Cell surface expression of C-terminally truncated CXCR4 mutants and CXCR4/CCR5 chimeras were downregulated by Nef almost as well if not better than their wild type counterparts.

<p>A) C-terminal sequence coordinates of the chemokine receptors (i) C-terminal sequence of wt CXCR4 and selected C-terminally truncated mutants, including the natural WHIM mutant, numbers within parentheses denote the position of respective deletion; (ii) C-terminal sequence of wt CCR5 and CCR5/CXCR4 chimeras swapping the respective C-terminal sequence. The swap position(s) are denoted on the left. In each case, CCR5 sequence is underlined. <b>B</b>) Nef effect on genetically engineered CXCR4 mutants, the naturally occurring WHIM mutant (WM CXCR4) and chimeras (X4R5 and R5X4) was evaluated in CHO cells or CXCR4 negative K562 cell line. CD4 was cotransfected in each case to monitor Nef effect (<b>B2</b>). Cells were transfected with an IRES plasmid encoding GFP and Nef or the null mutant. Expression of CXCR4 or CCR5 (in the case R5X4 chimera) and CD4 in GFP gated cells was evaluated by flow cytometry. Average MFVs for CXCR4 (and CCR5 for R5X4 transfection) and CD4 on null and Nef (+) cells are plotted in the histograms (with standard deviation) for CXCR4 (and CCR5) expression in the left panel and for CD4 in the right panel. MFVs for Nef (−) cells were set to 100 (n = 4). *** represents p<0.01) when Nef transfected cells are compared to plasmid transfected controls. Black bars indicate relative % downregulation in Nef (wt or mutant) expressers relative to control cells transfected with empty vector.