The overall goal of this procedure is to measure the in vitro integration activity of pre-integration complexes, or PICs, isolated from HIV-1 infected SupT1 cells. This method will answer some of the key questions in HIV biology, specifically how certain host factors and viral factors, for example, viral capsid, plays a role in HIV-1 DNA integration, and doing so will help us design or identify novel targets for antiretroviral therapy. The main advantage of this method is that it requires relatively small amount of the pre-integration complex, or PIC, preparation for measuring the integration activity in vitro.
The implications of this technique can extend towards diagnosis as well as therapy of HIV-1 AIDS, and this is because the earlier versions of this technique have been critical in the discovery of HIV-1 integrase inhibitors, which are one of the major anti-HIV drugs in clinical use today, so this method can not only be used to understand HIV-1 integration, but it can be also applied to study other retroviruses, such as RSV and MLV. Demonstrating this protocol will be Dr.Muthukumar Balasubramaniam, who is a post-doctorate fellow in the laboratory. To infect SupT1 with HIV-1, in a BSL-2 lab, maintain SupT1 cells between one and two times 10 to the sixth cells per milliliter in Roswell Park Memorial Institute or RPMI medium supplemented with 10%FBS and Pen-Strep.
After generating high titers of infectious HIV-1 according to the text protocol, pool the SupT1 cells from the culture flasks, mix well by pipetting, and use trypan blue exclusion staining in a hemocytometer to determine the viable cell count. Aliquot eight times 106 cells per virus infection in 50 milliliter conical centrifuge tubes, then centrifuge the cells in a swinging bucket rotor at 500 times g and 25 degrees Celsius for five minutes. Without removing the cell culture medium, transfer the tubes containing pelleted cells to the BSL-3 lab for further processing, then carefully aspirate the medium from the tube without disturbing the pellet.
Next, add 30 milliliters of DNase I treated virus inoculum to the cells and mix by gently pipetting, then distribute the mixture evenly between two six well tissue culture plates. For spinoculation, place the culture plates in plate carriers and centrifuge in a swinging bucket rotor at 480 times g and 25 degrees Celsius for two hours, then incubate the cells at 37 degrees Celsius and 5%carbon dioxide for five hours. To harvest and lyse the infected cells, pool the cells corresponding to each infection into a 50 milliliter conical centrifuge tube.
Centrifuge the cells at 300 times g and 25 degrees Celsius for 10 minutes, then carefully aspirate or pipette the supernatant. To remove any residual virus particles, wash each pellet by adding two milliliters of buffer K minus minus and spin the sample again, then without disturbing the pellet, carefully remove the supernatant by aspiration or with a pipette before repeating the wash one more time. After carefully removing any residual buffer with a micropipette, use ice cold lysis buffer K plus plus to gently re-suspend each pellet and transfer to a two milliliter microcentrifuge tube.
To prepare cytoplasmic pre-integration complexes, or PICs, rock the sample at room temperature for 10 minutes, then place the tubes into a rotor pre-cooled to four degrees Celsius, and centrifuge at 1, 500 times g and four degrees Celsius for four minutes. Without disturbing the pellet, carefully transfer the supernatant to a fresh microcentrifuge tube and spin at 16, 000 times g and four degrees Celsius for one minute. Carefully transfer the supernatant to a fresh microcentrifuge tube, then add RNase A to a final concentration of 20 micrograms per milliliter, and incubate the contents at room temperature without rocking for 30 minutes.
Add sucrose to a final concentration of 7%and mix well by gently pipetting, then prepare aliquots of the PICs in microcentrifuge tubes. Flash freeze in liquid nitrogen and place in a negative 80 degrees Celsius freezer for long term storage. To prepare the target DNA used in the in vitro integration or IVI assay, assemble the PCR mixtures to amplify a 2.0 kilobase region from the phiX174 plasmid DNA using the custom made primers phiX174-Forward and phiX174-Reverse, and carry out PCR using the program according to the text protocol.
After gel purifying and aliquoting the sample, add 600 nanograms of target DNA to 200 microliters of PICs. Mix well and incubate at 37 degrees Celsius for 45 minutes. Stop the IVI reaction by adding SDS, EDTA and proteinase K, then thoroughly mix the reaction contents and incubate at 56 degrees Celsius overnight.
The following day, to purify the total DNA from the IVI reaction, add an equal volume of phenol to the deproteinized sample. Mix vigorously by vortexing and centrifuge for two minutes. Carefully remove the upper aqueous phase and transfer it to a fresh tube before repeating the phenol extraction, then add an equal volume of one to one phenol chloroform.
Mix the sample by vortexing and centrifuge the tube for two minutes, then transfer the upper aqueous phase to a fresh tube. After extracting with chloroform according to the text protocol, precipitate the DNA from the aqueous phase by adding 0.1 micrograms per microliter of glycogen, 0.3 molar sodium acetate and 2.5 volumes of ice cold 100%ethanol. Place the tube at negative 80 degrees Celsius overnight.
The next day, centrifuge the sample at 16, 000 times g and four degrees Celsius for 30 minutes, then carefully remove the supernatant without disturbing the pellet. To carry out PCR for quantifying PIC activity, begin with the first round of conventional PCR using primers targeting the viral LTR and the phiX174 DNA to amplify the junctions of the integrated HIV-1 DNA. Assemble the master mix minus the template DNA as outlined here, then dispense 45 microliter aliquots into separate PCR tubes and add five microliters of template DNA or nuclease free water as a control.
Run the reaction in a thermocycler using the program outlined in the text protocol, then carry out a second round of quantitative PCR and analyze the data according to the text protocol. In this IVI assay, SupT1 were spinoculated with DNAse I treated HIV-1, and the absence or presence of the HIV-1 integrase inhibitor, Raltegravir. After incubation, PICs were isolated and the purified DNA was analyzed for the integration of viral DNA.
As shown here, the HIV-1 PICs robustly integrate the associated viral DNA into the target DNA and the IVI efficiency of PICs were greatly reduced in the reactions supplemented with Raltegravir, which strongly implicates the integration activity measured in this assay to HIV-1 integrase. The control reactions did not show any integration activity. In addition, the control that excluded Taq polymerase in the first round of PCR did not amplify integration products.
Notably, the data from the PICs alone control indicated that the nested qPCR strategy specifically measured the integrated viral DNA, but not the PIC-associated viral DNA. Collectively, these data demonstrate the PIC-associated integration activity. Once mastered, virus preparation and PCR assays for quantifying the PIC activity can be completed in a week.
Infection of the SupT1 cells and isolation of the PICs from the infected cells can be done in a day. After watching this video, you should have a good understanding of how to isolate HIV-1 PICs and measure the in vitro integration activity. Don't forget that working with high titers of infectious HIV-1 can be extremely hazardous and appropriate precautions, including protective clothing such as aprons and gowns, double gloves, and protective eyewear, should always be taken while performing this procedure.
