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Fig. 2 | Virology Journal

Fig. 2

From: Reassessment of the capacity of the HIV-1 Env cytoplasmic domain to trigger NF-κB activation

Fig. 2

The EnvCD activates NF-κB when fused to the CD8-α chain. a Comparison of the ability of native Env and CD8-EnvCD chimeras to activate NF-κB. 1.2 × 105 HEK293T cells were cotransfected with 200 ng of NF-κB-Firefly-Luciferase vector, 50 ng of pGL4-Renilla-Luciferase and 500 ng of pcDNA-Env expressing vectors (EnvHXB2, EnvNL4.3, EnvNLAD8, EnvΔCD) or the following CD8-EnvCD chimeric constructs: CD8-EnvCDHXB2 (residues 707–856 of EnvHXB2), CD8STOP, CD8-EnvCDHXB2Δ3 (residues 707–760 of EnvHXB2), CD8-EnvCDHXB2Δ4 (residues 707–780 of CDHXB2), CD8-EnvCDSIVmac239 (residues 716–879 of EnvSIVmac239), CD8-EnvCDMLV (residues 640–665 of EnvMLV) and CD8-EnvCDHTLV-I (residues 466–488 of EnvHTLV-I). Transfections were performed in duplicate wells. Firefly and Renilla-Luciferase activities were recorded 37 and 48 h post-transfection. The Firefly-Luciferase signal was normalized to the Renilla-Luciferase signal. The empty pcDNA3.1 vector was used as negative control (mock) and was used for standardization. The mean of seven independent experiments is reported. Error bars represent standard error. NF-κB activation by different constructs was compared by a Kruskal-Wallis test followed by a Dunn’s post-test using Graph Pad Prism version 5.0 and differences were considered significant if p < 0.05. b Intracellular localization of EnvNL43, EnvHXB2 and CD8-EnvCD. 1.2 × 105 HEK293T cells were cotransfected with 200 ng of EnvNL4.3 or EnvHXB2 and CD8-EnvCD or with EnvΔCD and CD8STOP. After 48 h, cells were washed and fixed with cold absolute ethanol and stained with a polyclonal goat α-Env antibody (Abcam ab53937) and Rabbit anti-CD8α antibody (H-160, Santa Cruz), then sequentially incubated with donkey anti-goat IgG then goat anti-mouse and anti-Rabbit IgG secondary antibodies coupled to Alexa Fluor 488 and Alexa Fluor 568 (Invitrogen). Images were captured with a Zeiss LSM510 META confocal laser scanning microscope (Jena, Germany) equipped with a 63× Plan-NeoFluar oil immersion objective (numerical aperture 1.3)

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