A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

1. Amplification of the HIV-1 gag Gene from Infected, Frozen Plasma

1. Extract viral RNA from 140 µl thawed HIV-1 infected plasma using an extraction kit.
   1. When feasible, immediately proceed to cDNA synthesis after RNA extraction as unfrozen viral RNA yields the best amplification results. If possible, set up PCR master mix for first-round DNA amplification and store at 4 °C prior to viral RNA extraction.
2. Reverse-transcribe cDNA from RNA and amplify first-round DNA products using reverse-transcriptase and a thermostable DNA polymerase in a one-step RT-PCR.
   1. Take RNA out of -80 °C freezer (if freezing was necessary) and briefly thaw at room temperature then place in cold block. Transfer 5 µl of RNA to each PCR tube containing 45 µl of master mix to give a final volume of 50 µl. Immediately place remaining RNA samples into -80 °C freezer.
   2. After enzyme is added to the first-round PCR master mix (Table 1A), aliquot 45 µl into each thin-walled PCR amplification tube for the number of reactions desired. Make sure to use PCR tubes with individual caps and not strip caps to ensure minimal cross-contamination between samples. Place PCR tubes in a cold block to protect the temperature sensitive RT enzyme and RNA template. Note: Samples should be run in triplicate in order to adequately sample the viral quasispecies. It is also important to utilize a product with a high fidelity DNA polymerase enzyme in order to minimize PCR-introduced misincorporation. Please refer to the reagents list for the recommended product.
   3. After RNA template has been added to all reaction tubes, transfer PCR tubes to a thermocycler for amplification using the cycler program described in Table 1B. Note: The product of this amplification will be used in a second-round nested PCR.
3. Perform a nested second-round PCR amplification, using 1 µl of the first round PCR amplification (1.2) as the DNA template.
   1. Aliquot 49 µl of second-round PCR master mix (Table 2A) to each thin-walled PCR tube for the number of reactions desired. Transfer 1 µl from the first-round PCR amplification to each reaction tube to give a final volume of 50 µl. Note: This will serve as template DNA for the second-round amplification. It is also important to utilize a DNA polymerase with very high fidelity and proofreading capabilities in order to minimize PCR-introduced misincorporation. Please refer to the reagents list for the recommended product.
   2. Transfer second-round PCR reaction mix to thermocycler and run program described in Table 2B. Note: After completion of the program, the products of this reaction will be run on an agarose gel to confirm the production of product.
4. Add 3 µl of 5x loading dye to 5 µl of the 50 µl second-round reaction volume and run at 120 V on a 1% agarose-TAE gel containing a UV-fluorescent DNA stain until bands are resolved. Visualize 1.6 kb bands on a blue light illuminator (see Figure 1A).
5. Run the remaining 45 µl reaction volume on a 1% agarose-TAE gel containing a DNA stain, and excise the appropriate bands with a clean razor blade.
   1. Extract DNA from the gel slice using a gel purification kit, elute in nuclease-free water, combine three positive reactions per individual, and freeze product at -20 °C for subsequent use.

2. Preparing gag Amplicons for Cloning by Introduction of the Necessary Restriction Sites

1. Amplify the Long Terminal Repeat (LTR)/5’ UTR portion of the MJ4 plasmid (see Table 3).
2. Visualize 1.3 kb PCR products on illuminator, excise positive amplicons, gel purify and freeze as previously described (1.4,1.5; see Figure 1B).
3. Create fused MJ4 LTR-gag amplicons via "splice-overlap-extension" PCR (see Table 4).
4. Visualize, excise, purify, and freeze the 3.2 kb amplicons as in 1.4,1.5 (see Figure 1C).

