Masato Ikeda

PY motifs of epstein-barr virus LMP2A regulate protein stability and phosphorylation of LMP2A-associated proteins

ABSTRACT Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. We have demonstrated that Nedd4 family ubiquitin-protein ligases (E3s), AIP4, WWP2/AIP2, and Nedd4, bind specifically to two PY motifs present within the LMP2A amino-terminal domain. In this study, LMP2A PY motif mutant viruses were constructed to investigate the role of the LMP2A PY motifs. AIP4 was found to specifically associate with the LMP2A PY motifs in EBV-transformed lymphoblastoid cell lines (LCLs), extending our original observation to EBV-infected cells. Mutation of both of the LMP2A PY motifs resulted in an absence of binding of AIP4 to LMP2A, which resulted in an increase in the expression of Lyn and the constitutive hyperphosphorylation of LMP2A and an unknown 120-kDa protein. In addition, there was a modest increase in the constitutive phosphorylation of Syk and an unidentified 60-kDa protein. These results indicate that the PY motifs contained within LMP2A are important in regulating phosphorylation in EBV-infected LCLs, likely through the regulation of Lyn activity by specifically targeting the degradation of Lyn by ubiquination by Nedd4 family E3s. Despite differences between PY motif mutant LCLs and wild-type LCLs, the PY motif mutants still exhibited a block in B-cell receptor (BCR) signal transduction as measured by the induction of tyrosine phosphorylation and BZLF1 expression following BCR activation. EBV-transformed LCLs with mutations in the PY motifs were not different from wild-type LCLs in serum-dependent cell growth. Protein stability of LMP1, which colocalizes with LMP2A, was not affected by the LMP2A-associated E3s.

Itchy, a Nedd4 Ubiquitin Ligase, Downregulates Latent Membrane Protein 2A Activity in B-Cell Signaling

ABSTRACT Nedd4 family ubiquitin protein ligases (E3s) specifically associate with latent membrane protein 2A (LMP2A) of Epstein-Barr virus. Our previous studies analyzing LMP2A function in vitro have suggested that Nedd4 family E3s regulate LMP2A function. To determine the role of Nedd4 family E3s in LMP2A B-cell signaling, LMP2A transgenic (LMP2A+) mice were crossed with mice with the Itch-deficient (Itch−/−) background. Itchy, a mouse homologue of human AIP4, is a Nedd4 family E3 and is also the most abundant Nedd4 family E3 found in LMP2A affinity precipitates from B cells. There were significantly fewer B-cell receptor-positive B cells in spleen and bone marrow B cells in LMP2A+ Itch−/− mice than in LMP2A+ mice. In addition, LMP2A+ Itch−/− bone marrow B cells formed larger colonies in cultures treated with interleukin-7 (IL-7) than control bone marrow B cells did. Finally, there was a dramatic increase in tyrosine phosphorylation of LMP2A and Syk in IL-7-cultured LMP2A+ Itch−/− B cells. These results indicate that Nedd4 family E3s, in particular Itchy, downmodulate LMP2A activity in B-cell signaling.

Ubiquitinylation of Igβ Dictates the Endocytic Fate of the B Cell Antigen Receptor

In both infection and autoimmunity, the development of high-affinity Abs and memory requires B cells to efficiently capture and process Ags for presentation to cognate T cells. Although a great deal is known about how Ags are processed, the molecular mechanisms by which the BCR captures Ag for processing are still obscure. In this study, we demonstrate that the Igβ component of the BCR is diubiquitinylated and that this is dependent on the E3 ligase Itch. Itch−/− B lymphocytes manifest both a defect in ligand-induced BCR internalization and endocytic trafficking to late endosomal Ag-processing compartments. In contrast, analysis of ubiquitinylation-defective receptors demonstrated that the attachment of ubiquitins to Igβ is required for endosomal sorting and for the presentation of Ag to T cells, yet, ubiquitinylation is dispensable for receptor internalization. Membrane-bound Igμ was not detectably ubiquitinylated nor were the conserved lysines in the mu cytosolic tail required for trafficking to late endosomes. These results demonstrate that ubiquitinylation of a singular substrate, Igβ, is required for a specific receptor trafficking event. However, they also reveal that E3 ligases play a broader role in multiple processes that determine the fate of Ag-engaged BCR complexes.

The Effects of the Epstein-Barr Virus Latent Membrane Protein 2a on B Cell Function

Epstein-Barr Virus (EBV) infects B-lymphocytes circulating through the oral epithelium and establishes a lifelong latent infection in a subset of mature-memory B cells. In these latently infected B cells, EBV exhibits limited gene expression with the latent membrane protein 2A (LMP2A) being the most consistently detected transcript. This persistent expression, coupled with many studies of the function of LMP2A in vitro and in vivo, indicates that LMP2A is functioning to control some aspect of viral latency. Establishment and maintenance of viral latency requires exquisite manipulation of normal B cell signaling and function. LMP2A is capable of blocking normal B cell signal transduction in vitro, suggesting that LMP2A may act to regulate lytic activation from latency in vivo. Furthermore, LMP2A is capable of providing B cells with survival signals in the absence of normal BCR signaling. These data show that LMP2A may help EBV-infected cells to persist in vivo. This review discusses the advances that have been made in our understanding of LMP2A and the effects it has on B cell development, activation, and viral latency.

Nucleotide sequence involving murG and murC in the mra gene cluster region of Escherichia coli.

