Fig. 5: The most abundant 18-bp deletion in CCR5 is MMEJ-induced and results in a protein variant (CCR5^Δ55-60) with impaired cell-surface expression.

a Microhomologies found at the positions of the most frequent 18- and 10-bp deletions at the CCR5 locus indicating microhomology-directed repair. b–d CCR5-negative cells transduced with lentiviral vectors encoding BFP and either wildtype (wt) CCR5 or CCR5^Δ55-60 were analysed for CCR5, CD3 and BFP expression. b CCR5 cell-surface expression. >95% of BFP-positive cells expressing wtCCR5, but no BFP-positive cells containing the CCR5^Δ55-60 variant became positive after cell-surface staining with anti-human CCR5-PerCP-Cy5.5 antibody. c Fixed and permeabilised Jurkat cells expressing either wtCCR5 or CCR5^Δ55-60 were stained with anti-human CCR5-PerCP-Cy5.5 and anti-human CD3-APC antibodies. Over 1000 images obtained with imaging flow cytometry were used to estimate cell membrane localisation of both CCR5 variants. CCR5 signal localisation was related to BFP present in the cytoplasm. With the gate set at 1, cell membrane localisation of wtCCR5 and CCR5^Δ55-60 was found for 88.8% and 5.4%, respectively. d Examples of Image Stream images of the fixed and permeabilised Jurkat cells expressing either wtCCR5 or CCR5^Δ55-60 stained with anti-human CCR5-PerCP-Cy5.5 and anti-human CD3-APC. CCR5, as a membrane protein, should be localised mainly outside of the BFP with cytoplasmic localisation.