight dilutions) of the harvested virus (obtained when the virus spread reaches >80% of cells as described in step 5.). Add 100 µL/well (with eight replicates) of the diluted virus to infect the Huh7.5 cells and keep the plate in an incubator at 37 °C with 5% CO₂ for 3 days.
   3. After 3 days, wash the infected cells 3x with 0.1 mL of PBS each time, and fix and permeabilize the cells with 0.1 mL of ice-cold methanol at −20 °C for 20 min. Wash the wells with 1x PBS 3x and then 1x with PBS-T (1x PBS/0.1% [v/v] Tween-20).
   4. After removing the PBS-T, block the cells for 30 min at room temperature with 0.1 mL of 1% bovine serum albumin (BSA) containing 0.2% skimmed milk in PBST. Remove the blocking solution and treat the cells for 5 min with 0.1 mL of 3% H₂O₂ prepared in 1x PBS.
   5. Again, wash the cells 2x with 1x PBS and 1x with PBS-T, then add 50 µL/well of anti-NS5A 9E10 mAb (1:10000, stock 1 mg/mL; a kind gift from Charles Rice, The Rockefeller University), and incubate at room temperature for 1 h. Wash the wells again 3x with 1x PBS and 1x with PBS-T.
   6. Add 50 µL/well of HRP-conjugated goat anti-mouse secondary IgG (1:4000), incubate for 30 min at room temperature, and then remove the unbound antibody by washing the wells with 0.1 mL of 1x PBS.
   7. Add 30 µL of DAB (diaminobenzidine tetrahydrochloride) color substrate and incubate the plate with gentle rocking for 10 min at room temperature. The substrate develops a brown precipitate on the well surface that indicates HCV NS5A antigen.Remove the DAB solution and wash the wells 2x with 1x PBS and 1x with distilled water. Add 100 µL of PBS containing 0.03% sodium azide.
   8. Examine each well under an inverted light microscope using a 10x objective. Count the number of positive wells. If the well contains one or more NS5A-positive cells, then it is positive; if the well shows an absence of NS5A-positive cells, then it is negative. Use a Reed and Muench calculator to estimate the endpoint dilution that infects 50% of the wells (TCID₅₀)^14,15. A reciprocal of the dilution required to yield the TCID₅₀ is the virus infectivity titer (foci forming units) per unit volume.
      ​NOTE: Virus infectivity titers vary for different mutant libraries.

7. Drug-resistant viral variant selection

1. Dissolve pibrentasvir (PIB), an NS5A inhibitor, in 100% DMSO to a concentration of 1 mM and further dilute it in complete DMEM to a concentration of 10 nM.
2. To determine the 50% effective concentration of PIB, seed Huh7.5 cells at a density of 0.6 x 10⁶/well in a 6-well plate and incubate the cells at 37 °C in a 5% CO₂ incubator. After 16 h of cell incubation, add a virus dose of either ML50 or clonal JFH1 virus to infect 50% of cells, and then, at 12 h post-infection (h p.i.), treat the cells with a two-fold dilution series of PIB ranging from 0.5-100 pM. Measure the FFA and quantify FFU as in step 6.2. after 3 days of incubation.
3. Plot the dose-response curves using FFU-reduction assay values in statistical analysis software. From the sigmoidal curve, obtain the 50% effective concentration (EC₅₀; Figure 5).
   NOTE: The effective concentration range may vary depending on the class, between HCV antivirals of the same class, and depending on the mutant library.
4. For the experiment, infect naïve Huh7.5 cells at 70% confluence with an ML50 virus (variants derived from ML50 RNA) dose to infect 50% of the cells for 12 h, and then transfer the infected cells onto 6-well plates 24 h post-infection.
5. Add 1x EC₅₀ PIB (47.3 pM) to the infected cells after 16 h of cell split. Do this after every cell split for six consecutive passages (18 days), followed by three drug-free passage cycles, and monitor virus spread using FFAs as described in step 6.2. Scale-up FFA reagents as per the growth area. Harvest viral supernatants at each passage and store them at −80 °C until use.
   NOTE: Viral spread to greater than 50% of cells during treatment follow-up may be defined as a breakthrough, and viral spread during the drug withdrawal period may be defined as viral relapse¹⁶.
6. Extract viral RNA from the supernatant on day 18, as described in step 6.1.1., and then synthesize cDNA, as shown in step 3.1. Amplify the NS5A gene using 5 µL of 1:5 diluted cDNA and the PCR components described in step 3.2. Then, follow steps 3.3.-3.5. to determine NS5A drug resistance mutations in 6-8 positive bacterial plasmids using the NS5A sequencing primers 6186F, 6862F, and 6460R (Table 3 and Table 4).
   NOTE: Here in this study, we reported substitutions in NS5A of variants selected during PIB treatment at 1x EC₅₀. This will rule out the contribution of substitutions other than NS5A in reduced susceptibility to PIB.