The overall goal of these procedures is to screen for inhibitors of Zika virus replication in a high-throughput screening format, using a replicon-based and/or a viral enzyme-based assays. Antiviral drug discovery requires the development of reliable cellular and biochemical assays that can be performed in high-throughput screening formats. Replicon-based screenings and viral enzyme-based assays are two valuable strategies to test small molecule compounds as inhibitors of Zika virus.
Replicons are self-replicating subgenomic systems expressed in mammalian cells in which the viral structural genes are replaced by a reporter gene. In our lab, we developed a Zika virus replicon system, including the Renilla luciferase, which is stably expressed in baby hamster BHK-21 cell lines. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the luciferase activity.
To characterize the specific targets of identified compounds, we established in vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RNA-dependent RNA polymerase, the RdRp. Start by culturing Zika viral replicon cells until they reach 70 to 90 percent confluence. In a sterile laminar flow, remove and discard the media from the flask.
Add five mils of trypsin-EDTA to the flask. Spread the trypsin solution over the cells and leave the flask for five to 10 minutes for cells to detach. Harvest the resuspended cells and place it in the sterile 50 mil tube and centrifuge it for 125G during five minutes.
After discarding the supernatant, resuspend the cells in five mils of DMEM 10%FBS for counting. Adjust the cells to two times 10 to four per well and seed them in a 96-well plate. Then incubate the plate for 16 hours at 37 celsius in a CO2 humidifier incubator.
Next discard the medium and add 100 microliters per well of DMEM 2%FBS. Add one microliter of the compound per well to result in a final concentration of 10 micromolar and 1%DMSO in the assay medium. In the first and in the last column, prepare the positive and negative control.
Incubate the plate for 48 hours at 37 celsius in a CO2 humidifier incubator. Prepare Renilla luciferase lysis buffer in reagent according to the manufacture instructions. After discarding the supernatant from the cells add 25 microliters of Renilla luciferase lyse buffer per well.
Add 100 microliters of Renilla luciferase assay reagent per well to a white opaque 96-well plate. Transfer 20 microliters of the cell lysate to the white plate and then read the luminescence signals in luminometer. For the MTT assay, procedure has previously demonstrated and after compound incubation add 10 microliters per well of MTT solution to the cells and incubate the plate at 37 celsius in a CO2 humidifier for 24 hours.
Discard the supernatant and add 100 microliters of DMSO to each well, and then solubilize the formazan crystals by pipetting up and down. Read the absorbents at 570 nanometers in a spectrophotometer. After thawing materials on ice, prepare an appropriate amount of protein dilution to reach a final concentration of four nanomolar per well.
In a 96-well white plate, dispense 84 microliters of the assay buffer in each well. To prepare a control reaction, add one microliter of DMSO to the first column of the plate. Add one microliter of the tested compounds to reach a final concentration of 10 micromolar to the plate.
Dispense five microliters per well of protease solution, excluding controls and incubate the plate at four celsius for 30 minutes. To start the reaction, dispense 10 microliters of fluorogenic substrate stock solution to the wells. Follow their reaction using a fluorometer set in appropriate parameters.
For this session, all reagents must be prepared with DEPC treated water. Anneal the polyuridine RNA by incubating it at 55 Celsius for five minutes in a thermal cycler. Thaw materials on ice and dilute the protein to reach a final concentration of 250 nanomolar in the assay buffer.
Prepare the SYBR Green I solution assay buffer, and then add ATP and the anealed RNA to a final concentration of one millimolar and 300 nanomolar, respectively. In a PCR plate, add 24.5 microliters of the diluted protein in columns one to 11. Add the same volume of the assay buffer in the remaining wells.
Add 0.5 microliters per well of compounds and controls to reach a final concentration of 10 micromolar. After 15 minutes incubation, start the reaction by adding 25 microliters of the substrate solution and seal the plate. Follow the reaction in a real-time PCR system incubated at 30 Celsius using a FAM filter.
In figure 1A, we see a schematic representation of the RNA construct developed to obtain Zika viral replicon cell lines, showing that Rluc gene replacing the structural sequences and the IRES new cassette inserted at 3'end. It also includes a T7 promoter in the hepatitis delta virus ribozyme sequence for in vitro transcription and for generation of the authentic 3'end and the RNA transcript, respectively. In figure 1B, we see a schematic representation of the replicon-based cell assay.
In figure 1C, we see the dose-response curves and the cytotoxicity curves of a given compound obtained using the Rluc replicon system. In figure 2A, we see an schematic representation of the assay used to determine the activity of the NS2B-NS3 protease. In figure 2B, we see the dose-response curve of a given inhibitor targeting the NS2B-NS3 protease.
In figure 2C, we see an schematic representation of the elongation assay used to determine the NS5 RdRp activity. In figure 2D, we see the dose-response group of a given inhibitor targeting the Zika virus RdRp activity. The demonstrated procedures could be readily adapted for screenings in a 384 or 1, 536-well formats.
For the replicon-based high-throughput screenings, we developed a stable cell line harboring a replicative Zika virus replicon containing a Renilla luciferase sequence at the 5'end, and also a neomycin phosphotransferase gene driven by an internal ribosomal entry site at the 3'end. Due to the lack of structural genes, replicons do not produce progeny virions. Thus, eliminating the risk of laboratory acquired viral infection.
In addition, we also demonstrated the procedures used in viral enzyme-based assays for the recombinant NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using a fluorogenic peptide substrate, which contains the Zika virus protease recognition and cleavage sequences coupled with the fluorescence stock aminomethylcoumarin. Regarding the NS5 RNA-dependent RNA polymerase, its elongation activity is directly detected by the increase of the fluorescent signal of SYBR Green I.These implementation, direct measurement, and affordability are key points to the user's florescence-based methods as high-throughput screening platforms.
