Figure 8: Depletion of CIB proteins in primary CD4+ T-lymphocytes influences Env-mediated virus entry but not viral attachment.
From: CIB1 and CIB2 are HIV-1 helper factors involved in viral entry
(A) Viral binding assay. Activated CD4+ T-lymphocytes transduced with vectors leading to the expression of the indicated shRNAs were incubated with the indicated amounts of HIV-1NL43 (20 or 100 ng p24) in serum-free RPMI-1640 medium for 2 h at 4 °C. Cells were washed to remove unbound virus, pelleted, and lysed, after which cell-associated HIV p24 was measured by ELISA. Values represent mean ± SEM of 4 independent transductions. (B–D) Viral fusion and entry. CD4+ T-lymphocytes expressing the indicated shRNAs were exposed to HIV-1 particles (200 ng p24) containing BlaM-Vpr and pseudotyped with either an X4-tropic HIV-1 Env (B), R5-tropic HIV-1 Env (C), or VSV-G (D) for 4 hours, after which the CCF2 substrate was added. Cells containing the cleaved substrate were quantified by flow cytometry to assess viral entry. As a control, non-transduced cells were infected with a virus expressing a HIV-1 Env with the F522Y mutation, which prevents viral entry (F522Y NT in panel B). Values represent the mean ± SEM of at least 4 independent transductions and are expressed relative to results obtained for non-transduced (NT) cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus sh-SCRAM (Mann Whitney test). (E,F) Cell-mediated virus transmission. Non-transduced activated primary CD4+ T-lymphocytes previously infected using 100 to 500 ng of HIV-1NL43 and labeled with CFSE dye were used as donor cells. Untransduced cells and those transduced with the indicated shRNAs were co-cultured with the CFSE-labeled donor cells, such that targets could be distinguished from donors. The appearance of intracellular Gag positive (iGag+) target cells was measured 4 h (E) or 24 h (F) later, to quantify the efficiency of virus transfer and productive infection, respectively. To serve as controls, untransduced cells were treated with either nevirapine (NVP) or T-20. Values represent the mean ± SEM of 3 independent transductions and are expressed relative to the values observed for non-transduced cells. *P < 0.05, **P < 0.01 versus sh-SCRAM (Mann-Whitney test).
