Mapping of Vpr-Vpr interaction by FLIM. HeLa cells were co transfected with mutated Vpr-eGFP and its own counterpart fused to mCherry. FLIM was carried out 24 h posttransfection (see methods). Column A corresponds to the FLIM images of the Vpr-eGFP mutants alone, column B to the FLIM images of cells co expressing the mutant Vpr-eGFP and the mutant Vpr-mCherry. FRET efficiency (E) expressed in percentage represents the average value calculated over the entire cell (column C). The color scale used to create theses images is the same than the one used for figure 4. Note the drastic reduction of Vpr-Vpr interaction and the loss of Vpr nuclear envelope accumulation upon mutating residues L23, Q44, I60 and L67 (column B and C).