Quantitation of Virus by Plaque Assay

1. For each mutant, set up 13 60-mm plates of 3T6 cells to be approx 70% confluent at the time of infection.

2. For each mutant, aliquot exactly 0.9 mL of sterile PBS to seven small glass test tubes with caps and number and order these tubes from 2 to 8.

3. Warm to 37°C the 1-mL aliquot of each virus stock to be titrated (Subheading 3.4.2., step 2). Do not leave at 37°C for prolonged periods. Once the stock is thawed, store on ice.

4. Aliquot exactly 0.1 mL of virus into tube 2. Vortex and remove exactly 0.1 mL from tube 2, and deposit it into tube 3. Vortex and repeat for the remaining tubes (changing tips each time), creating a dilution series of the original virus stock. Repeat for each virus stock.

5. Label two plates per dilution (103-108) and one plate as mock. Remove medium from plates before adding PBS or virus solution. Add 0.1 mL of PBS to the mock plate.

6. Beginning with the highest tube number (108), the most dilute virus sample, add exactly 0.1 mL of the solution to each of the two plates labeled 108. Rock the plates to cover the cells with the liquid. Repeat sequentially in descending order, but omitting tube 2, as there will be too many plaques to count from this dilution (see Note 18). If you have worked from most dilute to least dilute, as described above, you do not have to change tips with each sample.

7. Incubate at 37°C for 1 h. Meanwhile, microwave sterile 1.6% agarose until it is boiling and place it in a 45°C water bath. Allow agarose to equilibrate at 45°C for at least 30 min. (You will need 2.5 mL per plate, but allow some extra.) Warm 2X plaque assay medium containing 4% HICS that is supplemented with glutamine, pen/strep, and nonessential amino acids all at a final concentration of 2X, as well as MgCl2 that is at a final concentration of 50 mM (see Note 19). (You will need 2.5 mL per plate, but allow some extra.)

8. After the 1 h incubation, mix equal volumes of warm 2X DMEM and agarose. Working quickly, overlay each plate with 5 mL of medium/agarose by gently applying the solution to the cells with a wide-mouth pipet by adding to the side of the plate, rather than dispensing the medium to the center of the plate. Try not to introduce bubbles onto the plates. Allow to stand about 30 min. Place the plates in the 37°C incubator.

9. The next day, and every second or third day thereafter, overlay each plate as described above with 2 mL per plate, i.e., 1 mL of 2X plaque assay medium mixed with 1 mL of agarose.

10. When plaques become visible (days 8-9), begin counting and recording the number of plaques. Continue counting daily and overlaying until no or only a few new plaques appear for 1-2 d (see Note 20). Always count the plaques prior to overlaying because the recent addition of medium/agarose tends to make the plaques very hard to see. If desired for added visibility of plaques, add neutral red to a final concentration of 1X to the medium each time the plates are overlaid once plaques are first visible.

11. When no or very few new plaques appear for 1-2 d, total the number of plaques for each plate and multiply that number by the dilution factor. This number is the titer of the virus in PFU/mL. The most accurate titer will be determined from at least two plates with 10-100 plaques per plate. For example, if two 106 plates yielded a total of 47 and 53 plaques, respectively, the average of 50 plaques is multiplied by the dilution factor (106) to yield a titer of 5 x 107 PFU/mL. In some cases titers from two dilutions can be averaged to give the titer of the virus stock (see Note 21).

4. Notes

1. pmE301 and pmE101 are MAV-1 wt variants that contain only one £coRI site in the genome. pmE301 contains a single £coRI site in the E1A region, whereas pmE101 contains a single site in the E3 region (1,4).

2. 1X DMEM contains 0.15 M salt, and because the volume of NaCl contributed from the 1X DMEM is not negligible, be sure to include it in the calculation.

3. Keep movement of the gradient tubes to a minimum to avoid disturbing the gradients. Make sure that a layer is visible in each one prior to loading your samples. If no layer is present, discard and make another gradient.

4. Because these are equilibrium density gradients, the centrifugation can go longer than the indicated time.

5. We use a Hoeffer dripper: follow the manufacturer's instructions for your dripper. Using the dripper can be tricky, so it is advisable to practice the procedure on a blank tube first. This allows you to work out the best way to puncture the tube and to control the flow rate. We also recommend cleaning the dripper needle after each gradient to prevent clogging. To clean, insert a needle through the opening a few times to free any plastic. The dripper needle should be washed by injecting ethanol and/or water into the opening with a syringe. We recommended cleaning the needle after every sample or every other sample and when you finish a procedure.

6. Purified MAV-1 DNA can be made by digesting the dialyzed virus in 0.5% SDS and 250 |g/mL of proteinase K at 50-55°C for 15 min. Extract with phenol one or two times and re-extract the first phenol phase with TE. Extract with chloroform twice to remove the phenol. Dialyze against TE, pH 8.0, at 4°C for four changes of at least 4 h each. Read the A260 and store at 4°C. Expect the yield to be <0.1 mg/mL.

