Epstein-Barr virus latent membrane protein 2A (LMP2A) employs the SLP-65 signaling module

Abstract

In latently infected B lymphocytes, the Epstein-Barr virus (EBV) suppresses signal transduction from the antigen receptor through expression of the integral latent membrane protein 2A (LMP2A). At the same time, LMP2A triggers B cell survival by a yet uncharacterized maintenance signal that is normally provided by the antigen receptor. The molecular mechanisms are unknown as LMP2A-regulated signaling cascades have not been described so far. Using a novel mouse model we have identified the intracellular adaptor protein Src homology 2 (SH2) domain-containing leukocyte protein (SLP)-65 as a critical downstream effector of LMP2A in vivo. Biochemical analysis of the underlying signaling pathways revealed that EBV infection causes constitutive tyrosine phosphorylation of one of the two SLP-65 isoforms and complex formation between SLP-65 and the protooncoprotein CrkL (CT10 regulator of kinase like). This leads to antigen receptor-independent phosphorylation of Cbl (Casitas B lineage lymphoma) and C3G. In contrast, phospholipase C-gamma2 (PLC-gamma2) activation is completely blocked. Our data show that in order to establish a latent EBV infection, LMP2A selectively activates or represses SLP-65-regulated signaling pathways.

Figure 1

Loss of the LMP2A developmental and survival phenotype when bred into the SLP-65^−/− background. (A) Mouse splenocytes (SP) and (B) bone marrow samples (BM) from WT, TgE SLP-65^−/−, and TgE SLP-65^−/− animals were subjected to flow cytometric analysis with at least 10,000 gated lymphocytes collected per animal using CD19-PE (CD19) and IgM-FITC (IgM) antibodies. Typical dot plot results of 10,000 gated lymphocytes from 5–7-wk-old animals (n = 5) are shown. The polygons represent distinct subpopulations of B cells with the average percentage of total gated lymphocytes indicated above each polygon. (A) The left polygon represents CD19⁺IgM⁻ B cells typical in TgE animals; the right polygon represents CD19⁺IgM⁺ cells. (B) The left rectangle represents CD19⁺IgM⁻ B cells; the right rectangle represents CD19⁺IgM⁺ B cells.

Figure 2

Impaired BCR signaling but elevated levels of pTyr-containing proteins in EBV-infected B cells. Two independently obtained LCLs (lanes 1–4 in panel a and 1–6 in panel b) and EBV-negative Ramos B cells (lanes 5 and 6 in panel a and 7–9 in panel b) were left untreated or stimulated through their BCR with F(ab′)₂ fragments of anti-IgM antibodies for the indicated time points (30 s or 3 min). (a) From the cleared cellular lysates, phosphoproteins were isolated by affinity purification (AP) with a GST fusion protein encompassing the tandem SH2 domains of Syk (GST-SYK[SH2]₂) and detected by anti-pTyr immunoblotting. (b) Lysates were subjected to immunoprecipitation (IP) with anti-pTyr antibodies and the material was analyzed by subsequent immunoblotting with antibodies to pTyr (top panel), Syk (middle panel), and SLP-65 (bottom panel). (c) Cellular lysates of the different LCLs used in this study (lanes 1–4) and Ramos B cells (lane 5) were immunoblotted with anti–SLP-65 antibodies. Relative molecular masses of marker proteins are indicated on the left in kD.

Figure 2

Impaired BCR signaling but elevated levels of pTyr-containing proteins in EBV-infected B cells. Two independently obtained LCLs (lanes 1–4 in panel a and 1–6 in panel b) and EBV-negative Ramos B cells (lanes 5 and 6 in panel a and 7–9 in panel b) were left untreated or stimulated through their BCR with F(ab′)₂ fragments of anti-IgM antibodies for the indicated time points (30 s or 3 min). (a) From the cleared cellular lysates, phosphoproteins were isolated by affinity purification (AP) with a GST fusion protein encompassing the tandem SH2 domains of Syk (GST-SYK[SH2]₂) and detected by anti-pTyr immunoblotting. (b) Lysates were subjected to immunoprecipitation (IP) with anti-pTyr antibodies and the material was analyzed by subsequent immunoblotting with antibodies to pTyr (top panel), Syk (middle panel), and SLP-65 (bottom panel). (c) Cellular lysates of the different LCLs used in this study (lanes 1–4) and Ramos B cells (lane 5) were immunoblotted with anti–SLP-65 antibodies. Relative molecular masses of marker proteins are indicated on the left in kD.

