The protocol described here allows generation of randomly mutagenized full length RNA libraries of single strand positive RNA viral genomes up to 10 kilobytes in length, and selection of phenotypes of interest under desired experimental conditions. This technique can create full length viral RNA libraries with varying levels of genetic diversity in a short time using a cloning-free approach. The technique utilizes inexpensive and widely available reagents for the synthesis of libraries.
Demonstrating the procedure will be Shaheen Khan, a doctoral student from the Virology Section, Department of Life Sciences, Shiv Nadar University. To begin, perform ep-PCR by preparing the master mix for four sets of experiments with forward and reverse primers along with the reaction components without the pJFH1 template, as described in the text manuscript. Aliquot the master mix into four tubes.
Add 100 nanograms, 50 nanograms, 25 nanograms, and 10 nanograms of the template into the individual tubes, and adjust the total volume of the reaction to 50 microliters. Apply the cycling conditions to amplify a 97 36-base pair fragment. Estimate the ep-PCR product amplified by loading five microliters of the PCR products and comparing it to known amounts of one kilobase DNA ladder by running a 0.8%TAE-based agarose gel electrophoresis.
Purify the product using a column purification kit. Estimate the concentration of the purified product by measuring the absorbance at 260 nanometers. Vacuum concentrate the ep-PCR product to obtain a product concentration equal to, or greater than 100 nanograms per microliter.
Set up an in vitro RNA synthesis reaction, and place it at 37 degrees Celsius for incubation. Then purify the synthesized product using a column purification kit. Set up a 20 microliters cDNA synthesis reaction by adding approximately one microgram of the viral RNA, five micromolar reverse primer, and 200 units of reverse transcriptase, as per the manufacturer's recommendations.
Using the cDNA, prepare a reaction mixture as described in the text manuscript, and run an amplification cycle. Run the product on a 0.8%TAE-based agarose gel to confirm the product size of 25 71-base pairs, and then purify the product using a column purification kit as demonstrated earlier. Elute in 40 microliters of sterile water.
To add a three prime A overhang, add 0.5 micromolar DATP and one unit of low fidelity Taq DNA polymerase, and incubate the entire PCR product at 70 degrees Celsius for 30 minutes, along with 1X PCR buffer and 1.5 millimolar magnesium chloride. Then purify the mixture using a column purification kit. Set up the ligation reaction, and incubate it at room temperature for three hours.
Then add 100 microliters of Escherichia coli DH5-Alpha to the ligated DNA, and heat shock the cells at 42 degrees Celsius for 35 seconds. Add one milliliter of LB medium to the cell suspension, and incubate with gentle shaking for one hour at 37 degrees Celsius. Centrifuge at 13, 800 RCF.
Discard the supernatant, and resuspend in 200 microliters of fresh LB medium. Plate 100 microliters of the transformed E.coli DH5-Alpha cells on an LB plate containing 50 micrograms per milliliter ampicillin, and incubate at 37 degrees Celsius for 16 hours. Set up mini preps of 25 to 30 colonies in five milliliters of LB medium containing 50 micrograms per milliliter ampicillin, and grow overnight at 37 degrees Celsius.
On the next day, extract the plasmids with a commercial kit and perform restriction enzyme digestion in 10 microliters volume with 200 nanograms of the isolated plasmids, two units of EcoR1 and 1X restriction buffer for all the colonies. After incubating the digestion mixtures at 37 degrees Celsius for three hours, load the products on 0.8%TAE-based agarose gel to confirm plasmid DNA insertion. Perform Sanger sequencing of the plasmids of 25 positive clones using the recommended primers.
One day before transfection, split the cells, and count the number of viable cells using a hemocytometer. Then seed the human hepatoma 7.5 cells in 35 millimeter dishes in two milliliters of complete DMEM, and incubate the cells at 37 degrees Celsius for 16 hours in a humidified incubator with 5%carbon dioxide. On the following day, prepare the transcript lipid complex by diluting 10 microliters of transfection reagent in 50 microliters of the minimal essential medium, and separately dilute five micrograms of viral transcripts in 50 microliters of the minimal essential medium.
Incubate both mixtures at room temperature for 10 minutes. Then mix them in a single sterile microcentrifuge tube, and incubate the mixture at room temperature for 30 minutes. After the cell incubation, remove the culture medium, wash the cells twice with pre-warmed 1X PBS, and add 1.5 milliliters of the minimal essential medium.
Slowly add the complex, and gently swirl the dish for uniform distribution. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 10 hours. Then remove the medium.