7. When making the gradient containing guanidine HCl, put the dialyzed virus onto two gradients to have a balance in the centrifuge.

8. To generate a plasmid with a mutation in an MAV-1 gene, first clone the wt gene of interest into a vector and mutagenize using oligonucleotide-directed mutagen-esis (Amersham or Stratagene), PCR mutagenesis, or by replacing genomic DNA sequence with cDNA sequence. To screen for potential mutants generated by oligonucleotide-directed mutagenesis, a unique restriction site. Mutagenesis is a unique restriction site that can be incorporated into the mutation. The presence of the site will distinguish mutant from wt in subsequent steps. To prevent digestion of the plasmid DNA by the enzyme used to digest DNA-protein complex (when the two are mixed together for the transfection), you can first treat the plasmid DNA with the appropriate methylase (£coRI methylase in this case), following the manufacturer's instructions (4).

9. A complementing cell line may be necessary to obtain a stock of some mutants.

10. Other transfection methods may also be used, but this method has been most consistently successful in our experience.

11. Alternatively, air can be bubbled into the 2X HEBS while the DNA mix is added.

12. Use general precautions to prevent contamination of samples that will be used for PCR. For example, always use plugged pipet tips and remove clean tubes and reagents from containers with forceps or with newly gloved hands that have not come in contact with any DNA. Also, use water that is sterile and used only for PCR to make solutions used for this procedure or in the PCR reaction itself.

13. DNA extraction methods of Shinagawa et al. (30) have also been successfully used to isolate MAV-1 DNA from infected cells (A. Kajon and K. Spindler, unpublished).

14. When inoculating cells with a plaque, the viral titer may be very low; thus, CPE may not begin to appear for up to 10-14 d. However, the cells should be checked daily to determine if CPE is present. Harvest the virus-infected cells after 10-14 d if no CPE is visible and use as a virus stock.

15. Do not use all of the purified plaque stock in case you need to repeat the procedure. Also, do not set up a wt virus infection as a control when growing up stocks of the mutant viruses because it will greatly increase the chance of the mutant stocks becoming contaminated with wt virus.

16. MAV-1 is released into the medium in a wt infection; thus only the medium is usually harvested to make a virus stock. However, the effect of the mutation(s) is unknown, so scraping the cells into the medium and freeze-thawing is recommended for mutants until it can be determined that the virus is released from the cells as in a wt infection.

17. Once there is a virus stock available, the DNA sequence of the virus should be confirmed to determine if the desired mutations are present and if the stock is pure. DNA for sequencing may be obtained from 60-mm plates using the Hirt method described in Subheading 3.3.2. We have been unable to sequence directly from these DNAs; therefore, we recommend PCR amplifying the regions of interest, removing the primers, and sequencing using the fmol Sequencing Kit (Promega) (see Subheading 3.3.4., step 5). Alternatively, the DNA may be easily obtained by the following protocol. Remove 100 |L of medium from cells that are in the late stages of infection and boil for 5 min to denature the viral structural proteins. Centrifuge the samples for 10 s. Mix the stock container of StrataClean™ resin (Stratagene) and add 10 |L of resin to each sample to bind the proteins. Flick the tube to mix and centrifuge for 30 s to pellet the resin. Transfer the supernatant to a clean tube and use 1-2 |L for PCR. Follow Subheadings 3.3.3. and 3.3.4. to PCR amplify and sequence these DNAs.

18. The tubes are numbered according to what their dilution on the plate (DOP) will be. The dilution in the first tube (no.2) is a 10-fold dilution. Only 0.1 mL of this will be used to inoculate the plate, and thus it is a 100-fold dilution; the DOP will be 102.

19. When overlaying cells with medium/agarose for a plaque assay, keep the medium and agarose separate until ready to overlay. Warm the medium in a container that is big enough to accommodate the agarose as well. When ready to overlay, add an equal volume of agarose to the aliquot of medium and use immediately. Have separate aliquots of medium/agarose for each mutant when overlaying and overlay the mock plate first, followed by the plates infected with the least amount of virus (108), continuing in order to those plates infected with the most amount of virus (103). Prepare enough medium/agarose to overlay one to two extra plates.

20. The plaques should be visible as holes in the monolayer of cells or 1- to 2-mm circular areas where the density of the monolayer appears lighter or heavier than the surrounding area when the plates are held up to the light. The formation of a plaque can be confirmed by circling the area with an ethanol soluble pen and viewing under the microscope using the 10X objective. The plaque will appear as a roughly circular area of dead cells. The plaques will be more difficult to see than human adenovirus plaques, and seeing them macroscopically is easier if observed against a black background. We have a piece of black paper on the ceiling near an overhead light and this seems to be crucial for seeing MAV-1 plaques.

21. Wild-type virus generally grows to a titer of approx 107 or 108 PFU/mL; however, some mutants, depending on the nature of the defect, may grow to 10- to 100-fold lower titers than wt virus. The titer of the stocks may also decrease on multiple freeze-thaws, so we recommend aliquotting the large stock of virus, especially if small amounts of virus are needed to obtain the desired multiplicity of infection.


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