Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis

Abstract

The latent membrane protein 1 (LMP-1) oncoprotein of Epstein-Barr virus (EBV) is a constitutively active, CD40-like cell surface signaling protein essential for EBV-mediated human B-cell immortalization. Like ligand-activated CD40, LMP-1 activates NF-kappaB and Jun kinase signaling pathways via binding, as a constitutive oligomer, to tumor necrosis factor receptor-associated factors (TRAFs). LMP-1's lipid raft association and oligomerization have been linked to its activation of cell signaling pathways. Both oligomerization and lipid raft association require the function of LMP-1's polytopic multispanning transmembrane domain, a domain that is indispensable for LMP-1's growth-regulatory signaling activities. We have begun to address the sequence requirements of the polytopic hydrophobic transmembrane domain for LMP-1's signaling and biochemical activities. Here we report that transmembrane domains 1 and 2 are sufficient for LMP-1's lipid raft association and cytostatic activity. Transmembrane domains 1 and 2 support NF-kappaB activation, albeit less potently than does the entire polytopic transmembrane domain. Interestingly, LMP-1's first two transmembrane domains are not sufficient for oligomerization or TRAF binding. These results suggest that lipid raft association and oligomerization are mediated by distinct and separable activities of LMP-1's polytopic transmembrane domain. Additionally, lipid raft association, mediated by transmembrane domains 1 and 2, plays a significant role in LMP-1 activation, and LMP-1 can activate NF-kappaB via an oligomerization/TRAF binding-independent mechanism. To our knowledge, this is the first demonstration of an activity's being linked to individual membrane-spanning domains within LMP-1's polytopic transmembrane domain.

FIG. 1.

Schematic of LMP-1 and LMP-1 variants used in this study: comparison of full-length LMP-1, TMD1,2, TMD1,2Δ55/mycHis, lyLMP-1, Δ25-132, and mycASGPR/LMP-1. TMD1,2 lacks TMDs 3 to 6; TMD1,2Δ55/mycHis is a Myc- and His-tagged deletion of TMD1,2 lacking residues 331 to 386 of LMP-1; lyLMP-1 lacks the cytoplasmic N terminus and first four TMDs (residues 1 to 128); Δ25-132 is essentially lyLMP-1 with the wild-type LMP-1 cytoplasmic N terminus fused to TMD 5 (15); mycASGPR/LMP-1 is a chimera in which LMP-1's cytoplasmic N terminus was replaced with a Myc-tagged heterologous N terminus from the H1 asialoglycoprotein receptor (ASGPR) (4).

FIG. 2.

Lipid raft association of TMD1,2. 293 cells were transfected with pCMV-based LMP-1, TMD1,2, lyLMP-1, Δ25-132, and mycASGPR/LMP-1 expression vectors. Two days posttransfection, cells were solubilized in a buffer containing 0.2% Triton X-100, homogenized, mixed with an equal volume of 80% sucrose, and overlaid with 30% and 5% sucrose in MNE buffer. Following centrifugation for 18 h at 200,000 × g, fractions were taken from the top of each gradient and resolved on SDS-PAGE gels. Gels were transferred to Immobilon and stained with anti-LMP-1 antiserum. A marker for each LMP-1 protein is shown on the right or left of each blot (transfected 293 cell whole-cell lysate, lane M), and arrows point to the indicated LMP-1 protein. (A) LMP-1, (B) TMD1,2, (C) lyLMP-1, (D) Δ25-132, (E) mycASGPR/LMP-1. The lipid raft, soluble, and pellet fractions are labeled beneath. These results are representative of three independent experiments.

FIG. 3.

TMD1,2-induced cytostasis. HEp2 cells were transfected with pCMV-LMP-1, pCMV-TMD1,2, or pCMV-lyLMP-1 and plated on coverslips at clonal density immediately following transfection. Four days postplating, cells were fixed and stained with anti-LMP-1 antiserum. (A) All colonies on a given coverslip were scored for the number of positive cells (LMP-1 immunoreactive) and data are expressed as number of positive cells per colony. Data shown as “1 LMP-1-positive cell per colony” include both individual immunoreactive cells (see part B, LMP-1) and single immunoreactive cells within a colony (see upper panel in part B, TMD1,2). A total of 500 colonies were scored for each transfection. (B) Immunofluorescent images of representative colonies are shown for each transfection; two representative images are shown for each of the introduced plasmids; the name of the introduced plasmid is shown above each equivalent set of images (upper and lower panels). These results are representative of three separate experiments. Both LMP-1- and TMD1,2-positive HEp2 colonies primarily have one to four LMP-1-immunoreactive cells, whereas lyLMP-1-positive colonies primarily have four to eight immunoreactive cells. LMP-1- and TMD1,2-positive cells tend to be multinucleated and much larger than lyLMP-1-positive cells.

