Simultaneous ZFN modification of ccr5 and cxcr4 protects SupT1-R5 T cells from infection with viruses that use either CCR5 or CXCR4. (A) Surface expression of CCR5 (R5) and CXCR4 (×4) on SupT1-R5 cells following delivery of increasing MOIs of the Ad R5- and X4-ZFNs. Percentage of cells lacking both coreceptors is labeled in red. (B) Proportion of cells lacking both surface R5 and X4 (double-negative cells) following simultaneous treatment with the R5 (MOI 600) and X4 (MOI 600) ZFNs as measured by FACS. (C) Dual ZFN-treated cells were challenged with a mix of R5- and X4-using HIV and surface expression of R5 and X4 was measured over time in infected and uninfected R5/X4-ZFN–treated cells. (D) The proportion of double-negative cells following R5/X4-ZFN treatment and subsequent HIV infection was measured by FACS 5 and 25 days postinfection. (E) Cell viability after infection of mock (no ZFN) or R5/X4-ZFN–treated supT1-R5 cells with a mix of R5- and X4-HIV. Viability was measured by FACS following treatment with a viability dye. (F) Dual ZFN-treated supT1-R5 cells previously challenged with HIV and no longer expressing either coreceptor (▪) as shown in panel C, were rechallenged with either R5-HIV, X4-HIV, or VSV-G-HIV pseudoviruses expressing GFP. HIV pseudovirus rechallenge of previously HIV-selected double-negative cells resulted in 170-fold and 92-fold decreases in infection by R5 and X4-HIV, respectively, whereas VSV-G pseudovirus infection was decreased only ∼1.7-fold. All graphs represent the mean (±SEM) of 4 independent ZFN treatments and 4 independent infection experiments; P values calculated using the Student t test.