The aim of this procedure is to investigate the impact of restriction factors on completion of the hepatitis C virus replication cycle in any given cell line, using two methods that rely on heca formation for the first method begin by culturing human liver cells as well as heal A and Hep 56 1D packaging cells next transfect hepatitis C virus or H-C-V-R-N-A into the liver cells by electroporation. Subsequently fsed the HCV replicating cell line to the respective packaging cell line by polyethylene glycol in the second fusion method. The first step is production of a high titer HCV stock.
Different cell lines are then co-culture and cell fusion is induced by transfection of prototype foamy virus glycoprotein. Next, the hetero carons are infected with HCV. Following either method of hetero carion formation, fluorescent microscopy is performed to confirm fusion and production of infectious particles is quantified.
The main advantage of this technique is that it's useful for screening a large panel of cell lines and primary cells for the expression of HV specific dominant restriction factors. This method can help answer key questions about the species specificity and noms of HCV. Visual demonstration of this method is critical to facilitate understanding of the underlying concepts of the two fusion systems Prior to induction of cell fusion by polyethylene glycol.
HUH 7.5 cells are transfected with H-C-V-R-N-A following standard protocols and cultured for 24 hours at 37 degrees Celsius and 5%carbon dioxide on the following day. Harvest Transfected HUH 7.5 cells harboring the sub genomic replicon as well as heal R and he 56 1D packaging cells. These packaging cells have been engineered to ectopically express HCV proteins core E one, E two P seven, and NS two after tryps ization resuspend cells in DMEM complete medium dilute the cell suspensions to the following concentrations, HUH 7.5 to five times 10 to the five cells per milliliter and heal and hep 56 1D cells to two times 10 to the five cells per milliliter.
Next, prepare a six well culture plate with two to three sterile glass cover slips per well. For further analysis of fusion efficiency, combine one milliliter of transfected HUH 7.5 cells with one milliliter of packaging cells and seed into one well of the six well plate incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide after 24 hours. Check on the cell density.
Cell density should range between 60 to 80%co fluency before induction of cell fusion in order for cells to be in close proximity for cell membranes to fuse aspirate medium from the co cultured cells and wash once with one milliliter PBS. Then carefully add 500 microliters of pre warmed 40%polyethylene glycol or PEG 1, 500. Add 500 microliters of PBS to another well of co-culture cells as a control incubate for five minutes at 37 degrees Celsius to induce hetero carion formation aspirate PEG and carefully add two milliliters of PBS after one minute aspirate the PBS and add two milliliters of fresh PBS wash with PB S3 to five times for one minute per wash to remove excess PEG.
Finally, add two milliliters of DMEM, complete medium to each well and incubate at 37 degrees Celsius and 5%carbon dioxide for 48 hours. 48 hours Post fusion. Collect each SNAT and filter through a 0.45 micron filter after collecting cell culture snat fusion efficiency can be determined by immunofluorescence wash the wells containing glass cover slips once with one milliliter of PBS for one minute.
Fix cells with 600 microliters of 3%paraldehyde in PBS for 15 minutes. Wash three times with PBS for one minute per wash after the final wash. Use forceps to carefully transfer cover slips into a 24 well plate permeable cells with 500 microliters of 0.5%Triton X 100 in PBS for five minutes.
Subsequently wash three times with PBS apply 250 microliters of the primary antibody solution to each. Well use anti NS five a antibody as a concentration of 0.5 micrograms per milliliter and human monoclonal anti E two antibody as a concentration of 6.4 nanograms per milliliter. Incubate for 45 minutes of room temperature after 45 minutes, wash three times with PBS.
Next, add 250 microliters of secondary antibodies to each well secondary goat anti mouse or go anti-human IgG specific antibodies conjugated to Alexa floor 5 46 or Alexa floor 4 88 are used as a concentration of two micrograms per milliliter. Incubate for 30 minutes at room temperature in the dark after 30 minutes. Wash once with PBS.
Subsequently stain cell nuclei with 250 microliters of DPI diluted one to 3000 in PBS. Incubate for one minute at room temperature in the dark wash four times with PBS and once with water. Finally mount cover slips upside down onto a drop of flora mount on a microscope slide and let them dry in the dark.
To begin this procedure, harvest and prepare single cell suspensions of the following cells. Luna NA human liver cell line that lacks endogenous expression of CD 81. He r packaging cells and Hep 56 1D cells that stably express human CD 81 co cultivate Luna n cells with he r cells at a ratio of one to one and with Hep 56 1D HCD 81 cells at a ratio of one to two, yielding a total cell density of 1.5 times 10 to the five cells per 12.
Well incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide on the following day. Transiently transfect cells with a highly FU genic PFE envelope protein by using lipectomy 2000 according to the manufacturer's instructions. 30 hours after transfection with PFE glycoprotein inoculate hetero carons with 350 microliters of a previously prepared virus stock at an MOI of around 2.3.