The mra (murein synthesis cluster a) region at 2 min on the Escherichia coli chromosome map carries the genes for peptidoglycan synthesis (1, 2): genes murC, murD, murE, murF and ddl, coding for five enzymes synthesizing UDP-Nacetylmuramyl-pentapeptide (L-alanyl-D-glutamyl-mesodiaminopimelyl-£>-alanyl-Z)-alanine) from UDP-N-acetylmuramic acid by sequential addition of appropriate amino acids. The genes pbpB and ftsW (3) that code for septum-peptidoglycan synthetic proteins, murG (4) which produces a protein of unknown function and an open reading frame ORF-Y (5) were also located in the mra region. In the present study, we determined the base sequences of the murG and murC genes located between JisW and ddl and thus completed the sequencing of the total 12 kb mra region which is flanked by a 5 kb region involving JisQ, JisA,ftsZ and envA. An open reading frame of 1065 bp capable of encoding a moderately hydrophobic peptide with 355 amino acid residues (Mw 37,814) was found for murG, and one of 1473 bp encoding a peptide with 491 amino acid residues (Mw 53,625) was found for murC. The proteins MurG and MurC were detected on SDS/PAGE in an in vitro protein synthesis system. Overlapping of the open reading frames of the murG-murC area and its flanking genes ftsWand ddl was observed. Considerable homologies were found in the deduced amino acid sequences of the product proteins of murC, D, £and F, i.e., four ligases that synthesize UDP-N-acetylmuramyl-pentapeptide, including putative ATP-binding domains GXXGKT/S (126-131 of MurC). MurG showed considerable homology with the corresponding region of the Bacillus subtilis chromosome.

Latent Membrane Protein 2A, a Viral B Cell Receptor Homologue, Induces CD5+ B-1 Cell Development

The latent membrane protein 2A (LMP2A) of EBV plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking a constitutively active B cell Ag receptor. When expressed as a B cell-specific transgene in mice, LMP2A drives B cell development, resulting in the bypass of normal developmental checkpoints. In this study, we have demonstrated that expression of LMP2A in transgenic mice results in B cell development that exclusively favors B-1 cells. This switch to B-1 cell development occurs at the pre-B-cell stage of normal B cell development in the bone marrow, a B cell stage much earlier than appreciated for B-1 commitment. This finding indicates that all pre-B cells have the capacity to assume a B-1 cell phenotype if they encounter the appropriate signal during normal development. Furthermore, these studies offer insight into EBV latency and pathogenesis in the human host.

The c-Cbl proto-oncoprotein downregulates EBV LMP2A signaling

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking signals induced by the B-cell receptor (BCR) altering normal B cell development. As c-Cbl ubiquitin ligase (E3) is a critical negative regulator in the BCR signal pathway, the role of c-Cbl in the function and formation of the LMP2A signalosome was examined. c-Cbl promoted LMP2A degradation through ubiquitination, specifically degraded the Syk protein tyrosine kinase in the presence of LMP2A, and inhibited LMP2A induction of the EBV lytic cycle. Our earlier studies indicated that LMP2A-dependent Lyn degradation was mediated by Nedd4-family E3s in LMP2A expressing cells. Combine with these new findings, we propose a model in which c-Cbl and Nedd4-family E3s cooperate to degrade target proteins at discrete steps in the function of the LMP2A signalosome.

A fusion protein library: an improved method for rapid screening and characterization of DNA binding or interacting proteins

A rapid method for screening and characterization of DNA binding or protein-interacting molecules is described. The method relies on a fusion protein library in which randomized DNA fragments are inserted into pGEX-3X and its derivatives to generate collections of GST-fusion proteins. After inducing the expression of the fusion proteins by addition of IPTG, the colonies can be screened either with radioactively labeled DNA/RNA fragment for specific clones encoding DNA/RNA binding proteins or with an antibody for clones encoding proteins of interest. They can also be screened with a radioactively labeled protein for cloning of interacting molecules. The fusion proteins encoded by the isolated clones can be readily purified by conducting the lysis of the cells and an affinity column in the presence of an alkyl anionic detergent, N-laurylsarcosine (sarkosyl), and can be further characterized.

Amino Acid Substitution Analyses of the DNA Contact Region, Two Amphipathic α-Helices and a Recognition-Helix-Like Helix outside the Dimeric β-Barrel of Epstein-Barr Virus Nuclear Antigen 1

Objectives and Methods: Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), which is essential for EBV latency, homodimerizes and binds to the EBV replication origin, oriP. We analyzed the dimerization/DNA-binding domain of EBNA-1 by random and site-directed amino acid substitution. Results: Random point mutations that resulted in reduced DNA binding clustered in the DNA contact region (a.a. 461–473) and at or near the termini of α-helix II (514–527). Three substitutions of Gly in the DNA contact region each greatly reduced binding to a single binding site oligonucleotide. Substitutions at and near the termini of α-helix II diminished DNA binding. A helix-deforming substitution in α-helix I (477–489) blocked DNA binding. A helix-deforming substitution in α-helix III (568–582) abolished dimerization and DNA binding. Similarities in surface electrostatic properties and conserved amino acids were found between α-helix II and recognition helices of papillomavirus E2 proteins. Conclusions: The basic DNA contact region is crucial for the specific interaction of EBNA-1 with a single binding site. α-Helix I477 is indispensable for oriP binding, and α-helix III568 contributes to the homodimeric structure of EBNA-1. α-Helix II514 contributes to oriP binding, perhaps changing its alignment with DNA.

Pre-B-cell colony formation assay.

Latent membrane protein 2A (LMP-2A) of Epstein-Barr virus (EBV) mimics a constitutively active B-cell receptor (BCR) and plays a key role in viral latency and EBV pathogenesis. By functioning as a BCR mimic, LMP-2A drives B-cell development, resulting in the bypass of normal B-cell developmental checkpoints. To assess the function of LMP-2A, we have utilized a colony formation assay for the progenitor B cells that uses the B-cell proliferation factor IL-7.