The CTGC motifs of TASHET are essential for activated HIV transcription in single-round infection assays. (A) Mutations have been introduced into the 3’LTR of pNL4-3-LucE- (in red). Viral proteins whose expression is deficient in this construct are shown in grey (env, nef). The mutated pNL4-3-LucE- constructs were co-transfected together with a plasmid expressing VSV-G envelope into HEK293 packaging cells for virion production. Culture supernatant was titrated for viral content by ELISA p24-antigen titration to normalize the quantity of virus for PBMC infection. Activated PBMCs were infected, allowing genomic integration of the virus. Luciferase expression corresponding to retroviral promoter activity was measured 48 h later. (B) RLU were measured and normalized to proviral content as measured by qPCR of an LTR sequence in infected cells extract. Results were expressed as a percentage of wt HIV luciferase activity.