Fig 2.

Generation of an HIV-sensing cell line. (A) Design of HIV reporter construct. An mCherry gene was cloned downstream of an HIV LTR and HIV TAR. Downstream of mCherry, a Rev response element (RRE) and internal SV40 promoter driving a puromycin resistance gene were added. A polypyrimidine tract (PPT) and a 3′ LTR were also included to allow generation of a packageable lentiviral RNA. (B) Overall scheme for generation of an HIV-sensing cell line shown. HOS cells were transduced with lentiviral particles containing the construct from A and selected with puromycin. Cells containing an HIV-responsive insert were enriched by transfection of Tat mRNA and sorting mCherry+ cells using an AIR cell sorter. (C) Flow cytometry of puromycin-resistant HOS cell population in the presence and absence of infection with an HIV-GFP reporter virus. (D) Flow cytometry showing the infection of a highly responsive HOS cell clone (A5) with HIV-GFP. (E) Flow cytometry showing stable expression of HIV receptors CD4, CXCR4, and CCR5 in the HIV-responsive A5 clone.