3. Cloning Amplified gag Genes into the MJ4, Subtype C, Infectious Molecular Clone

1. Digest 1.5 µg of MJ4 plasmid and 1.5 µg of purified LTR-gag PCR product with the BclI (methylation sensitive) restriction endonuclease for 1.5 hr at 50 °C (see Table 5A).
2. Add 1 µl of NgoMIV to the digestion reaction and incubate at 37 °C for 1 hr.
3. Add 5x loading dye to restriction digest reactions and slowly electrophorese the total volume on a 1% agarose-TAE gel containing a DNA gel stain for 1-2 hr at 100 V. Visualize, excise, and purify indicated bands for cloning as previously described (see Figure 2).
4. Prepare ligation reactions using purified LTR-gag insert and MJ4 vector DNA at a 3:1 insert to vector ratio. Incubate ligation reactions overnight at 4 °C (see Table 5B).
5. Transform JM109 chemically competent bacteria with ligation products and spread on LB agar plates with 100 µg/ml ampicillin and grow at 30 °C for 22+ hr.
   1. Thaw JM109 competent cells on ice for 15 min. Label 1.5 ml microcentrifuge tubes and chill on ice.
   2. Aliquot 50-100 µl of JM109 cells to pre-chilled 1.5 ml microcentrifuge tubes. Add 2.5-5 µl of ligation reaction to JM109 cells, flicking tube lightly to mix and immediately returning to ice. Incubate competent cells with ligation product on ice for 30 min.
   3. Heat shock 1.5 ml microcentrifuge tubes containing JM109 competent cells and ligation reaction in a 42 °C heat block for 45 sec and return to ice for at least 3 min.
   4. Add 50 µl of SOC media to each microcentrifuge tube and plate the entire transformation reaction onto room temperature LB-agar plates supplemented with 100 µg/ml of ampicillin.
   5. Transfer plates to a 30 °C incubator and leave for 20 hr, or until colonies are clearly formed.
6. Pick isolated colonies and grow in 4 ml of LB with 100 µg/ml ampicillin at 30 °C for 22+ hr.
7. Spin down cultures at 3,200 x g for 15 min, pour off broth, and extract plasmid DNA.
8. To confirm cloning fidelity, cut miniprep DNA with NgoMIV and HpaI restriction enzymes in a double digest at 37 °C for 2 hr. Analyze on 1% agarose-TAE gel (see Table 5C).
9. Sequence the LTR-gag insert region of each plasmid using the following primers: GagInnerF1, GagF2, Rev1, Rev3, and GagR6 (Table 6) to confirm sequence identity.
10. Compare the cloned sequences obtained with the population sequences derived from the initial purified amplicon from 1.5.1. Note: It is important to ultimately assess the replication capacity of two independent clones in order to ensure that any in vitro replication phenotypes are not due to backbone errors introduced during the cloning process.

