Epstein-Barr Nuclear Antigen Leader Protein Research Articles

Loop-mediated isothermal amplification linked a nanoparticles-based biosensor for detecting Epstein-Barr virus

Epstein-Barr virus (EBV) is a ubiquitous gamma herpesvirus that maintains a lifelong latent association with B lymphocytes. Here, a rapid and reliable diagnosis platform for detecting EBV infection, employing loop-mediated isothermal amplification (LAMP) combined with a gold nanoparticles–based lateral flow biosensors (AuNPs-LFB) (termed LAMP Amplification Mediated AuNPs-LFB Detection, LAMAD), was developed in the current study. A set of specific LAMP primers targeting the Epstein-Barr nuclear antigen (EBNA) leader protein (EBNA-LP) gene was designed and synthesized. Subsequently, these templates extracted from various pathogens and whole blood samples were used to optimize and evaluate the EBV-LAMAD assay. As a result, the limit of detection (LoD) of the EBV-LAMAD assay was 45 copies/reaction. The EBV-LAMAD assay can detect all representative EBV pathogens used in the study, and of note, no cross-reactions were observed with other non-EBV organisms. Moreover, the whole workflow of the EBV-LAMAD assay can be completed within 70 min, including rapid EBV template preparation, EBV-LAMP amplification, and AuNPs-LFB-mediated detection. Taken together, the EBV-LAMAD assay targeting the EBNA-LP gene is a rapid, simplified, sensitive, reliable, and easy-to-use detection protocol that can be used as a competitive potential diagnostic/screening tool for EBV infection in clinical settings, especially in basic laboratories in resource-limited regions.Key points• A novel, simplified, and easy-to-use AuNPs-LFB biosensor was designed and prepared.• LAMP combined with an AuNPs-LFB targeting the novel EBNA-LP gene was established.• EBV-LAMAD is a rapid, sensitive, and reliable detection protocol for EBV infection.Graphical

New role of Epstein-Barr virus in pathogenesis of acute and chronic lymphocytic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common mortal cancer in children. Chronic lymphocytic leukemia (CLL) is the most prevalent form of adult leukemia in western countries. B lymphocytes dominate the origin of both diseases. We have found a possible role for Epstein-Barr virus (EBV) in the origins of both ALL and CLL. We applied our strategy (Nat Genet 50:699, 2018) to determine whether the binding of EBV transcription factors (TFs) was concentrate at the 84 known risk loci for CLL and 16 loci for ALL. We evaluated 52 virally encoded TF ChIP-seq (chromatin immunoprecipitation with DNA sequencing) datasets and complemented this analysis with the results from 1535 human TF ChIP-seq datasets. We found that Epstein-Barr nuclear antigen leader protein (EBNALP), EBNA3C and EBNA2 were concentrated in the CLL loci by 3.7, 3.7 and 3.5-fold with p=4.89*10−19, p=2.74*10−11 and p=1.07*10−8, respectively. The viral (n=3) and human TFs (n=40) cluster together in an optimal subset of ~15 of the 84 known loci in CLL at p<10−6. Eighty percent of the most highly associated viral and human TF ChIP-seq datasets were collected from EBV transformed B cell lines in the Latency III program of viral expression, for which EBNALP, EBNA3C and EBNA2 are viral gene products. In ALL 3 of 16 loci were occupied by EBNA3ABC by a 17.7-fold enrichment with p=2.0*10−10. Among human TFs only c-MYC reached statistical significance binding 2 non-overlapping risk loci with 28.4-fold enrichment with p=2.9*10−10. These results nominate EBV for a role in the pathogenesis of CLL and ALL by a mechanism operating in transformed B cells through the EBV Latency III program of viral expression.

Correction: Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006890.].

Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300.

Epstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCE Epstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells.

New Interactors of the Truncated EBNA-LP Protein Identified by Mass Spectrometry in P3HR1 Burkitt's Lymphoma Cells.

The Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) acts as a co-activator of EBNA-2, a transcriptional activator essential for Epstein-Barr virus (EBV)-induced B-cell transformation. Burkitt’s lymphoma (BL) cells harboring a mutant EBV strain that lacks both the EBNA-2 gene and 3′ exons of EBNA-LP express Y1Y2-truncated isoforms of EBNA-LP (tEBNA-LP) and better resist apoptosis than if infected with the wild-type virus. In such BL cells, tEBNA-LP interacts with the protein phosphatase 2A (PP2A) catalytic subunit (PP2A C), and this interaction likely plays a role in resistance to apoptosis. Here, 28 cellular and four viral proteins have been identified by mass spectrometry as further possible interactors of tEBNA-LP. Three interactions were confirmed by immunoprecipitation and Western blotting, namely with the A structural subunit of PP2A (PP2A A), the structure-specific recognition protein 1 (SSRP1, a component of the facilitate chromatin transcription (FACT) complex), and a new form of the transcription factor EC (TFEC). Thus, tEBNA-LP appears to be involved not only in cell resistance to apoptosis through its interaction with two PP2A subunits, but also in other processes where its ability to co-activate transcriptional regulators could be important.

