Quantification of integrated HIV-1 DNA in mouse and human genome

The analysis of HIV-1 integration level in human cells was reported in detail previously [56]. To quantify integrated EcoHIV in mouse cells, we (i) prepared the integration standard curve and used two-step PCR amplification including first (ii) a 12-cycle nonkinetic preamplification followed by (iii) nested real-time PCR assay. The procedure is depicted schematically in Fig 1A. (i) Preparation of the integration standard (IS) DNA. Mouse RAW 264 cell line was infected with high-titer stock of VSV-G pseudotyped EcoHIV-ΔENV virus and cultured in vitro for 30 days to ensure degradation of extrachromosomal forms of viral DNA. The number of integrated EcoHIV DNA copies per cells was then determined by quantifying the amount of total HIV-1 DNA by real-time PCR [55] and it was found to be 1 copy/cell. The IS DNA was then diluted with spleen cell DNA from uninfected mice to keep the number of B1 sites per reaction constant. (ii) 12-cycle non-kinetic preamplification. In this step, the IS and the unknown samples were amplified for 12 cycles. In addition, for each unknown sample, a control reaction in which the mouse repeat element [57] primer pair (B1-F and B1-R) was omitted was run in parallel. Standard curves were prepared by making serial 5-log dilutions of IS DNA. The preamplification primers were: fNef, 5’-TACAAAGAAGCTGTTGATCTTAGCC-3’; murine cellular anchor primer B1-R, 5’-CCGAGTGCTGGGATTAAAGG-3’; and B1-F, 5’-CCAGGGCTACAGAGAAACCCTGTC-3’. The conditions for first-round PCR reaction were: a 2-min hot start at 94°C followed by 12 steps of the following: denaturation at 94°C for 30s, annealing at 50°C for 30s, and extension at 68°C for 16 min. (iii) Nested real-time PCR. The 2 μl preamplification product including both IS standard and unknown samples was supplemented with the following primers and a probe: LTRF, 5’-CTGGCTAATTAGGGAACCCACTG-3’; LTRR, 5’-GGACTAAACGGATCTGAGGGATCTC-3’; and the LTRP, 5’-(FAM)-TTACCAGAGTCACACAACAGACGGGCA-(MGBNFQ)-3’. The thermal program was 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C. Data analysis was performed with the 7500 System Software (ThermoFisher Scientific). The number of proviral copies in the unknown sample is the number of the total number of copies minus the number of copies in the control reaction. All samples were run in duplicate. HIV integrants were normalized to the number of cells in the sample determined in a parallel amplification of murine β-globin DNA.