4. Generation and Titering of Replication Competent Gag-MJ4 Chimeric Viruses

1. Generate replication competent virus by transfecting 1.5 µg of the chimeric MJ4 plasmid miniprep DNA into 293T cells using a 4:1 ratio of Fugene HD as described by Prince et al³⁹.
2. Titer harvested virus stocks on TZM-bl cells as described below and in Prince et al³⁹.
   1. Plate TZM-bl cells 24 hr before infection with virus in order to have a 30-40% confluent cell monolayer on the following day. This can usually be achieved by adding 5 x 10⁴ cells in a total volume of 800 µl per well in a 24-well plate.
   2. After adding cells to the well, gently move the 24-well plate forward and back, then side to side in order to efficiently distribute cells. Never swirl – this will result in cells accumulating in the center of the plate.
   3. The following day, prepare 1% FBS in DMEM; this will be used to dilute DEAE-Dextran and to dilute virus stocks. Stock DEAE-Dextran is 10 mg/ml or 125x, and a final concentration of 80 µg/ml or 1x is desired.
   4. Take out virus stock to be tested from -80 °C freezer and place on shaker to thaw at room temperature.
   5. Using a multichannel pipette, serially dilute virus stocks in a round-bottomed tissue culture treated 96-well plate with lid. This is a 3-fold dilution protocol. See Table 7 for the dilution scheme.
   6. Remove media from seeded TZM-bl 24-well plates using a vacuum aspirator making sure not to disturb cell monolayer. Only remove the media from one 24-well plate at a time so that cells do not dry out.
   7. Add 150 µl of the 1x DEAE-Dextran + 1% FBS in DMEM mixture to each well using a 1,000 µl pipette. Do not use repeat pipette as this disrupts the monolayer.
   8. Add 150 µl of each virus dilution to the appropriate well. Add virus to the center of the well and swirl gently to evenly distribute virus across the cell monolayer. Incubate infected cells for 2 hr in a tissue culture incubator at 37 °C and 5% CO₂.
   9. After the 2 hr incubation, add 0.5 ml 10% FBS in DMEM to each well and return plates to incubator for an additional 48 hr.
   10. After 48 hr, cells should be 100% confluent. Because MJ4 is a replication competent virus, there will be some cell death, but this is to be expected.
   11. Remove media from each well and add 400 µl/well of fixing solution (see Table 8A for recipe). Fix only one plate at a time. Add fixing solution using a 1,000 µl pipette, and let sit for 5 min at room temperature.
   12. Remove fixing solution and wash 3x with PBS with Ca²⁺/Mg²⁺. Use a squirt bottle to gently add PBS to the side of the well to avoid disrupting the monolayer. After the third wash, blot plate dry on absorbent paper.
   13. Add 400 µl/well of staining solution (see Table 8B for recipe) to each well of the 24-well plate, and incubate at 37 °C for at least 2 hr. Longer incubation times are acceptable, but incubation times should be kept consistent between titering experiments.
   14. Wash 2x with PBS with Ca²⁺/Mg²⁺ and score for infection, or add PBS and store at 4 °C for scoring later. Plates can be kept at 4 °C for up to four days, as long as the monolayer is kept hydrated.
   15. To score for infection, use a permanent marker to divide wells of the 24-well plate into quadrants. Count all blue cells within a field of view, using a total magnification of 200X, once in each of the four quadrants of a well.
   16. Compute Infectious Units per µl as follows: [(# blue cells/4) x 67] / (µl virus added) = IU/µl. Refer to Table 8C for volume in µl of virus added to each well. Average several wells together; the numbers should be relatively similar for an accurate titration.

5. Preparation of Culture Media and Propagation of GXR25 Cells for in vitro Replication Assay

1. Prepare complete RPMI (cRPMI) for propagation of GXR25 cells. NOTE: It is extremely important to culture GXR25 cells at least 4 months prior to planned replication experiments (see Table 9).
2. Split cells 1:10 twice per week and maintain cell density between 1 x 10⁵ to 2 x 10⁶ cells/ml.

6. In vitro Replication of Gag-MJ4 Chimeras in GXR25 (CEM-CCR5-GFP) Cells

1. The day before the infection, split cells to a concentration between 2-3 x 10⁵ cells/ml so that they are in logarithmic growth on the day of the infection.
2. On the day of infection, remove virus stocks from -80 °C freezer and thaw. Calculate the volume of virus needed from each stock based on the IU/µl titer from TZM-bl cell assay in order to infect 5 x 10⁵ GXR25 cells at a multiplicity of infection of 0.05, using the formula:
   Volume of virus for infection (µl) = 1 µl/X IU x 0.05 x 500,000
   NOTE: We have chosen an MOI of 0.05 as this results in a logarithmic growth phase for all of the viruses that we have tested. It is important to conduct initial experiments in order to determine the optimal MOI to capture logarithmic growth for the virus to be tested.
3. Dilute virus in cRPMI to a volume of 100 µl (in order to achieve a 0.05 MOI) and add into a well of a V-Bottom tissue culture treated 96-well plate. Note: Include both a positive (MJ4 WT infected) and a negative (mock infected cells) control in each set of replication experiments. Set up the plate such that every other column is blank to limit cross-contamination between wells. The positive control is imperative, as it will be used later as a standard to normalize the replication slopes, which are a measure of the replication rate of experimental replicates.
4. Count GXR25 cells using an automated cell counter. Calculate the number of cells needed in total for all infections (5 x 10⁵ x # infections). Aliquot the required volume into a 50 ml conical tube and centrifuge to pellet cells. Always calculate for 25% more infections than needed.
5. Aspirate media carefully and resuspend in cRPMI at a concentration of 5 x 10⁵ cells/100 µl.
6. Pipette cells into a sterile trough and mix thoroughly. Using a multi-channel pipette, add 100 µl of cells into each well of the 96-well plate containing the diluted virus. Mix thoroughly.
7. Add 2 µl of 5 mg/ml (100x) solution of polybrene to each well and mix cells thoroughly.
8. Incubate at 37 °C in tissue culture incubator with 5% CO₂ for 3 hr.
9. In order to wash infected cells, centrifuge 96-well V-bottom plate to pellet cells. Then carefully remove 150 µl of the medium and replace with fresh 150 µl of cRPMI without disturbing the cell pellet. Repeat 2 more times (including centrifugation) to sufficiently wash cells.
10. After the last centrifugation and addition of 150 µl cRPMI, resuspend the cell pellet with a multichannel pipette and add the entire cell/virus mixture (approximately 200 µl) from each well independently to 800 µl of cRPMI in a well of a 24-well tissue culture plate.
11. Place plate in 5% CO₂ tissue culture incubator at 37 °C.
12. Every two days, remove 100 µl of supernatant from surface of culture well and transfer to a 96-well U-bottom plate and store frozen at -80 °C until running virus quantitation assay. NOTE: Careful arrangement of supernatants in 96-well plates will allow for multi-channel pipette transfer of culture supernatants during virion quantification via a radiolabeled reverse transcriptase (RT) assay. Every plate should contain samples from an infection standard in order to normalize inter-plate variation in the RT assay readout.
13. After removing each 100 µl sample, split cells 1:2 by thoroughly resuspending cells and removing half of the remaining volume (450 µl). Restore original volume (1 ml) by adding 550 µl of fresh cRPMI to each well.

