The overall goal of this procedure is to identify the stages where viral gene expression is affected by chemical treatment. To do this, tissue culture cells are plated and incubated. Until confluent, the cells are then infected with either a triple reporter vaccinia virus or a control promoter list virus.
Next treatment compounds are applied and the cells are incubated for 18 hours to allow the completion of all stages of viral gene expression. The resulting fluorescence is then determined using a plate reader. Ultimately, results can be obtained that show the relative changes in expression caused by the experimental compounds.
The main advantage of this technique over infection with a virus that expresses a single fluorescent fusion protein is that this set of viruses provides more information about where in the viral lifecycle a specific therapeutic drug is acting, providing insight into its mechanism of action. Although this method can be used to identify inhibitors of viral infection, it can also be used to detect the stage of viral replication completed in non permissive cell lines or in RNAi screens, identifying cellular factors necessary for infection. This protocol utilizes the triple virus or Trip V multi-stage reporter virus, in which three spectrally distinct fluoro fours are incorporated into the doublet stranded genome of a single venia virus.
Each of these fluoro fours is controlled by a well-defined stage specific promoter. The C 11 R promoter for early expression of Venus, the G eight R intermediate promoter for expression of m cherry and the F 17 R promoter for late stage expression of tag BFP. Thus, Venus m Cherry and tag BFP are sequentially expressed during infection.
As a control, A promoter list virus is used. PLV contains a similar venous construct to that in the Trip V.However, it does not have a functional transcription promoter. Begin this protocol by dissociating.
Hela cells grown on a 10 centimeter dish, dilute the cells in growth medium to approximately 2.0 times 10 to the fifth cells per milliliter. Then dispense 100 microliters in each well of a black walled clear flat bottom 96 well plate incubate the cells for 24 hours in a 37 degrees Celsius incubator with 5%carbon dioxide until they are confluent to dilute the viruses thaw Tripp V and PLV in a 37 degrees Celsius water bath, then sonicate for five minutes to disaggregate. Next, dilute the virus stock to 1.0 times 10 to the seventh PFU per milliliter in infection medium that has been prewarm to 37 degrees Celsius to infect the cells.
Replace growth media with 50 microliters of the diluted virus to each well for each treatment. Infect three replicate wells with Tripp V and another three with PLV to account for background fluorescence specific to each treatment. This is defined as time equals zero hours.
Post-infection, for example, make 350 microliters for 6 96 wells with 50 microliters each. Each compound should be diluted to twice the final concentration, since it will be added to the inoculum volume already in the well. Next, for each treatment condition, make a two x master mix by diluting the experimental compounds or vehicle control solvents such as PBS or DMSO into enough of the infection medium for all replicates.
Plus one, Note that the concentration of solvent in both your experimental and vector only controls should be the same. For example, if you're using IBT at one microliter per mil in DMSO, you should also use DMSL alone at one microliter per mil as your vector control. Immediately after adding the virus, add 50 microliters of infection medium containing the desired treatment and control compounds to each well as shown in this illustration.
Note that each compound is tested with Tripp V and PLV in triplicate, and a vehicle control is run in triplicate for each virus. Incubate for 18 hours in a 37 degrees Celsius incubator plus 5%carbon dioxide. The next day, fix the cells by adding 100 microliters of 8%paraform aldehyde to the infection medium already in each well incubate the plate at room temperature for 15 minutes protected from light.
Adding two x or 8%paraform aldehyde directly to the infection medium will prevent aerosolization of the virus, which could otherwise occur if the unfixed virus is directly inverted into the waste dish. After 15 minutes have passed, remove the fixative by inverting the plate into the waste dish. Then add 100 microliters of room temperature PBS and seal the plate with optically clear adhesive film.
If the plate will not be read immediately, store it at four degrees Celsius. Be sure to return the sealed plates to room temperature before reading them to prevent condensation from distorting spectrophotometer measurements. To quantify virus growth, use a spectrophotometer to measure endpoint fluorescence.
Take four measurements per well using optimized gain settings for each channel. The TAN Infinite M 1000 Pro plate reader used in this video exports raw fluorescence measurements to a Microsoft Excel file. After the readings have been obtained, open the raw data in Microsoft Excel.
Then copy and paste it into a GraphPad prism. Results sheet in Prism determine the mean of replicate Tripp v and PLV wells. Then subtract the mean PLV value from the mean Tripp V value for each treatment.
To facilitate comparison with replicate experiments, normalize the data by dividing the background subtracted data by the vehicle only Tripp v Wells. Once an experiment has been repeated multiple times, perform a one-way analysis of variance or one-way a NOVA test and multiple comparison post-test to determine whether any of the treatments are significantly different from the DMSO treatment. For each channel to perform kinetic assays infect the cells and apply test compounds as before, it is particularly important to use a buffered medium to maintain proper pH without addition of 5%carbon dioxide.
After sealing the plate with adhesive film, place it in the plate reader chamber equilibrate to 37 degrees Celsius. Special care must be taken to keep plates consistently at 37 degrees Celsius. This will prevent undue cell stress and condensation.
Manually set the plate reader gain to prevent saturation of later time points. Acquire hourly readings for eight to 24 hours following infection and normalize. As for the endpoint assay, individual time points can be analyzed for statistical significance using similar methods as the previously described endpoint assay.
To compare points of inhibition, heela cells infected with TPV or PLV vaccinia were grown in the presence of the pox virus inhibitors, Aris, CIBT, Rifampicin, and ST 2 46. This time, lapse movie shows the sequential expression of green, red, and blue fluoro fours during virus growth in control cells when infected in the presence of the DNA replication blocking drug aee. The early green fluoro four is produced as before, but intermediate and late production of red and blue fluorescence does not occur.
Quantitative analysis by spectrometry showed a 210%increase in early gene expression, but a lack of intermediate and late expression in cells treated with a C cells infected in the presence of IBT, which is thought to promote. Read through transcription had intermediate and late expression levels that were 24.7%and 2.9%of control levels cells infected in the presence of ST 2 46 and rifampicin, which inhibit after late gene expression during virion assembly and maturation were not significantly different from controls. These results are consistent with the understood mechanism of action of these compounds and suggest that the Trip V reporter virus can be used to identify the specific stage of viral inhibition for unknown compounds.
While attempting this procedure, it's important to remember to include appropriate controls. Not only does this allow for the proper normalization of results, but also to determine a potential mechanism for your experimental compound. Don't forget that working with vaccinia virus can be extremely hazardous, always wear proper personal protective equipment and follow recommended bsl.
Two containment techniques.
