Amphiphysin 1 and 2 interact with latent membrane protein 2 A of Epstein-Barr virus and regulate its exosomal secretion

Aim. Latent membrane protein 2A (LMP2A) of Epstein-Barr virus is implicated in the regulation of viral latency. The aim of the current study was to identify proteins interacting with proline-rich motifs of LMP2A. Methods. In silico prediction with Scansite allowed to recognize amphiphysin 1 (Amph1) as a binding partner of LMP2A. Molecular cloning techniques, site-directed mutagenesis, in vitro binding assay made it possible to study the interaction interface of Amph1/LMP2A complex. Sequential centrifugation steps were used to isolate an exosomal fraction. Results. LMP2A but not LMP2∆NT mutant has been found to bind the SH3 domain of Amph1 via three distinct proline-rich motifs located in the N-terminal tail. All three motifs seem to be interchangeable as the presence of at least one of them was sufficient to mediate LMP2A/Amph1 interaction. Furthermore, the binding of LMP2A to Amph1 and related protein amphiphysin 2 was demonstrated by co-immunoprecipitation of endogenous complexes. We have found that inability of LMP2A mutant to bind Amph1 leads to the vanishing of the viral protein from the exosomal fraction. Conclusions. The latent membrane protein 2A of Epstein-Barr virus forms complexes with endocytic adaptor proteins Amph1 and Amph2. Described interaction might be involved in the regulation of intracellular traffic and secretion of LMP2A.

Introduction.Epstein-Barr virus (EBV) is a member of the herpesvirus family and one of the most common human viruses [1].EBV is associated with a number of human malignancies, such as Burkitt's lymphoma [2], Hodgkin's lymphoma [3] and nasopharyngeal carcinoma [4].Only restricted set of viral genes is expressed within the latent phase: LMP1, LMP2A, LMP2B, EBNAs and EBERs.Latent membrane proteins (LMPs) are key players in transformation and survival of infected cells [5].EBV latency is regulated by LMP2A and LMP2B [5].LMP2A is a transmembrane protein comprising 12 transmembrane segments and two cytosolic tails.The N-terminal tail is responsible for LMP2A signalosome assembly and signaling, while C-terminal one mediates clusterization of LMP2A molecules [6,7].Cytosolic Nand C-terminal tails of LMP2A contain phosphotyrosine-containing (pY) and proline-rich motifs (PRMs).Through the pY-motifs LMP2A interacts with SH2and PTB-containing proteins [8].Due to the binding of tyrosine kinases Syk and Lyn, LMP2A mimics activated receptors and establishes its own signaling [9][10][11].
LMP2A-induced signaling events were uncovered in B-cells, epithelial cells and fibroblasts [5,10].LMP 2A activates AKT kinase providing anti-apoptotic and pro-survival signals [11,12].Much attention was paid to mitogenic signaling but knowledge about LMP2A internalization and traffic in EBV-positive cells is unclear so far.
Despite the presence of five putative PRMs no interactions with the SH3 domains have been reported.Here we report the identification of amphiphysins 1 and 2 as protein partners of LMP2A.Inability of LMP2A mutant to interact with Amph1 affects the secretion of LMP2A on exosomes produced by HEK293 cells suggesting a putative role of Amph1 in LMP2A traffic.
The SH3 domains of Amph 1, PI3Kp85a, Src and endophilin were described previously [14][15][16].Fulllength CDS of Amph 1 and Amph 2 were amplified by PCR from human embryonal brain cDNA using the High Fidelity PCR enzyme mix.The PCR products were cloned into the pcDNA4 His/MaxC vector to generate Omni-Amph 1 Omni-Amph 2 respectively.All PCRgenerated DNA fragments were sequenced to confirm fidelity.
Protein expression, pull-down assays and Western blot analyses were carried out as described previously [14].
Exosome preparation.Exosomes were isolated as previously described [17] with minor modifications.In brief, HEK293 cells were grown to 60-70 % confluence on 10 cm plates, washed twice with 1 ´ PBS to remove secreted exosome and metabolites and supplied with fresh complete medium.Then cells were transfected with 15 mg of plasmid DNA.24 h post-transfection culture medium was collected and cleared by centrifugation at 10,000 g for 10 min to remove apoptotic cells and cell fragments.Exosomes were further isolated by centrifugation at 70,000 g for 2 h (exosome fraction).Cells were lysed as described above; pelleted exosome fraction was solubilized in Laemmli buffer and analyzed by Western blotting.