7. Analysis of Reverse Transcriptase (RT) in Cell Culture Supernatants

Protocol adapted from Ostrowski et al⁴⁰.

1. Set up biological safety hood in a BSL-3 facility for use with radioactive materials.
   1. Place absorbent paper in hood and replace aspirator for aspirator designated for radioactive use. Note: All radioactive waste must be properly disposed of with accordance to safety regulations.
2. Add 1-2 µl of 10 mCi/ml of [α-³³P] dTTP and 4 µl of 1 M dithiothreitol (DTT) to each 1 ml aliquot of RT master mix (see Table 10 and Ostrowski et al.⁴⁰).
3. Carefully mix each 1.5 ml microcentrifuge tube of RT master mix, DTT, and [α-³³P] dTTP with a 1,000 µl pipette and transfer to a sterile trough.
4. Dispense 25 µl of labeled RT mix into each well of 96-well thin-walled PCR plate. Note: Include space for a positive control (MJ4 WT infection) and negative control (Mock infection).
5. Add 5 µl of each supernatant to the PCR plate containing the RT master mix.
6. Seal PCR plate with adhesive foil cover and incubate for 2 hr at 37 °C in a thermocycler. For safety reasons, rinse pipette tips with Amphyl and dispose of tips in small container containing Amphyl waste, which will later be disposed of in radioactive waste.
7. After incubation, make small holes in the foil cover using a 200 µl multi-channel pipette. Note: If pipette tips touch liquid in well, replace tips before moving to the next column or row.
8. Mix samples 5x and transfer 5 µl of each sample to the DE-81 paper. Air dry 10 min.
9. Wash blots 5x with 1x SSC (sodium chloride, sodium citrate), and then 2x with 90% ethanol at 5 min per wash. Allow to air dry.
   1. Place each blot in a separate washing container, such as a sandwich storage box, add enough wash buffer (1x SSC or 90% EtOH) to cover blot, and shake for 5 min.
   2. Pour off wash buffer into separate container and repeat. Note: The first 3 washes are considered radioactive and should be disposed of properly. The last 2 washes can be disposed of regularly.
10. Once dry, carefully wrap blot in Saran wrap and expose to a phosphoscreen in a tightly sealed cassette overnight at room temperature.
11. Analyze phosphoscreens with a phosphorimager and quantify radioactive transcripts.
12. Draw a circle around each radioactive signal using OptiQuant software. Graph the Digital Light Unit (DLU) values to generate replication curves.
13. In order to generate replication capacity scores, DLU values were log₁₀-transformed and slopes were calculated using the day 2, 4, and 6 time points. Replication slopes are then divided by the slope of WT MJ4 in order generate replication capacity scores. It is important to normalize based on the MJ4 WT values obtained from the same RT plate to reduce intra-assay variability.