Overview of the Quantitative Measurement of Viral Sequences In Vivo

We have developed a fully quantitative method for measuring integrated proviruses in infected cells in vivo using a combination of Alu PCR and Fluorescence-monitored quantitative PCR technology.

PDF Download S. L. Butler, M. S. T. Hansen, and F. D. Bushman . (2001) A Quantitative Assay for HIV cDNA Integration In Vivo. Nature Med. 7, 631-634.

DNA samples are generated by various Experimental Infections

Real-Time Fluorescence Monitored PCR:

The method in general:
assay The assay involves a forward, reverse, and Probe primer for each target sequence. The probe oligo has two fluorofores (reporter-yellow and quencher-green). The exonuclease activity of Taq polymerase releases the reporter yielding a signal proportional to the level of amplification plot Raw data of an experiment plots fluorescent signal (X-axis) against cycle number (Y-axis). A threshold is set to measure when a reaction enters the exponential phase of amplification (cycle of threshold).

Specific primers used for HIV sequences are HERE (PCR parameters too).

Target sequences we have studied in detail: primers The products of reverse transcription and 2-LTR circles can be measured and quantitated against reliable copy number standards. These are generated simply by cloning each target sequence into a plasmid of known size.
In order to detect integrated proviruses, the reverse primer was designed to a consensus of the Alu element, the most repeated sequence in the human genome. Quantitation of these products had an added challnge: Since HIV integration can occur virtually anywhere in the genome, the target sequence is never the same. As a result the amplicon size (and thus PCR efficiency) varies from one integration event to the next. Some integration events will be too far from the nearest Alu to amplify at all. aluamps

At this point we took advantage of the single round nature of the vector system: The southern blot shown represents a vector infection of SupT1 cells. Total DNA was isolated and cut either once or twice to and probed with an internal sequence to distinguish the unintegrated forms (1 cut) from total viral DNA (2 cut). south

Between 3 and 5 days post-infection, the unintegrated forms have either degraded or been diluted away by several rounds of cell replication. The total DNA however, levels out to a signal that carries out to later points as well. This signal represents integrated proviruses that are now being replicated along with the cell genome.

If such an infection is carried out for several weeks, We can be certain, as confirmed by southern blot, that the only viral sequences present are integrated proviruses. This is how the "Alu Standard" DNA is made: chart

This standard curve, run along with every Alu experiment, yields the number of proviruses present even in the presence of other viral sequences.

Quantitation of Viral cDNA by Real Time PCR

Reaction mixtures:

Reactions contained 1X Taqman universal master mix (Perkin-Elmer), 300 nM forward primer, 300 nM reverse primer, 100 nM probe primer, and 250 or 500ng of template DNA in a 30 µl volume.

Primer sequences
(See the method overview to see general location of each of the primers):

  • Late RT forward, MH531: 5'-TGTGTGCCCGTCTGTTGTGT-3'
  • Late RT reverse, MH532: 5'-GAGTCCTGCGTCGAGAGAGC-3'

  • 2-LTR circle forward, MH535: 5'-AACTAGGGAACCCACTGCTTAAG-3'

  • Alu forward, MH535 (above)
  • Alu reverse, SB704: 5'-TGCTGGGATTACAGGCGTGAG-3'
  • Alu probe, MH603 (above)

  • Mitochondrial forward primer, MH533: 5'-ACCCACTCCCTCTTAGCCAATATT-3'
  • Mitochondrial reverse primer, MH534: 5'-GTAGGGCTAGGCCCACCG-3'
  • Mitochondrial probe, Mito-Probe: 5'-(TET) CTAGTCTTTGCCGCCTGCGAAGCA (TAMRA)-3'

PCR parameters:

After initial incubations at 50°C for 2 min. and 95°C for 10 min, 40 cycles of amplification were carried out at 15 sec at 95°C followed by 1 min at 60°C (1 min 30 sec for Alu PCR). Reactions were analyzed using the ABI Prism 7700 sequence detection system and the SDS software package (PE-Applied Biosystems).

Experimental Infection of Non-Adherent Cells for quantitative PCR

Experimental infections can be carried out with as little as 10^6 cells at an MOI of less than 0.2. (Most of our experiments use 5x10^6 cells and a targetr MOI of 1 to 10.)

This protocol is designed around a six point timecourse experiment on dividing cells (3,6,12,24,48,72 hours).