Results and discussion.The aim of the current study was to identify proteins that bind directly PRMs located in LMP2A cytosolic tails.The canonical binding site for SH3 domains is PXXP (where X-any amino acid) [18].Five motifs fitting PXXP consensus were found in the primary structure of LMP2A protein: four motifs (designated P1-P4) in the N-terminal domain and one (P5) in the C-terminal one.In silico analysis with Scansite service (www.scansite.mit.edu)evidenced for the presence of at least four motifs (P2, P3, P4 and P5) for binding the SH3 domain of endocytic adaptor protein Amph1.To validate this interaction we performed GST pull-down assay.We have found that the SH3 domain of Amph1 bound Omni-LMP2A, while it was unable to interact with LMP2ADNT variant that represents the LMP2B isoform, an important negative regulator of LMP2A-dependent signaling (Fig. 1, B).Thus, P5 motif is dispensable to mediate interaction between LMP2A and Amph1.We also tested ability of LMP2A to bind the SH3 domains of other proteins: endophilin, Src kinase and p85 regulatory subunit of PI3K.As Fig. 1, C, shows, LMP2A failed to interact with the proteins mentioned above.This fact could evidence for the specificity of LMP2A/Amph1 interaction.
Further, we decided to determine which of four potential PRMs found in the N-terminus of LMP2A mediates this interaction.For this aim, prolines in motifs P2, P3 and P4 individually or in combinations P3 + + P4, P2 + P3 + P4 were substituted for alanines.The P1 motif was considered less probable for binding because the sequence of this PRM differs significantly from the consensus.The core motif in P1 is flanked with numerous glycines that is very unusual for classic PRMs, which are typically surrounded with charged amino acid residues [18].The individual mutations in motifs P2, P3 and P4 had moderate effect on LMP2A binding to the SH3 domain of amphiphysin 1 (Fig. 1, D). P3 + P4 LMP2A mutant also bound to the SH3 domain of Amph1 (Fig. 1, D, lane 5) whereas P2 + P3 + P4 mutant of LMP2A was unable to interact with Amph1 (Fig. 1, E).Thus, one can suggest that Amph1 can bind any of three PRMs (P2-P4) in the N-terminal tail of LMP2A.All three motifs seem to be interchangeable as presence of at least one of them was sufficient to mediate LMP2A/Amph1 complex formation.
Moreover, LMP2A was shown to form a complex in vivo with Amph1 as well as with a highly related Amph2 (Fig. 2, A) in HEK293 cells transiently transfected with corresponding recombinant plasmids.The data obtained may link LMP2A to the endocytic compartment, enabling viral protein to be effectively internalized from the cell surface.Previously, LMP2A was detected in the cytoplasm in the association with different types of vesicles presumably derived from the plasma membrane.LMP2A is secreted on exosome pathway by different cell types [17].Exosomes are membrane vesicles of endocytic origin after they have passed through multivesicular body [19].We decided to determine whe- ¬ Omni-LMP2A ther inability of LMP2A to interact with Amph1 affects secretion of LMP2A on exosomes produced by HEK 293 cells.The wild type LMP2A was found in the exosomal fraction as well as inside of cells, while LMP2A P2 + P3 + P4 had not been detected on exosomes (Fig. 2, B).These data imply a role of interaction between LMP2A and Amph1 for intracellular traffic of LMP2A.The current comprehension of composition of the LMP2A-mediated signalosome and mechanisms of its traffic through the cell remains unclear.Amphiphysins are adaptor proteins that have been implicated in clathrin-mediated endocytosis, regulation of actin cytoskeleton and cellular signaling [20][21][22][23].Amph2 was shown to bind Myc oncoprotein and to function as onco-suppressor [24].It is possible to speculate that the role of LMP2A in lymphogenesis is not restricted to providing pro-survival stimuli for the cells but also involves deregulation of Amph2/BIN function that leads to the inhibition its tumor-suppressing activity.
Answers for this question might help to improve therapy of EBV-associated lymphomas and make our knowledge deeer in the field of host-pathogen interactions.