Epstein–Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation

Abstract

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

Epstein‐Barr virus LMP2A utilizes Syk and PI3K to activate NF‐κB in B‐cell lymphomas to increase MIP‐1α production

“…In addition, the LMP2A ITAM motif that activates Syk is critical for LMP2A‐mediated effects . To determine the importance of the ITAM region that binds Syk for the LMP2A‐dependent increase in MIP‐1α production, we utilized B‐cell lines that express either wild‐type LMP2A, or LMP2A containing phenylalanine substitutions at tyrosines 74 and 85 of the ITAM region that bind Syk . These cell lines were incubated for 24 hours and supernatants were analyzed by ELISA to assess MIP‐1α levels.…”

“…These findings indicate that LMP2A not only directly promotes the survival of HL survival, but may also indirectly promote HL tumor survival through the production of IL-10 24 32 The ITAM mutant cell line and the corresponding control cell lines were generated by transducing BJAB cells with a retrovirus containing the pIEGZ retroviral vector (generous gift of Dirk Lingemann, Germany) that encoded GFP fluorescence and Zeocin resistance, LMP2A (wild-type LMP2A) or LMP2A with mutations in the ITAM region (Syk-mutant) as described previously. 33 LMP2A expression in all cell lines was determined by immunofluorescence by fixing the cells in IC Fixation buffer (eBioscience, SanDiego, CA) and incubated for 20 minutes at room temperature. Cells were then immediately resuspended in permeabilization buffer (eBioscience) and incubated for 20 minutes at room temperature with anti-LMP2A (14B71-1) Rat mAb (Abcam, Cambridge, UK).…”

“…17 To determine the importance of the ITAM region that binds Syk for the LMP2A-dependent increase in MIP-1α production, we utilized B-cell lines that express either wild-type LMP2A, or LMP2A containing phenylalanine substitutions at tyrosines 74 and 85 of the ITAM region that bind Syk. 17,18,33 These cell lines were incubated for 24 hours and supernatants were analyzed by ELISA to assess MIP-1α levels. As shown in Figure 1C, Taken together, these data demonstrate that LMP2A increases MIP-1α in multiple B-cell lines and that this increase is dependent on the ability of Syk to bind LMP2A.…”

Revisión sistemática: estrategias virales para la inducción de cáncer “virus de Epstein-Barr: latencia y mecanismos asociados a la oncogénesis viral”

“…La cola amino terminal se encuentra constitutivamente fosforilada dentro de múltiples sitios, incluyendo dos regiones ricas en tirosina (Y74/Y85) que constituyen motivos de inmunoreceptores activables por tirosina (ITAM), donde la proteína Syk es secuestrada y activada; una tirosina en 112 que une y activa la tirosina quinasa Lyn 32,44 , y dos motivos ricos en prolina (PY), responsables de la unión de Ubiquitina-ligasas U3 y la activación de la β-catenina en células epiteliales al inhibir la GSK-3 por medio de la proteína quinasa C, señalizando e inhibiendo la diferenciación de células epiteliales 45 .…”

“…LMP2A mimetiza la función del receptor de célula B (BCR), uniendo inicialmente a Lyn, seguida de Syk y desencadenando la activación de la proteína de unión a célula B (BLNK), y las vías NF-κβ, MAPK/ERK y Ras/PI3K/Akt, a través de las cuales promueve la supervivencia de células B, al inducir los genes antiapoptóticos Bcl-2 y Bcl-xL, lo que parece ejercer una función crítica en la infección temprana (4 a 7 días) 44,46 . Por tanto, al actuar como un BCR activado, LMP2A aumenta la activación temprana, proliferación y supervivencia de células B, favoreciendo el establecimiento de LCLs.…”