Fig 1. SIVcol and SIVolc Nef fail to efficiently downmodulate the T cell receptor CD3.

(A) Human PBMCs were transduced with VSV-G pseudotyped NL4-3 constructs coexpressing the indicated Nef proteins and eGFP via an IRES. 72 hr post-transduction, CD3 surface expression was quantified by flow cytometry. Mean values of six infections ± SEM are shown on the left. The panel on the right shows examples for primary flow cytometry data. (B) Due to the lack of an antibody detecting SIVcol and SIVolc Nef, counteraction of human SERINC5 was analyzed to verify functional Nef expression. HEK293T cells were cotransfected with increasing amounts of a SERINC5 expression plasmid and the NL4-3-based proviral constructs also used in (A). 40 hr post-transfection, infectious virus yield was quantified by infection of TZM-bl reporter cells. Mean values of three independent experiments in triplicates ± SEM are shown. (C) HEK293T cells were cotransfected with the proviral constructs described in (A) and expression vectors for CD3-CD8 fusion proteins comprising the cytoplasmic domain of human or colobus CD3ζ and the extracellular and transmembrane domain of human CD8. 40 hr post transfection, CD3ζ downmodulation was determined by monitoring CD8 surface levels via flow cytometry. Mean values of three to four independent experiments ± SEM are shown. In (A) and (C), asterisks indicate statistically significant differences compared to the nef-defective control (**p < 0.01; ***p < 0.001).