Incubate for 12 hours at 37 degrees Celsius and 5%carbon dioxide on the following morning. Wash once with PBS and add one milliliter of DMEM complete medium per well incubate for 48 hours at 37 degrees Celsius and 5%carbon dioxide after 48 hours, harvest the supinate of the hetero carion cultures, filter the supinate through a 0.45 micron filter. Next, use the supinate to inoculate HUH 7.5 cells that were seeded in 96 well plates the day before in a limiting dilution assay incubate for 72 hours before quantifying the production of infectious progeny particles.
The procedure to visualize fusion events must be completed six hours before co-culture wash adherence cells once with five milliliters of PBS and then add four milliliters of the appropriate DI solution per 10 centimeter dish dish incubate cells for 45 minutes at 37 degrees Celsius and 5%carbon dioxide wash cells once with five milliliters of PBS for one minute and then add DMEM complete medium incubate for six hours at 37 degrees Celsius and 5%carbon dioxide before seeding. Add a cover slip to each well of the 12 well plate after that seed and transfect stain cells as shown previously. 30 hours post transfection wash cells carefully with one milliliter of PBS for one minute and then fix cells with 250 microliters of 3%paraldehyde for 15 minutes at room temperature.
Wash cells once with one milliliter of PBS for one minute and then stain with dappy for one minute after washing cells once with PBS and once with water mount cover slips, sunglasses, slides in the first approach to investigate the impact of restriction factors on completion of the HCV replication cycle. In hetero carons, HUH 7.5 cells were first transiently transfected with a sub genomic replicon expressing a luciferase transgene and HCV non-structural proteins and then co cultured and fused to cell lines expressing HCV structural proteins core and accessory proteins. A representative result is depicted in this figure cells or immunostain using monoclonal antibodies against the E two structural protein and NS five A non-structural protein.
The images in the upper panels show that upon treatment with PEG signals for both MS five A and D two were detected simultaneously in the same cytoplasm indicating self fusion. In contrast, the in the lower panels show that when co cultured cells were treated with PBS, no fusion was induced. Thus no colocalization of signals was observed to quantify viral infectivity produced from hetero carons snet were collected from the co cultures at 48 hours post fusion induction and used to inoculate naive HUH 7.5 cells for subsequent luciferase assays.
In this graph, the meme values of three independent experiments are displayed. The gray horizontal bar represents the background for relative light units or RLU determined in uninfected HUH 7.5 cells. Notably, when HUH 7.5 replicant cells were either fused with naive cell lines or treated with PBS as a control, no infectivity was detected in the culture fluids.
However, when cell fusion between HUH 7.5 replicon and packaging cell lines was induced by PEG, infectious viral particles were released into the culture fluids. This indicates that virus production requires cell fusion and expression of viral proteins in the packaging cell lines strikingly not only the hetero carons of HUH 7.5 human hepatoma cells, but also hetero carons with human non-live and mouse liver cells allowed virus release indicating that assembly and release are not dominantly inhibited by possible restriction factors in these cell types. In an alternative approach, somatic cell fusion was induced by the expression of a genic glycoprotein after cultivation of the cells.
Hetero carons were visualized by staining separate cell lines with cell tracker dyes prior to co cultivation and transfection with the genic protein cells were fixed and stained with DPI 30 hours post transfection and a representative result is shown in this figure. The fusion of two cell types is indicated by a hetero carryon with homogeneously distributed dyes within the cytoplasm as illustrated here. In contrast, the images in the lower panel show the absence of cells with a double stain cytoplasm indicating the absence of cell fusion.
This final graph shows that all HCV entry factors are expressed only in hetero carriers of Luna N cells with hela or Hep 56 1D cells expressing human CD 81. Here the cultures were inoculated with infectious HCV particles. Culture fluids were collected 48 hours later and de novo particle release from hetero carons was determined by limiting dilution assay using naive HUH 7.5 target cells mean values of three independent experiments are shown and the gray horizontal bar represents the detection limit of the assay.
A as a control lunar N cells were fused with lunar N cells, which resulted in very low levels of infectious HCV close to the detection limit. Similar results were obtained when lunar N cells were fused with naive HEP 56 1D cells. In both cases, human CD 81 was absent, so HCV could not productively infect the heteros.
In contrast, de novo production of infectious viral progeny approximately tenfold above the background of the assay was observed in lunar 10 cells views with heer and in lunar hand cells fused with HEP 56 1D cells expressing human CD 81. This indicates that HCV successfully completed its replication cycle in RetroCars, thus ruling out expression of dominant restriction factors in these cell lines. Once master, the complete experimental procedure can be done in one week if it's performed properly.
After watching This video, you should have a good understanding on how to generate hetero carryon for the identification of possible HV restriction factors. Don't forget that working with Hepatitis C virus can be extremely dangerous and precautions such as high biosafety should always be taken into account while performing this procedure.
