Mutational analysis of Nef O8 identifies functional importance of the VGF region which links the acidic cluster and the proline-rich motif. (A) Alignment of the sequence of Nef O8 relative to a subtype O reference allele (O.BE.87.ANT70) obtained from the Los Alamos HIV sequence database reveals that the VGF motif is deleted in Nef O8. In addition, the first proline of the PxxP motif changed into alanine. A panel of mutants was generated in which the different mutations were sequentially reverted to their consensus variant (marked in red) or to a variant in which VGF was changed into triple alanines (AAA; blue). As reference, boxes below reverted sequences mark the acidic cluster (Ac) and proline-rich motif (Proline-motif) (B) Jurkat CD4-CCR5 cells were transduced with retroviral vectors expressing either eGFP alone or together (Nef-IRES-eGFP) with the indicated nef mutant and were analyzed by flow cytometry. Bivariate dot plots show expression of CD4, MHC-I, CXCR4 as a function of eGFP (Nef) expression. Dot plots shown are representative for results obtained in 3 independent experiments. (C) To determine relative surface levels, the MFI of transduced cells (eGFP+) was divided by the MFI of untransduced cells (eGFP-) in the same culture. Data represent the mean relative expression and standard deviations from 3 independent experiments. * over a bar indicates a statistically significant difference between the experimental construct compared with the eGFP control (P < 0.01).