FIG. 4.

Activation of NF-κB by TMD1,2 in 293 cells. 293 cells were cotransfected with RSV-LMP-1 expression vectors encoding LMP-1, lyLMP-1, TMD1,2, or TMD1,2Δ55/mycHis (2 μg and 7 μg), together with an NF-κB-responsive luciferase reporter as described previously (4) and in Materials and Methods. (A) Extracts were assayed for luciferase activity 48 h posttransfection with a Dual Light assay kit (Tropix). Data are expressed as a percentage of maximal LMP-1 activation, with maximal activation by LMP-1 occurring at 2 μg of input DNA (423,310 relative light units [RLU]). The activation detected in the presence of 7 μg of empty vector control (pRC-RSV) is denoted by the dotted line. Solid circles, LMP-1; open circles, TMD1,2; shaded circles, TMD1,2Δ55/mycHis; open squares, lyLMP-1. (B) Extracts from A were assayed for LMP-1 expression by Western blot. Each lane was loaded with 5 × 10⁴ cell equivalents. The expressed protein is shown above the blot, and lanes are marked below the blot with input DNA amounts (2 μg or 7 μg). The amount of LMP-1 protein detected in each lane is representative of duplicate transfections in a single experiment. Arrows point to LMP-1 and lyLMP-1. 721 cells (5 × 10⁵ cells) and tetradecanoyl phorbol acetate- and butyrate-induced B95-8 cells (10⁵ cells) (EBV-positive lymphoblastoid cell lines) were loaded on either side of the blot and served as markers for LMP-1 and lyLMP-1, respectively. Shown is a representative of three independent experiments. TMD1,2 activates NF-κB with a different concentration dependence than LMP-1 via a mechanism involving LMP-1 residues 331 to 386, which include CTAR2.

FIG. 5.

Oligomerization of TMD1,2. 293 cells were electroporated with the indicated CMV-based expression vectors and harvested 48 h posttransfection, and cell lysates were immunoprecipitated with an anti-Myc antibody (9E10, Santa Cruz). Immunoprecipitates (IP) and whole-cell lysates (WCL) from transfected cells were resolved on SDS-10% PAGE gels and visualized by Western blot with anti-LMP-1 antiserum as described previously (4) and in Materials and Methods. The introduced LMP-1 protein is shown above each blot, and arrows on the sides of the blots mark each protein's migration. (A) Immunoprecipitation of individually expressed proteins with anti-Myc antibody. (B) Coimmunoprecipitation of LMP-1myc with CΔ55 (LMP-1 deletion mutant lacking 55 amino acids from the C terminus, included as an LMP-1-interacting partner [positive control]), NΔ25 (LMP-1 deletion mutant lacking the cytoplasmic N terminus, included as a noninteracting control), or TMD1,2. (C) Coimmunoprecipitation of TMD1,2 with TMD1, 2Δ55mycHis (a C-terminally truncated TMD1,2 lacking 55 amino acids from the C terminus in pcDNA3.1/mycHis [Invitrogen]). TMD1,2 does not detectably interact with LMP-1 or with itself.

FIG. 6.

Interaction of TMD1,2 with TRAF1, TRAF2, and TRAF3. 293 cells were transfected with CMV-based expression vectors encodingLMP-1 or TMD1,2, with or without pCMV-TRAF1/HA-1 (A), pCMV-TRAF2/HA-1 (B), or pCMV-TRAF3/HA-1 (C). Cells were harvested 24 h posttransfection, and extracts were immunoprecipitated with anti-HA-1 antibodies (F-7; Santa Cruz). TRAF immunoprecipitates (IP) and whole-cell lysates of transfected cells (WCL) were resolved by SDS-10% PAGE and analyzed by Western blot with anti-LMP-1 antiserum. The introduced LMP-1 proteins are shown below each blot, and inclusion of the indicated TRAF in the transfection is noted by a + above each blot (top row). Lanes containing HA-1 immunoprecipitates are noted by + above each blot, and whole-cell lysates are noted by − (bottom row). Individual LMP-1 proteins (LMP-1, TMD1,2, and lyLMP-1) are identified by arrows to the sides of each blot. LMP-1-immunoreactive bands migrating faster than LMP-1 are the result of degradation. LMP-1 and not TMD1,2 or lyLMP-1 is detected in TRAF1, TRAF2, and TRAF3 immunoprecipitates.