Figure 2

Impaired BCR signaling but elevated levels of pTyr-containing proteins in EBV-infected B cells. Two independently obtained LCLs (lanes 1–4 in panel a and 1–6 in panel b) and EBV-negative Ramos B cells (lanes 5 and 6 in panel a and 7–9 in panel b) were left untreated or stimulated through their BCR with F(ab′)₂ fragments of anti-IgM antibodies for the indicated time points (30 s or 3 min). (a) From the cleared cellular lysates, phosphoproteins were isolated by affinity purification (AP) with a GST fusion protein encompassing the tandem SH2 domains of Syk (GST-SYK[SH2]₂) and detected by anti-pTyr immunoblotting. (b) Lysates were subjected to immunoprecipitation (IP) with anti-pTyr antibodies and the material was analyzed by subsequent immunoblotting with antibodies to pTyr (top panel), Syk (middle panel), and SLP-65 (bottom panel). (c) Cellular lysates of the different LCLs used in this study (lanes 1–4) and Ramos B cells (lane 5) were immunoblotted with anti–SLP-65 antibodies. Relative molecular masses of marker proteins are indicated on the left in kD.

Figure 4

Syk and SLP-65 associate in vivo. Anti–SLP-65 immunoprecipitates from unstimulated and BCR-stimulated LCLs (lanes 1–4) or Ramos B cells (lanes 5 and 6) were analyzed by anti-Syk immunoblotting. Relative molecular mass of marker protein is indicated on the left in kD. Note that a constitutive Syk/SLP-65 complex formation was observed in all LCLs that have been investigated (n = 4), but some variability in the amount of Syk present in SLP-65 immunoprecipitates was observed (compare lanes 1 and 4).

Figure 3

Constitutive tyrosine phosphorylation of the p70 isoform of SLP-65 in EBV-infected B cells. Two independent LCL clones (lanes 1–4) and Ramos B cells (lanes 5 and 6) were left untreated (lanes 1, 3, and 5) or stimulated through their BCR for 3 min (lanes 2, 4, and 6). From the cleared lysates of these cells, anti–SLP-65 immunoprecipitates (IP) were prepared and analyzed by anti-pTyr immunoblotting. Relative molecular mass of marker protein is indicated on the left in kD.

Figure 5

Block of PLC-γ2 phosphorylation in LCLs. Anti–PLC-γ2 immunoprecipitates from unstimulated and BCR-stimulated LCLs (lanes 1–6) or Ramos B cells (lanes 7 and 8) were analyzed by immunoblotting with antibodies to pTyr (top panel), SLP-65 (middle panel), or PLC-γ2 (bottom panel). Relative molecular masses of marker proteins are indicated on the left in kD.

Figure 6

Association of the protooncoprotein CrkL with SLP-65. (a) Anti-CrkL immunoprecipitates (IP) were prepared from cleared cellular lysates of untreated or BCR-stimulated LCLs (lanes 1–4) and Ramos B cells (lanes 5 and 6), and analyzed by subsequent immunoblotting with antibodies to SLP-65 (top panel), Cbl (middle panel), and C3G (bottom panel). (b) Anti-pTyr immunoprecipitates from the same cells were analyzed by anti-CrkL or anti-C3G immunoblotting (top and bottom panel, respectively). Relative molecular masses of marker proteins are indicated on the left in kD.

Figure 6

Association of the protooncoprotein CrkL with SLP-65. (a) Anti-CrkL immunoprecipitates (IP) were prepared from cleared cellular lysates of untreated or BCR-stimulated LCLs (lanes 1–4) and Ramos B cells (lanes 5 and 6), and analyzed by subsequent immunoblotting with antibodies to SLP-65 (top panel), Cbl (middle panel), and C3G (bottom panel). (b) Anti-pTyr immunoprecipitates from the same cells were analyzed by anti-CrkL or anti-C3G immunoblotting (top and bottom panel, respectively). Relative molecular masses of marker proteins are indicated on the left in kD.

Figure 7

EBV-positive B cells are unresponsive to treatment with pervanadate/H₂O₂. Two independent LCL clones (lanes 1–4) and Ramos B cells (lanes 5 and 6) were left untreated (lanes 1, 3, and 5) or incubated with 20 μM pervanadate/H₂O₂ for 3 min at 37°C (lanes 2, 4, and 6). From the cleared cellular lysates, tyrosine-phosphorylated proteins were purified by immunoprecipitation (IP) and analyzed by subsequent immunoblotting with antibodies to pTyr (top panel), Syk (middle panel), and SLP-65 (bottom panel). Relative molecular masses of marker proteins are indicated on the left in kD.