Fig 6. CD4 addressing by the ORF2 ARM.

Schematic representation of ORF2wt and CD4wt proteins. SP ORF2 residues are shown in blue. ARM residues are highlighted in red. CD4 sequences are in green. (A) and (C), H7-T7-IZ cells were transfected with pTM plasmids expressing CD4wt or chimeric C4 and C5 proteins. Twenty-four hours post-transfection, cells were fixed and processed for CD4 staining (in green). Nuclei are in blue. Representative confocal images are shown together with CD4/DAPI merge images. Blue dots observed in some pictures are DAPI-stained transfected plasmids. A schematic representation of each construct is shown on the left. Scale bar, 20μm. (B) Nuclear-to-cytoplasmic CD4 staining ratio in H7-T7-IZ cells expressing CD4wt or chimeric proteins. Quantification was done using ImageJ software (mean ± S.D., n ≥ 30 cells, Kruskal-Wallis with Conover’s test). ****p < 0.0001. (D) Subcellular fractionation of H7-T7-IZ cells expressing CD4wt or chimeric proteins at 24h post-transfection. Fractionation was done using the subcellular protein fractionation kit for cultured cells. CD4 proteins were detected by WB with a rabbit polyclonal anti-CD4 Ab. Tubulin, Calnexin (CNX) and Lamin B1 were also detected to control the quality of fractionation. Molecular mass markers are indicated on the right (kDa).