Genetic Diversity of EBV-Encoded LMP1 in the Swiss HIV Cohort Study and Implication for NF-Κb Activation
Figure 1
(A) NF-κB activation by LMP1 variants. 293T cells were transfected with 50 ng of expression vector coding for LMP1 prototype (B95-8) and variants (P1, A3, A1, A2, CAO) and 50 ng of Firefly luciferase reporter plasmid. An analogous strategy of subcloning was applied to variants, prototype and CAO LMP1, to ensure appropriate comparisons. NF-κB activity was measured twenty hours after transfection using luciferase assay (Promega). Data are mean ± SD of triplicates and shown is a representative of three independent experiments with similar results. (B) Expression of LMP1 B95-8, A2 and P1 was visualized by SDS-PAGE and Western blotting with anti-LMP1 8G3 antibody. Detection against tubulin was used as internal control. (C) Measure of the toxicity of LMP1 variants. Cells were transfected with 50 ng of LMP1 B95-8, P1 or A2. Untransfected cells and cells transfected with empty vector were used as experimental controls. ATP amount was measured 24 hours after transfection using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Shown is a representative experiment of three independent experiments with similar results. (D) Amino acid sequence alignment of B95-8, P1 and A2 LMP1. Only amino acids that differ from the sequence of prototype B95-8 LMP1 are indicated. Transmembrane segments are indicated by light gray boxes and deletions by dashes.