Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

The overall goal of this procedure is to determine the relative replication fitness of HIV one strains of interest. This is accomplished by first constructing viral molecular clones or DNA plasmids containing full length HIV one sequences of interest. The second step is to generate viral stocks for each molecular clone by DNA transfection.

Next, the growth kinetics and the exponential growth phase of each HIV one strain are established by longitudinal sampling of mono infected cell cultures. The final step is to perform pairwise growth competition or dual infection in cell cultures. Ultimately reverse transcription, quantitative real-time PCR and DNA sequence Chromatogram Peak heights obtained from Sanger DNA sequencing are used to determine viral ratios, which are then used to calculate relative viral replication fitness.

The main advantage of this technique over existing methods like paraminol infection or to DU infection, is that it provides more sensitive and robust measurements of the replication finnet difference between two viral strains. The protocols presented in this paper encompass constructing HIV one recombinant clones, introducing mutations of interest by site directed mutagenesis, making viral stocks and performing fitness assays to establish viral growth kinetics and to determine relative viral fitness. This video will demonstrate to the growth competition and two different methods.

To determine the viral ratio for calculating fitness, please refer to the protocol text for all other procedures to prepare for the growth competition assay seed three times 10 to the fifth PHA stimulated peripheral blood mononuclear cells or P BMCs in a total volume of 500 microliters per well in a 48 well flat bottom plate. Keep the plate at 37 degrees Celsius in a 5%carbon dioxide atmosphere until inoculation. Next, prepare three milliliters of inoculum containing 6, 000 IU for each virus.

In a typical experiment, multiple mutants are tested, but for the purpose of this demonstration, only one mutant will be tested against the prototype virus. Proper preparation of the viral in alum is a critical step in this procedure. A serial dilution may be needed with the viral stalk with a very high infectious title Transfer, 1.5 milliliters of each viral inoculum to a sterile tube to create the dual infection inoculum.

Add 500 microliters of the dual inoculum to the PHA stimulated pbmc. In each well of the 48 well plate. The final culture volume is one milliliter per well aliquot, 200 microliters per well of the dual inoculum to 2 96 well plates.

For RNA isolation save one plate as a backup. Incubate the inoculated cells at 37 degrees Celsius with a 5%carbon dioxide atmosphere for 16 to 24 hours. 16 to 24 hours after inoculation wash the cultures, remove and discard 750 microliters of the culture supernatant add 750 microliters of fresh complete is scarves, modified docos medium or C-I-M-D-M.

Wrap the plate in plastic wrap and spin for 10 minutes. At 300 gs, remove and discard 750 microliters of the supernatant add 750 microliters of fresh C-I-M-D-M incubate the plates at 37 degrees Celsius with a 5%carbon dioxide atmosphere. Sampling times should be selected to include at least three time points within the viral exponential growth phase.

To collect a sample transfer 500 microliters of culture supernatant to a 1.8 milliliter centrifuge tube and spin for one minute. At 3000 Gs, transfer 200 microliters of the cell-free supernatant to 2 96. Well sample plates for RNA isolation, again, saving one plate as a backup store the super natan at negative 80 degrees Celsius until RNA isolation.

Add 500 microliters of fresh C-I-M-D-M to each culture and return the plate to the incubator until the next sampling time. Subsequently, RNA is isolated from the samples, followed by CDNA synthesis. Two methods for determining viral ratios will be demonstrated.

The first using reverse transcription, quantitative real-time PCR or R-T-Q-P-C-R and the second using Chromatogram Peak Heights. For QPCR, prepare a standard serial dilution series in triplicate of the plasmid PNL four three VIFA diluting tenfold in each step from three times 10 to the six copies per microliter to 30 copies per microliter. Set up a 96 well QPCR reaction plate.

Each plate should contain at least one negative control the standard dilution series in triplicate and duplicates of each CD NA sample. Use A-V-I-F-A primer probe to detect signals in the negative controls in the standard dilution series and with one duplicate of the CDNA sample. Use the VIFB primer probe with the other duplicate of the CD NA sample for each QPCR reaction use 12.5 microliters of the QPCR master mix.

0.2 micromolar of the QPCR probe. 0.8 micromolar each of the forward and reverse primers and one microliter of CD NA for the standard dilution series, add the serial dilution of PNL four three VIFA instead of CDNA for the negative controls, use water or buffer instead of CDNA, set the PCR cycling parameters following the manufacturer's instructions for operating the QP qPCR R machine. Calculate the standard curve using amplification data from the standard dilution series.

Compare amplification data of the CD NA samples to the standard curve to determine copy number. Use the viral growth rate calculation or GRC web tool to calculate relative viral fitness input. The CDNA copy number obtained from the RT qPCR R step in the specified format.

Shown selecting calculate will output the net growth rate difference between two competing viruses to determine viral ratios using chromatogram peak heights. PCR amplify, HIV one VIF fragments containing the VIF AB sequence tag using VIF forward and VIF reverse primers. For each PCR reaction, use one microliter of CDNA one XNH four buffer, 1.5 millimolar of M gcl two 0.2 millimolar of DTPs, 2.5 U of TAC polymerase, and 0.45 micromolar of each primer.

Add water to a final volume of 50 microliters. Set the PCR cycling parameters to three cycles at 94 degrees Celsius for one minute. 55 degrees Celsius for one minute and 70 degrees Celsius for one minute, followed by 34 cycles at 94 degrees Celsius for 15 seconds.

58 degrees Celsius for 30 seconds and 70 degrees Celsius for one minute. And finally, a hold at four degrees Celsius. Subsequently purify the PCR products using a commercially available kit and submit the purified PCR products to A DNA sequencing service provider for Sanger sequencing.

Check the average read quality score provided by the sequencing service. If the average base call accuracy is less than 85%repeat the PCR amplification purification of PCR products and Sanger sequencing. Use the Chromat quantitative web tool to calculate the viral ratio at each time point.

The inputs required are the leader sequence and the sequence chromatogram file. In AB one format. The tool measures the peak intensity at the specified site.

The ratio of the peak intensity corresponds to the ratio of the two viruses. Use the GRC web tool to calculate relative viral fitness. The input is the sequence chromatogram peak height obtained using the Chromat quantitative web tool in the specified format.

Shown the GRC web tool will calculate the net growth rate difference between the two competing viral strains, a prototype and three HIV mutant Viruses were constructed and their growth in pbmc was characterized. The TCID 50 ranged from 10 to the fourth to 10 to the fifth IU per milliliter. All viruses grew exponentially between day two and day four.

In the exponential growth phase, all three mutants had a slower growth rate than the prototype virus. Each mutant was competed against the prototype virus in growth competition assays at a total MOI of 0.005, viral growth kinetics in dually infected cultures was similar to that of mono infection. The viral exponential growth phase was between days two and four, and viral growth reached a plateau around day five.

Viral growth rate differences were derived from the change in the viral ratio over time. The viral ratio was calculated based on CDNA copy number of the reference and the mutant viruses using R-T-Q-P-C-R and by comparing peak heights in sequence chromatograms at nucleotide sites, distinguishing the two viruses, the T 2 42 N mutant is compared to the prototype. In this example, the growth rate differences determined using the two methods yielded similar results.

All three mutants had lower replication fitness than the prototype virus With the I 2 56 V mutant having the lowest fitness. After watching this video, you should have a good understanding of how to perform PWIs growth competition essays to determine the relative replication fitness of hiv.One.