Figure 4

HA-CycT1-V107E mutant inhibits HIV transcription in T cells and primary CD4(+) cells. a) HA-CycT1-V107E inhibits HIV replication in human T cell line - Jurkat T cells that harbor an integrated LTR-Tat-Rev-BFP provirus and express low levels of BFP (J-LTR-Tat-BFP), thus serve as model for viral latency, were transduced with lentiviruses expressing either HA-CycT1-wild type or HA-CycT1-V107E. Transduced cells stably expressing human CycT1 were generated following puromycin selection. J-LTR-Tat-BFP cells that did not express CycT1 were used as control. Cells were analyzed by FACS for their LTR-BFP expression. Presented is a FACS spectra dot-blot analysis for J-LTR-Tat-BFP cells expressing HA-CycT1-V107E (J-LTR-Tat/HA-CycT1-V107E; dark grey), or cells that expressed HA-CycT1-wild type (J-LTR-Tat/HA-CycT1 wild type; light grey spectra). The P3 gate was set based on J-LTR-Tat-BFP cells that did not express CycT1. Data is a representative of three separate experiments. Bottom panel shows HA-CycT1 protein expression levels in Jurkat cells as determined by anti-HA-WB. b) HA-CycT1-V107E inhibits HIV replication in primary CD4(+) T cells - Human CD4(+) T primary cells were isolated from naïve PBMCs and stimulated with CD3/CD28 beads for 48 hr. Isolated cells were then transduced with either HA-CycT1-V107E or HA-CycT1-wild type expressing lentivirus. 48 hr. post transduction, cells were infected with HIV-LTR-BFP, or CMV-BFP lentivirus at MOI of 1. BFP-mediated viral replication was analyzed by FACS to monitor effects of HA-CycT1 expression. Expression of HA-CycT1-wild type or HA-CycT1-V107E was confirmed by anti-HA WB.