Figure 6.

Resistance to R5-tropic HIV infection of HR5 cells transduced with anti-CCR5 mirGE lentivector. (a) Representative images of control parental HeLa cells (HeLa), untransduced HR5 (HR5), HR5 cells transduced with a lentivector expressing GFP alone (GFP), and HR5 cells transduced with a lentivector expressing GFP and three hairpins of anti-CCR5 mirGE (mirGE). Pictures of these four cell lines were taken at day 0, day 5, and day 7 of HIV-R5 infection. Only parental HeLa cells and mirGE-transduced cells survive the infection. Note that mirGE 1 µl indicates cells transduced with the lowest volume of vector corresponding to a 7% transduction rate. (b) Values of HIV p24 antigen in supernatants of cells showed in Figure 6a, collected over 6 days post-R5 HIV infection. (c) Values of HIV p24 antigen collected over 6 days post-R5 HIV infection, in supernatants of mirGE cells that survived the first HIV challenge in Figure 6b. Negative and positive controls for HIV replication were provided by naive HeLa and HR5 cells, respectively. (d) Values of HIV p24 antigen collected over 6 days post X4 HIV infection, in supernatants of mirGE cells that survived the first and second HIV challenges in Figure 6b,c. Negative control for X4 HIV infection was provided by naive CD4-negative parental HeLa cells and a fresh batch of naive HR5 cells served as the positive control for infection. (e) Phenotypic analysis of HR5 cells transduced with GFP control lentivector (no miRNA). (f) Phenotypic analysis of HR5 cells transduced with GFP-anti-CCR5 mirGE lentivector prior to R5-tropic HIV infection. Within the untransduced cells in gate R2 (89%), the CCR5 and GFP MFIs are 144 and 2.4, respectively. Within the transduced cells in gate R3 (6%), the CCR5 and GFP MFIs are 14 and 18, respectively. (g) Phenotypic analysis of HR5 cells from (F) 7 days post infection with R5-tropic HIV. The majority of cells (81%) are CCR5-negative. The CCR5 and GFP MFIs in this population (gate R3) are 3 and 48, respectively.