High rates of mutation and recombination help human being immunodeficiency virus (HIV) to evade the immune system system and develop resistance to antiretroviral therapy. overall recombination rate. This indicates that the difference in rates is a general feature of HIV DNA synthesis during macrophage infection. In contrast to HIV recombination, we found that T-cells have a 30% higher mutation rate than macrophages (< 0.001) and that the mutational profile is similar between these cell types. Unexpectedly, we found no association between mutation and recombination in macrophages, in contrast to T-cells. Our data highlights some of the fundamental difference of HIV recombination and mutation amongst these two major target cells of infection. Understanding these differences will provide invaluable insights toward HIV evolution and how the virus evades immune surveillance and anti-retroviral therapeutics. 1.46/1000nt) in macrophages compared to T-cells respectively, but interestingly a lower mutation rate (0.091/1000nt 0.12/1000nt respectively). Although we found similar recombination hotspots between the two cell types, we were unable to observe an association between mutation and recombination in macrophages. 2. Materials and Methods 2.1. Molecular Clones The HIV-1 molecular clones have been previously described [8,16,18]. Briefly, the wild-type (WT) molecular clone PROML1 pDRNLAD8 is based on the prototypic HIV strain pNL43, engineered to remove 1.5 kb of cellular DNA flanking the HIV-1 genome in the pNL43 construct [19], and to express the R5-tropic AD8 envelope. The marker (MK) molecular clone pDRNLAD8MKhigh is a modified pDRNLAD8 clone engineered to AZD5438 contain 15 and 34 silent marker points in and and cooled to 12 C with a counter-centrifugal flow of FACS wash being pumped through the chamber at 12 mL/min. Smaller lymphocytes (T-cells, B-cells and natural killer (NK) cells) and residual platelets were collected into 50 mL conical tubes by allowing 1L of DPBS- to flow through the chamber and increasing the flow of the DPBS- to 15 mL/min. The flow of DPBS- was increased to 17 mL/min and monocyte enriched fractions were collected into 50 mL conical tubes. During collection the flow was increased by 1 mL/min every 100 mL until the monocytes were depleted from the chamber. Collection tubes were centrifuged at 320 for 10 min at 4 C and monocytes were pooled. Elutriation of PBMC from buffy coats typically yielded 5C10 107 monocytes. The purity of PBL and monocyte fractions was assessed by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA) and estimated to be 90%C95% pure based on forward scatter (FSC) and side scatter (SSC) characteristics. PBLs were stimulated in RF10 media (2 106 cells/mL) supplemented with 10 g/mL polyhydroxyalkanoate (PHA) and 10 units/mL human interleukin-2 (IL-2) (Roche Applied Science, Sydney, NSW, Australia) for two days in Teflon-coated jars. PBLs were then resuspended in fresh medium containing 10 units/mL human IL-2 (Roche Applied Science) and incubated for a further two days before infection. Monocytes were cultured in IH10 medium, adherent to plastic, and allowed to differentiate into monocyte-derived macrophages (MDMs) for seven days before infection. 2.3. Virus Production AZD5438 Viruses were produced by AZD5438 co-transfecting 293T-cells with HIV-1 molecular clones using polyethylenimine (PEI) [20]. PEI stocks were prepared at 1 mg/mL by dissolving PEI in water, adjusting the pH to 7.0, followed by filtration with a 0.2 m sterile syringe filter. 2.5 106 293T-cells were seeded into 100 mm2 tissue culture plates 24 h prior to transfection. Transfection mix was prepared by adding 3 g total HIV-1 proviral DNA to 500 L of serum-free DMEM and 27 L of PEI, vortexed and incubated for 5 AZD5438 min before addition to cells. 12 h post infection, cells were washed twice in.