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. 2012;7(2):e32168.
doi: 10.1371/journal.pone.0032168. Epub 2012 Feb 22.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation

Emilie Zuercher  1 , Christophe Butticaz, Josiane Wyniger, Raquel Martinez, Manuel Battegay, Emmanuelle Boffi El Amari, Thanh Dang, Jean-François Egger, Jan Fehr, Esther Mueller-Garamvögyi, Andrea Parini, Stephan C Schaefer, Franziska Schoeni-Affolter, Christine Thurnheer, Marianne Tinguely, Amalio Telenti, Sylvia Rothenberger; Swiss HIV Cohort Study
Collaborators, Affiliations

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation

Emilie Zuercher et al. PLoS One. 2012.

Abstract

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: JFE is employee of Laboratoire Viollier Weintraub SA. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Analysis of LMP1 variants.
(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.
Figure 2
Figure 2. NF-κB activation levels by LMP1 chimeras and mutants.
(A, C) Schematic representation of LMP1 chimeras split at amino acid 231 (A) and 118 (C). The six transmembrane segments are represented by boxes. (B, D–F) NF-κB activation by LMP1 chimeras split at amino acid 231 (B) and 118 (D), and LMP1 mutants on B95-8 background (E) and on A2 background (F). HEK cells were transfected with 50 ng of LMP1 vector and 50 ng of NF-κB reporter plasmid. Empty vector was used as control. NF-κB activity was measured twenty-four hours after transfection using luciferase assay (Promega). Shown are representative of three independent experiments with similar results. Data are given as mean ± SD of triplicates. Statistical analysis was done using one-way ANOVA with Bonferroni posttest using GraphPad Prism, n = 9 triplicates of three experiments. **** P<0.0001 relatively to the NF-κB activation of B95-8 LMP1. RLU: relative light units.
Figure 3
Figure 3. Analysis of LMP1 variants amplified from blood of HIV-infected individuals.
(A) Frequency of polymorphisms from 31 LMP1 variants compared to the B95-8 prototype reference sequence. Dashes represent insertion of amino acids in direct repeats region compared to B95-8. Deletion of 10 (aa 343 to 352) or 23 (aa 332 to 354) amino acids are present in 13 and 2 variants, respectively. (B) Phylogenetic tree was built on LMP1 nucleotide sequences by employing neighbor-joining method using Geneious software. (C) Percentages of NF-κB activation induced by the 31 LMP1 variants. HEK cells were transfected with 50 ng of LMP1 vector and 50 ng of NF-κB reporter plasmid. NF-κB activity was measured twenty four hours after transfection using luciferase assay (Promega). Values were normalized to the B95-8 activation value fixed at 100%. Data are given as mean ± SD of percentages of triplicates of three independent experiments. Statistical analysis was done using one-way ANOVA with Bonferroni posttest using GraphPad Prism. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 relatively to the NF-κB activation of B95-8 LMP1. RLU: relative light units. ND: not done. (D) Typing based on EBNA-2 gene was performed on genomic DNA according to Telenti and coworkers . B95-8 and Raji are EBV type 1 references and AG876 is EBV type 2 reference.
Figure 4
Figure 4. Flow chart of the study.
(A) Comparison of LMP1 polymorphisms in blood samples from HIV-infected individuals with or without HL. * patients enrolled in the SHCS at the beginning of the study by March 2009 . (B) Comparison of LMP1 polymorphisms in blood and corresponding biopsy from HIV-HL.
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