Fig. 1
From: Reassessment of the capacity of the HIV-1 Env cytoplasmic domain to trigger NF-κB activation
The HIV-1 and SIV CDs do not trigger NF-κB activation. a Induction of NF-κB by a panel of HIV-1 subtype B and C Envs. 1.2 × 105 HEK293T cells were cotransfected in duplicate wells with 500 ng of pcDNA-Env expressing vector, 200 ng of NF-κB-Firefly-Luciferase vector and 50 ng of pGL4.74-Renilla-Luciferase for normalization using the Calcium Phosphate precipitation method. We used a panel of full-length Envs cloned in pCDNA3.1: Env of pNL4.3, Env of pNLAD8, 5 primary subtype B Envs (EnvSVPB5, EnvSVPB11, EnvSVPB12, EnvSVPB18, EnvSVPB8), 5 primary subtype C Envs (EnvSVPC3, EnvSVPC7, EnvSVPC10, EnvSVPC13, EnvSVPC17) and EnvΔCD as negative control. All Env vectors express the two Rev exons. As a positive control, NF-κB was triggered with 100 ng/ml TNF-α 31 or 42 h post-transfection. After 37 and 48 h, Firefly- and Renilla-Luciferase were measured in cell lysates using the Dual-Glo Luciferase kit (Promega) and the Firefly-Luciferase signal was normalized using the Renilla-Luciferase. Results are expressed as Fold-Change in NF-κB induction with respect to the empty pcDNA3.1 vector (mock). The mean of at least two independent experiments is reported. Error bars represent standard error. b Induction of transcription from the viral LTR by HIV-1 subtype B and C Envs. 8 × 104 TZM-bl cells were cotransfected with 1 μg of pcDNA-Env expressing vector and 100 ng pGL4.74-Renilla-Luciferase in duplicate wells. LTR-driven transcription (Firefly-Luciferase) was assessed in cell lysates after 48 h (no signal was detected 37 h post-transfection) and normalized using the Renilla-Luciferase. As a positive control, Env expression vectors containing Tat were used. The empty pcDNA3.1 vector (mock) was used for standardization. The mean of three independent experiments is reported. Error bars represent standard error. Statistical analyses for a and b were performed with GraphPad Prism (version 5). NF-κB induction (a) and LTR activation (b) were compared using a Kruskal-Wallis test followed by a Dunn’s post-test and differences were considered significant if p < 0.05