Wash the transfected cells twice with one milliliter pre-warmed 1X PBS, and add two milliliters of complete DMEM. Isolate viral RNA from 140 microliters of culture supernatants using a viral RNA isolation kit. Set up a 10 microliter qRT-PCR reaction using a commercial qRT-PCR kit.
Use the forward and reverse primers and probe for HCV RNA quantification. Set the reaction cycle to 48 degrees Celsius for 20 minutes, 95 degrees Celsius for 10 minutes, and 45 cycles of 95 degrees Celsius for 15 seconds, and 60 degrees Celsius for one minute. Run the reactions, and set up negative controls.
In parallel, generate a standard curve using a tenfold serially diluted known copy number of HCV transcripts to quantify viral RNA. Perform in triplicate. Plate human hepatoma 7.5 cells in a 96-well plate, and incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator approximately 16 hours before adding the virus.
In a biosafety Class II cabinet, perform tenfold serial dilutions of the virus. Add 100 microliters of the diluted virus per well to infect cells, and incubate them at 37 degrees Celsius with 5%carbon dioxide. After three days, wash the infected cells thrice with 0.1 milliliters of PBS, and fix and permeabilize the cells with 0.1 milliliters of ice cold methanol at minus 20 degrees Celsius for 20 minutes.
Wash the wells with 1X PBS thrice, and then 1X with PBST. After removing the PBST, block the cells for 30 minutes at room temperature with 0.1 milliliters of 1%BSA containing 0.2%skimmed milk in PBST. Remove the blocking solution, and treat the cells for five minutes with 0.1 milliliters of 3%hydrogen peroxide prepared in 1X PBS.
Again, wash the cells twice with 1X PBS, and 1X with PBST. Add 50 microliters of anti-NS5A 9E10 monoclonal antibody per well, and incubate at room temperature for one hour. Wash the wells thrice with 1X PBS and once with PBST.
Add 50 microliters of HRP conjugated goat anti-mouse secondary IgG per well, incubate for 30 minutes at room temperature, and then remove the unbound antibody by washing the wells with 0.1 milliliters of 1X PBS. Add 30 microliters of DAB, and incubate the plate with gentle rocking for 10 minutes at room temperature. Remove the DAB solution, and wash the wells twice with 1X PBS, and once with distilled water.
Add 100 microliters of PBS containing 0.03%sodium azide. Examine each well under an inverted light microscope using a 10X objective. Count the number of positive wells.
Use a Reed and Muench calculator to estimate the endpoint dilution that infects 50%of the wells. Dissolve pibrentasvir, an NS5A inhibitor, in 100%DMSO to a concentration of one millimolar and further dilute it in complete DMEM to a concentration of 10 nanomolar. Then infect naive Huh-7.5 cells at 70%confluence with an ML50 virus dose to infect 50%of the cells for 12 hours, and then transfer the infected cells onto six-well plates 24 hours post-infection.
Add 1X EC50 PIB to the infected cells after 16 hours of cell split. Do this after every cell split for six consecutive passages, followed by three drug-free passage cycles, and monitor virus spread using a focus-forming assay. Harvest viral supernatants at each passage, and store at minus 80 degrees Celsius.
Extract viral RNA from the supernatant on day 18 and the synthesized cDNA. Amplify the NS5A gene using five microliters of diluted cDNA. Then determine NS5A drug resistance mutations in six to eight positive bacterial plasmids using the NS5A sequencing primers.
Full genome mutant libraries were synthesized using clonal pJFH1 in decreasing amounts from 100 to 10 nanograms. Average yields of ep-PCR products ranged from 3.8 to 12.5 nanograms per microliter. The proportion of mutations in mutant libraries increased with decreasing input of pJFH1 in the ep-PCR reaction.
ML50 synthesized using 50 nanograms of the template had four substitutions per 10, 000 base pair copied, whereas ML25 synthesized using 25 nanograms of template harbored nine substitutions. No substitutions within the number of nucleotides sequenced were found in clonal pJFH1. ML50 viral variants were less susceptible to pibrentasvir compared to the clonal JFH1 virus.
Of the eight NS5A clones, four had a combination of D7V+F28C, whereas V8A+F28C and F36L occurred in one clone each. These mutations were at the N terminus region of NS5A, which is known to harbor clinically relevant NS5A resistance mutations. The final concentration of each component of ep-PCR is critical, especially template amount.
Lack of KB Extender will greatly reduce the yield of the ep-PCR product.
