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. 2008 Sep;82(17):8520-8.
doi: 10.1128/JVI.00843-08. Epub 2008 Jun 25.

Epstein-Barr virus latent membrane protein 2A preferentially signals through the Src family kinase Lyn

Mark Rovedo  1 , Richard Longnecker
Affiliations

Epstein-Barr virus latent membrane protein 2A preferentially signals through the Src family kinase Lyn

Mark Rovedo et al. J Virol. 2008 Sep.

Abstract

Latent membrane protein 2A (LMP2A) is a viral protein expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor signal transduction and provides survival and developmental signals to B cells in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, we crossed LMP2A transgenic mice (TgE) with Lyn(-/-), Fyn(-/-), or Blk(-/-) mice. TgE Lyn(-/-) mice had a larger immunoglobulin M (IgM)-positive B-cell population than TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. Both TgE Fyn(-/-) and TgE Blk(-/-) mice have an IgM-negative population of splenic B cells, similar to the TgE mice. LMP2A was also transiently transfected into the human EBV-negative B-cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates, whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. From these results, we conclude that LMP2A preferentially associates with and signals through Lyn compared to its association with other SFKs. This preferential association is due in part to the SH2 domain of Lyn associating with LMP2A.

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Figures

FIG. 1.
FIG. 1.
Splenic B cells from Lyn−/− TgE mice contain an IgM-positive population. Representative flow cytometry dot plots from multiple experiments are shown. Splenic cells were harvested from 5-week-old mice. Red blood cells were lysed, and the remaining splenic cells were counted using a hemocytometer. Cells were then washed, stained with fluorescently labeled antibodies (1 million cells stained with IgM-FITC and CD19-PE), and analyzed by flow cytometry. The x axis represents IgM expression, and the y axis represents CD19 expression. IgM-positive and IgM-negative populations are marked by boxes.
FIG. 2.
FIG. 2.
Splenic B cells from Lyn−/− TgE mice contain an IgM-positive population that is statistically significant compared to the IgM-positive B-cell populations in TgE, Fyn−/− TgE, and Blk−/− TgE mice. Flow cytometry data tabulated from multiple experiments are shown. IgM-positive populations were calculated as the percentage of total CD19-positive cells. Dots represent individual mice. The bar in each column represents the average of the results.
FIG. 3.
FIG. 3.
Lyn levels are lower in LMP2A-expressing cells. (A) Lysates were prepared using 1 ml Triton X-100 lysis buffer and 10 million BJAB cells stably expressing LMP2A or vector control (previously described). Lysates were separated by SDS-PAGE and immunoblotted with rabbit anti-Lyn, rabbit anti-Fyn, rat anti-LMP2A, or mouse anti-GAPDH antibody. Lyn was detected at a lower level in LMP2A-positive BJAB cells than in wt BJAB cells. Similar amounts of Fyn were detected in both LMP2A-positive BJAB cells and wt BJAB cells. GAPDH served as a loading control. +, present; −, absent. (B) B cells were purified from disassociated mouse spleens by using CD19-coated magnetic beads. Cell lysates were prepared from the purified splenic B cells of TgE and wt mice. Lysates were separated by SDS-PAGE and immunoblotted with rabbit anti-Lyn, rabbit anti-Fyn, rat anti-LMP2A, or mouse anti-GAPDH antibody. Lyn was detected at a lower level in TgE mouse splenic cells than in wt mouse splenic cells. Similar amounts of Fyn were detected in both TgE and wt mouse cells. GAPDH served as a loading control.
FIG. 4.
FIG. 4.
LMP2A preferentially associates with Lyn. Ten million BJAB cells were electroporated with either 12 μg of an HA-tagged LMP2A-expressing plasmid or empty vector. Cells were incubated at 37°C and 5% carbon dioxide for 14 h. Lysates were prepared by using 1 ml of Triton X-100 lysis buffer per 10 million cells. (A) Whole-cell lysates (WCL) were separated by SDS-PAGE and immunoblotted with rabbit anti-Lyn, rabbit anti-Fyn, rat anti-LMP2A, or mouse anti-GAPDH antibody. (B) Lysates were immunoprecipitated (IP) with mouse anti-HA antibody, separated by SDS-PAGE, and immunoblotted with rabbit anti-Lyn or rabbit anti-Fyn antibodies. Lyn was detected in the LMP2A immunoprecipitate, but Fyn was not. +, present; −, absent.
FIG. 5.
FIG. 5.
Expression of the YEEL LMP2A mutant results in lower Lyn and Fyn levels. Two separate LMP2A mutants were derived by using a previously described retroviral construct (pMP2). Cells infected with the parental construct, the YEEL mutant, the YMEM mutant, or the control empty vector were selected in hygromycin to generate four different cell lines. Lysates were prepared by using 1 ml of Triton X-100 lysis buffer per 10 million cells from each of the four cell lines. Whole-cell lysates were separated by SDS-PAGE and immunoblotted with rabbit anti-Lyn, rabbit anti-Fyn, rat anti-LMP2A, or mouse anti-GAPDH antibody. Lyn levels were lower in the LMP2A-expressing cells than in cells with vector control and lower in the YEEL mutant cell line than in LMP2A-expressing cells. There was no significant difference in Lyn levels in the YMEM cell lysates and in lysates of cells with the vector control. Fyn levels in the vector control, LMP2A-expressing, and YMEM-expressing cell lines were equivalent, but they were lower in the YEEL-expressing cell line. Densometric analysis was used to quantify protein levels normalized to the levels in the control.
FIG. 6.
FIG. 6.
YEEL and YEEI mutants associate with both Lyn and Fyn. Ten million BJAB cells were electroporated with 12 μg of an HA-tagged LMP2A-expressing plasmid, one of three HA-tagged mutant LMP2A-expressing plasmids (YEEI, YEEL, or YMEM), or empty vector. Cells were incubated at 37°C and 5% carbon dioxide for 14 h. Lysates were prepared by using 1 ml of Triton X-100 lysis buffer per 10 million cells. (A) Whole-cell lysates were separated by SDS-PAGE and immunoblotted with rabbit anti-Lyn, rabbit anti-Fyn, rat anti-LMP2A, or mouse anti-GAPDH antibody. (B) Lysates were immunoprecipitated with mouse anti-HA antibody, separated by SDS-PAGE, and immunoblotted with rabbit anti-Lyn or rabbit anti-Fyn antibody.
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