For quantitative strand transfer assays, donor DNA substrate is formed by annealing HPLC grade oligonucleotides 5'-GACTCACTATAGGGCACGCGTCAAAATTCCATGACA and 5'-ATTGTCATG GAATTTTGACGCGTGCCCTATAGTGAGTC. Reactions (40 μL) contains 0.75 μM PFV IN, 0.75 μM donor DNA, 4 nM (300 ng) supercoiled pGEM9-Zf(?) target DNA, 125 mM NaCl, 5 mM MgSO₄, 4 μM ZnCl₂, 10 mM DTT, 0.8% (vol/vol) DMSO, and 25 mM BisTris propane-HCl, pH 7.45. Raltegravir is added at indicated concentrations. Reactions are initiated by addition of 2 μL PFV IN diluted in 150 mM NaCl, 2 mM DTT, and 10 mM Tris-HCl, pH 7.4, and stopped after 1 hour at 37°C by addition of 25 mM EDTA and 0.5% (wt/vol) SDS. Reaction products, deproteinized by digestion with 20 μg proteinase K for 30 minutes at 37°C followed by ethanol precipitation, are separated in 1.5% agarose gels and visualized by staining with ethidium bromide. Integration products are quantified by quantitative real-time PCR, using Platinum SYBR Green qPCR SuperMix and three primers: 5'-CTACTTACTCTAGCTTCCCGGCAAC, 5'-TTCGCCAGTTAATAGTTTGCGCAAC, and 5'-GACTCACTATAGGGCACGCGT. PCR reactions (20 μL) contained 0.5 μM of each primer and 1 μL diluted integration reaction product. Following a 5-min denaturation step at 95°C, 35 cycles are carried out in a CFX96 PCR instrument, using 10 seconds denaturation at 95°C, 30 seconds annealing at 56°C and 1 minutes extension at 68°C. Standard curves are generated using serial dilutions of WT PFV IN reaction in the absence of INSTI.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Human MT-4 cells are infected for 2 hours with the SIVmac251, HIV-1 (IIIB) and HIV-2 (CDC 77618) stocks at a multiplicity of infection of, approximately, 0.1. Cells are then washed three times in phosphate buffered saline, and suspended at 5 × 10⁵/mL in fresh culture medium (to primary cells 50 units/mL of IL-2 are added) in 96-well plates, in the presence or absence of a range of triplicate raltegravir concentrations (0.0001 μM-1 μM). Untreated infected and mock-infected controls are prepared too, in order to allow comparison of the data derived from the different treatments. Viral cytopathogeniciy in MT-4 cells is quantitated by the methyl tetrazolium (MTT) method (MT-4/MTT assay) when extensive cell death in control virus-infected cell cultures is detectable microscopically as lack of capacity to re-cluster. The capability of MT-4 cells to form clusters after infection. Briefly, clusters are disrupted by pipetting; and, after 2 hours of incubation at 37°C, the formation of new clusters is assessed by light microscopy (100× magnification). Cell culture supernatants are collected for HIV-1 p24 and HIV-2/SIVmac251 p27 core antigen measurement by ELISA. In CEMx174-infected cell cultures, which show a propensity to form syncytia induced by the virus envelope glycoproteins, syncytia are counted, in blinded fashion, by light microscopy for each well at 5 days following infection.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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