This uses Qiagen Dneasy kit for DNA prearation. Begin with 6x10^6 cells per infection, duplicate or triplicate per condition. If inhibitors are being used: Prepare 5x or 10x stocks of the inhibitors in RPMI beforehand. Minimize the concentration of DMSO or other solvents as much as possible. Also control infection should have the solvent present at the same concentration. Pellet cells and resuspend in 1ml of 1x medium and incubate for 60¹ in the conical tube. Have virus solution ready to add, with all necessary components. You may also want an infection enhancer, DEAE dextran for vector or polybrene for wildtype virus. If virus/vector is high enough titer, you can add directly to exhisting medium (1.6ml virus and 400µl 5x inhib. for example). Otherwise spin cells down again and bring up in the same solution. Incubate with virus for 2 hours. Spin down cells, resuspend in 5ml of 1x medium. At 3 hours post-infection, collect first time point: Transfer 1ml of each culture to an eppi tube (remember to homogenize the culture whenever taking cells and remember they sink pretty quick). Spin at 5000rpm for 1min. Aspirate medium (be VERY careful not to aspirate the cells!!!) and wash with 1ml of PBS. Spin down again andresuspend cells in 200µl of PBS. Add 200µl lysis solution (Buffer AL) and pump mix until schlering stops. Store all timepoints at ­80°C until timecourse is complete. After collecting the 6hr timepoint, add back 1ml of fresh medium (+inhibitor). After 12hr timepoint, add 2ml more fresh medium (+inhibitor). Try to maintain similar concentration of cells for remainder of the experiment (+/­ 2-fold).

Prepare total cellular DNA using the Qiagen Dneasy protocol. On the final elution, 2x100µl is recommended to maintain concentrations high enough for Taqman experiments. Measure the concentration of each sample by OD260 of a 1:10 dilution. Calculate the dilution needed to bring each to 25ng/µl and make a second set of samples that are all normalized to that concentration. A standard sample for Taqman is 250ng, 10µl of these samples.

Sample Alu-PCR Data

This example presents a comparison of results from different methods for preparations of the cellular DNA.

DNA was extracted from SupT1 cells that had been infected with SM2 vector and grown for several weeks to eliminate unintegrated DNA. DNA was purified using one of two protocols –

  1. Using phenol extraction and ethanol precipitation as outlined in the Maniatis "Molecular Cloning" Manual.
  2. Using the DNEasy kit obtained from Qiagen.

Serial dilutions of the DNA were made using uninfected SupT1 DNA for dilution such that total DNA concentration in all samples was maintained constant at 25 ng/ul. Samples ranged from 25ng/ul to 0.04 ng/ul of infected SupT1 DNA (5 fold dilution series).

Standards were analyzed using the Taqman method. Primers and probe sequences (Alu-PCR set), experimental methods and cycling parameters used for determining number of integrated provirus copies are reported in Butler et al, (Nature Medicine 2001). A primer ratio of 50nM forward primer to 900nM of reverse primer per well was found to produce improved signals (unpublished data). This ratio was used in the analysis. Probe concentration was 100nM per well. 10 ul of DNA was added to each well for a total of 250 ng per reaction.

The figure below shows the data obtained from Alu PCR analysis of three sets of dilutions of infected SupT1 DNA (obtained from three different DNA preparations).

The curves with the lowest cycle of threshold and highest ΔRn correspond to the standards with highest concentration of long term infected SupT1 DNA (i.e. 250 ng/reaction).

Blue – DNA extracted using phenol/chloroform/ethanol pptation.
Yellow – DNA extracted using the DNEasy kit from Qiagen.
Red – DNA from an earlier prep extracted using phenol/chloroform/ethanol pptation.

Concentration of proviruses per genome

The average numbers of integrated proviruses per cell in the DNA samples analyzed are listed below. This was calculated by analyzing the same DNA preparations using primers and a probe that both bound within the HIV cDNA ("late RT" or "total cDNA" amplicon). This illustrates the concentration of proviruses needed per human genome equivalent to obtain a robust signal.

SB stock DNA US EtOH/CHCl3 US DNEasy Average
0.4 ng/well 0.004876222 0.004304616 0.005310313 0.00483038
2 ng/well 0.024272948 0.021925936 0.026663775 0.02428755
10 ng/well 0.141501244 0.166373417 0.16164729 0.15650732
50 ng/well 0.738368327 0.833222569 0.800920672 0.79083719
250 ng/well 4.130883799 4.869782087 4.76489155 4.58851915