A Protocol for Analyzing Hepatitis C Virus Replication

The overall goal of this procedure is to investigate various stages of the Hepatitis C virus replication cycle. This is accomplished by first generating HCV genomic RNA transcripts from Linearized HCV plasmid constructs. The second step is to transfect the cells with H-C-V-R-N-A. Next, the cells are plated for various time points and assays. The final step is to collect the cell culture supernatant for measuring viral titer and harvest transfected cells for protein and RNA. Ultimately reverse transcription-PCR, Western blotting, immunofluorescence assay and HCV titer are performed and demonstrate a robust HCV infectious cell culture system.

The main advantage of this infectious hepatitis C virus cell culture system over the HCV replicon system is that viral entry, virion assembly and egress steps can be investigated. This protocol can help answer the key questions in the hepatitis C virology field, such as virion-morphogenesis, and host a pathogen interaction. The implications of this technique extend toward vaccine development as it allows characterization of attenuated viral strains that can be evaluated as potential vaccine candidates.

Though this method can provide insight into hepatitis C virus, it can also be applied to other RNA viruses in the family of Flaviviridae. Generally, individuals new to this method will be faced with the challenge of generating high quality HCV genomic RNA. Studying the complete replication cycle of HCV became feasible in cell culture following the discovery of a genotype-2A HCV Japanese fulminant Hepatitis one isolate. Visual demonstration of virological assay is critical because the assay will require expertise in both molecular and cellular techniques.

Using an intra-genotype 2A chimeric virus, F-N-X-H-C-V and F-N-X-H-C-V pol-null HCV for evaluation of viral replication, linearize previously generated viral plasmids by digestion with XBA-one restriction enzyme in a two milliliter tube. Then treat the plasmids with mung bean nuclease to generate blunt ends.

Purify the digested plasmids by anion exchange chromatography. Verify the integrity of the linearized plasmid by subjecting DNA to agarose gel electrophoresis. Next, add the linearized plasmid DNA template into a 0.2 milliliter tube containing T-seven RNA polymerase reaction components to synthesize HCV genomic RNA transcripts.

Purify the newly synthesized DNase treated RNA using an RNA purification kit. Then verify the RNA production by agarose gel electrophoresis. Following this, quantify the RNA by spectrophotometry.

Detach Huh-7 based adherent cells using trypsin enzyme treatment and collect the cells into a 50 milliliter conical. Two following centrifugation resuspend the cell pellet with cold low serum media. After repeating the centrifugation, resuspend the cells in low serum media at one times 10 to the seven cells per milliliter.

Next, mix a total of 10 micrograms of transcribed viral RNA with 400 microliters of resuspended cells into a pre-chilled 0.4 centimeter electroporation cuvette. Deliver the viral RNA into the cells using an electroporation at 270 volts, 100 ohms and 950 microfarads. When finished, resuspend the electroporated cells in 10 milliliters of complete growth media with 15%FBS. At this point, plate the cells in both T 25 flasks at about 1.2 times 10 to the six cells per flask and 48 well plates at one times 10 to the fourth cells per well for 4, 48, and 96 hour time points. Replace the media at four to eight hours post transfection with fresh supplemented growth media with 10%FBS to remove dead cell debris from the cultured flasks and plates.

Using a serological pipette harvest the cell culture supernatants at the 48 and 96 hour time points into a 15 milliliter conical tube. Then remove cellular debris from the collected samples by centrifugation at 15, 000 rpm for 10 minutes at four degrees Celsius. Following centrifugation, store the cell-free supernatants at negative 80 degrees Celsius.

Lyse the cells for protein and RNA analysis by western blot and reverse transcriptase quantitative PCR or RT-qPCR at the indicated time points. Reverse transcribe one microgram of total cellular RNA using reverse transcriptase enzyme and a specific primer for the HCV sense strand that binds to the five prime untranslated region in a 0.2 milliliter tube. Also reverse transcribe F-N-X-H-C-V-R-N-A of known genome copies using an HCV sense strand primer. Using a real-time PCR system carry out qPCR by using 50 nanograms of the resulting transcribed CDNA using specific HCV primers and DNA binding green dye containing qPCR super mix. Use the following conditions when running qPCR to determine the H-C-V-R-N-A copy number. Following qPCR resolve the cell lysate from the viral RNA transfected at 96 hours post transfection using SDS page. Then transfer the resolved proteins in the gel to a polyvinylidene difluoride membrane by trans-blot turbo method. Block the membrane using a blocking solution containing 5%skim milk and 0.2%tween-20 in PBS and placed in a container.

