The wild-type and fusion Nef proteins can induce CD4 down-regulation in HEK-293 cells. (A) One million cells were cotransfected with 0.5 μg pcDNA3/CD4 and 4.5 μg pCi-Neo, pCi-Neo/NefLai, pCi-Neo/NefLai-Rluc, or pCi-Neo/NefLai-EYFP. The CD4 was labeled the day after with the RT-PA-PC5 antibody, and the cells were analyzed by flow cytometry. Abbreviations for each panel: dotted line, pcDNA3/CD4 plus pCi-Neo plus isotypic antibody; thin line, pcDNA3/CD4 plus pCi-Neo plus RPA-T4-PC5 antibody; bold line, pcDNA3/CD4 plus pCi-Neo plasmid expressing one of the Nef proteins indicated above the panels plus RPA-T4-PC5. (B) The same experiment was performed with the pCML/CD4 414AA plasmid.

The Nef/CD4 or Nef/CD4 414AA complexes can be directly detected by BRET in living HEK-293 cells. Ten million HEK-293 cells were transfected with 10 μg pCi-Neo/Nef-Rluc and 10 μg pHIS/β2AdrR-EYFP or pCi-Neo/CD4-EYFP. Twenty-four hours posttransfection, the BRET experiments were performed and the emission signals were measured (A). After subtraction of the negative control spectrum from the sample one, a distinct EYFP fluorescence emission can be observed (B). In parallel, a similar experiment was done by replacing pcDNA3/CD4-EYFP with pCi/Neo-CD4 414AA-EYFP (C and D).

The CD4 and CD4 414AA molecules can abolish Nef-Rluc/CD4-EYFP and Nef-Rluc/CD4 414AA-EYFP luminescence transfer. (A) Ten million cells were transfected with 15 μg of plasmid coding for CD4, CD4 414AA, or the pCi-Neo empty plasmid and 2.5 μg pCi-Neo/Nef-Rluc and 2.5 μg pCi-Neo/CD4-EYFP; as a negative control, cells were transfected with 15 μg pCi-Neo, 2.5 μg pCi-Neo/Nef-Rluc, and 2.5 μg pHIS/β2AdrR-EYFP. Two days after transfection, BRET experiments were realized. After subtraction of the negative control spectrum from the sample one, the previously observed EYFP signal in cells expressing Nef-Rluc and CD4-EYFP was abrogated in the presence of the nonfluorescent receptors CD4 and CD4 414AA. (B) The same experiment was performed with the pCi-Neo/CD4 414AA-EYFP plasmid. The EYFP signal generated with Nef-Rluc/CD4 414AA-EYFP pair was not detected anymore when CD4 or CD4 414AA was coexpressed.

The different fusion proteins are correctly addressed in HEK-293 cells. (A) HEK-293 cells (6.5 × 10⁴) were cotransfected with 1 μg pHIS/β2Adr-EYFP, pCi-Neo/Nef-Rluc, pCi-Neo/Nef-EYFP, pcDNA3/CD4-EYFP, or pCi-Neo/CD4 414AA-EYFP. Twenty-four hours posttransfection, the cells expressing proteins fused to EYFP were fixed and observed with a Zeiss LSM 510 confocal microscope. The cells producing Nef-Rluc were fixed and permeabilized. The Nef protein was primary marked with maTG020 and labeled with an FITC-conjugated goat anti-mouse immunoglobulin G antibody. (B) To assess the colocalization of Nef or Nef-Rluc with the two CD4 variants at the plasma membrane, HEK-293 cells were cotransfected with 0.5 μg pCi-Neo/Nef-Lai or pCi-Neo/Nef-Rluc and the same amount of pcDNA3/CD4 or pCML/CD4 414AA. Forty-eight hours posttransfection, the cells were fixed and permeabilized. The Nef proteins were marked as previously. The CD4 molecules were labeled with the RPA-T4-PC5 antibody. The colocalization panels represent the overlay of CD4-PC5 and Nef-FITC fluorescences. Bars correspond to 10 μm.

The Nef dimerization can be visualized in HEK-293 cells, using the BRET technology. Ten million HEK-293 cells were transfected with 20 μg pCi-Neo/NefLai-Rluc/EYFP, 10 μg pCi-Neo/NefLai-Rluc, and 10 μg pHis/β2AdrR-EYFP or with 10 μg pCi-Neo/NefLai-Rluc and 10 μg pCi-Neo/NefLai-EYFP. (A) Two days after transfection, the signals obtained in the presence of 10 μM coelenterazine-f were detected using a PTI spectrofluorimeter. The emission spectra obtained are represented as follows: cells expressing NefLai-Rluc and EYFP, thin line; cells expressing NefLai-Rluc and β2AdrR-EYFP, dotted line; and cells expressing NefLai-Rluc and NefLai-EYFP, bold line. (B) The transfer spectrum (dotted line) is obtained by subtracting the negative control emission spectrum (NefLai-Rluc/β2AdrR-EYFP) from the spectrum obtained in cells producing NefLai-Rluc and NefLai-EYFP. This transfer signal is compared to the EYFP emission spectrum (bold line). One representative result obtained from three independent experiments is presented.

Nef interacts in situ with the CD4, CD4 414AA, CD4-EYFP and CD4 414AA-EYFP receptors. (A) Proteins corresponding to CD4 or CD4-EYFP are specifically coimmunoprecipitated with the Nef antibody only in lysates of cells coexpressing Nef. One million HEK-293 cells were transfected with 5 μg pCi-Neo, pCi-Neo/NefLai, pcDNA3/CD4, pCML/CD4 414AA, pcDNA3/CD4-EYFP, or pCi-Neo/CD4 414AA-EYFP or cotransfected with 2.5 μg pCi-Neo/Nef-Lai and 2.5 μg pcDNA3/CD4, pCML/CD4 414AA, pcDNA3/CD4-EYFP, or pCi-Neo/CD4 414AA-EYFP. The next day, the cells were metabolically labeled, treated with the DSP cross-linker, and immunoprecipitated with the Nef antibody. The proteins of the immune complexes were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and revealed by fluorography. (B) The proteins coimmunoprecipitated with the Nef antibody, in the presence of Nef, are recognized by the anti CD4 antibody. Six million HEK-293 cells were transfected with 20 μg pcDNA3/CD4 or pcDNA3/CD4-EYFP or cotransfected with 10 μg pCi-Neo/Nef-Lai and 10 μg pcDNA3/CD4, pCML/CD4 414AA, pcDNA3/CD4-EYFP, or pCi-Neo/CD4 414AA-EYFP. Two days later, the cells were treated with the DSP cross-linker and lysed. The cell lysates were incubated with the Nef antibody or the CD4 antibody as indicated (IP). The proteins of the immune complexes were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-CD4 rabbit polyclonal antibody and then with a polyclonal antibody coupled to HRP and directed against rabbit immunoglobulin G. The proteins were revealed by fluorography in the presence of the enzyme substrate.