Incubate the membrane with primary mouse monoclonal antibody NS3 at a one in 1000 dilution and beta-actin at a one in 5, 000 dilution in a four degrees Celsius cold room. After incubation, add goat anti mouse immunoglobulin-G conjugated to horse-radish peroxidase at a one in 5, 000 dilution and detect by chemiluminescence.

At this point, fix the H-C-V-R-N-A transfected cells using methanol for 30 minutes at negative 20 degrees Celsius for the immunofluorescence assay. When finished, wash the cells with PBS three times. After blocking with immunofluorescence assay blocking buffer use rabbit polyclonal anti-NS5A primary antibody and mouse monoclonal anti-DSRNA antibody J2 at a dilution of one to 200 and incubate for five hours to overnight in a four degrees Celsius cold room. Following incubation, wash the cells with PBS three times after the primary antibody. Then add goat anti-rabbit immunoglobulin-G 488 polyclonal secondary antibody and goat anti- mouse immunoglobulin 594 polyclonal secondary antibody at a one in 1000 dilution and incubate for one hour at room temperature on a tabletop rocker. After washing the cells with PBS three times stain the nuclei using Hoechst dye and view using a fluorescent microscope.

Next plate naive Huh7.5.1 cells at approximately three times 10 to the third cells per well using a 96 well plate. The next day, perform tenfold serial dilution of cell-free culture supernatant harvested from H-C-V-R-N-A transfected cells using growth media and inoculate in triplicate onto Huh7.5.1 cells. Fix the cells at 72 hours post-infection using methanol for 30 minutes at negative 20 degrees Celsius. After removing the cells from the freezer amino-stain for H-C-V NS5A protein using the previously described conditions. Using a fluorescent microscope, count the NS5A positive cell foci in the well with the highest viral dilution and calculate the average number of focus forming units per milliliter.

The linearized HCV plasmid quality was assessed by gel electrophoresis. The H-C-V-D-N-A was subjected to T-seven RNA polymerase mediated in vitro transcription, which yielded a single RNA product at 9.6 Kilobases. RT-qPCR results indicated that the wild type virus replicated the genome efficiently. The wild type exhibited a one to three log higher level of genome replication compared to pol-null virus.

The wild type virus produced the NS3 protein involved in viral protein cleavage and genome replication. The wild type virus also expressed NS5A protein and the NS5A protein and double stranded RNA co-localized in the cellular cytoplasm of wild type transfected cells suggesting active viral replication. Virus titer measurements indicated that the wild type virus is infectious and produces over 10, 000 foci forming units per milliliter of infectious particle. At six hours post transfection both wild type and pol-null viruses had similar luciferase activities indicating a similar input level of transfected RNA had been translated. However, at 48 hours and 96 hours post transfection the wild type virus exhibited increased genome replication levels compared to pol-null virus. The wild type virus also produced viral NS3 protein. The poll null virus had base level luciferase activity, whereas the wild type virus had a two to three log higher level of replication compared to pol-null reporter virus. Once mastered Hepatitis-C virological assays can be done in two weeks if they're performed properly. While attempting this procedure, it's important to have robust cells and high quality HCV genomic RNA transcripts. Following this procedure, characterized HCV strains can be tested in animal model systems to investigate in vivo fitness and host pathogen interactions.

The development of an infectious HCV cell culture system paved the way for researchers to explore the virion-morphogenesis and viral egress steps of the HCV replication cycle. After watching this video, you should have a good understanding of how to perform various virological assays for characterizing different stages of the Hepatitis-C virus replication cycle. Don't forget that working with Hepatitis-C virus can be extremely hazardous and safety precautions such as wearing appropriate personal protective equipment should always be taken while performing this procedure.