Heterogeneity of the Epstein-Barr Virus (EBV) Major Internal Repeat Reveals Evolutionary Mechanisms of EBV and a Functional Defect in the Prototype EBV Strain B95-8.

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous pathogen of humans that can cause several types of lymphoma and carcinoma. Like other herpesviruses, EBV has diversified through both coevolution with its host and genetic exchange between virus strains. Sequence analysis of the EBV genome is unusually challenging because of the large number and lengths of repeat regions within the virus. Here we describe the sequence assembly and analysis of the large internal repeat 1 of EBV (IR1; also known as the BamW repeats) for more than 70 strains. The diversity of the latency protein EBV nuclear antigen leader protein (EBNA-LP) resides predominantly within the exons downstream of IR1. The integrity of the putative BWRF1 open reading frame (ORF) is retained in over 80% of strains, and deletions truncating IR1 always spare BWRF1. Conserved regions include the IR1 latency promoter (Wp) and one zone upstream of and two within BWRF1. IR1 is heterogeneous in 70% of strains, and this heterogeneity arises from sequence exchange between strains as well as from spontaneous mutation, with interstrain recombination being more common in tumor-derived viruses. This genetic exchange often incorporates regions of <1 kb, and allelic gene conversion changes the frequency of small regions within the repeat but not close to the flanks. These observations suggest that IR1—and, by extension, EBV—diversifies through both recombination and breakpoint repair, while concerted evolution of IR1 is driven by gene conversion of small regions. Finally, the prototype EBV strain B95-8 contains four nonconsensus variants within a single IR1 repeat unit, including a stop codon in the EBNA-LP gene. Repairing IR1 improves EBNA-LP levels and the quality of transformation by the B95-8 bacterial artificial chromosome (BAC).IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the world population but causes illness in only a small minority of people. Nevertheless, over 1% of cancers worldwide are attributable to EBV. Recent sequencing projects investigating virus diversity to see if different strains have different disease impacts have excluded regions of repeating sequence, as they are more technically challenging. Here we analyze the sequence of the largest repeat in EBV (IR1). We first characterized the variations in protein sequences encoded across IR1. In studying variations within the repeat of each strain, we identified a mutation in the main laboratory strain of EBV that impairs virus function, and we suggest that tumor-associated viruses may be more likely to contain DNA mixed from two strains. The patterns of this mixing suggest that sequences can spread between strains (and also within the repeat) by copying sequence from another strain (or repeat unit) to repair DNA damage.

Regulation of Sp100A Subnuclear Localization and Transcriptional Function by EBNA-LP and Interferon

Epstein-Barr virus (EBV) efficiently immortalizes human B cells and is associated with several human malignancies. The EBV transcriptional activating protein EBNA2 and the EBNA2 coactivator EBNA-leader protein (EBNA-LP) are important for B cell immortalization. Recent observations from our laboratory indicate that EBNA-LP coactivation function is mediated through interactions with the interferon-inducible gene (ISG) Sp100, resulting in displacement from its normal location in promyelocytic leukemia nuclear bodies (PML NBs) into the nucleoplasm. The EBNA-LP- and interferon-mediated mechanisms that regulate Sp100 subnuclear localization and transcriptional function remain undefined. To clarify these issues, we generated a panel of Sp100 mutant proteins to ascertain whether EBNA-LP induces Sp100 displacement from PML NBs by interfering with Sp100 dimerization or through other domains. In addition, we tested EBNA-LP function in interferon-treated cells. Our results indicate that Sp100 dimerization, PML NB localization, and EBNA-LP interaction domains overlap significantly. We also show that IFN-beta does not inhibit EBNA-LP coactivation function. The results suggest that EBNA-LP might play a role in EBV-evasion of IFN-mediated antiviral responses.

Epstein–Barr nuclear antigen leader protein coactivates transcription through interaction with histone deacetylase 4

Epstein-Barr nuclear antigen (EBNA) leader protein (EBNALP) coactivates promoters with EBNA2 and is important for Epstein-Barr virus immortalization of B cells. Investigation of the role of histone deacetylases (HDACs) in EBNALP and EBNA2 promoter regulation has now identified EBNALP and EBNA2 to be associated with HDAC4 in a lymphoblastoid cell line. Furthermore, a transcription-deficient EBNALP point mutant did not associate with HDAC4. HDAC4 and 5 overexpression repressed EBNA2 activation and EBNALP coactivation, whereas other HDACs had little effect. Moreover, EBNALP expression decreased nuclear HDAC4. Expression of 14-3-3 anchors HDAC4 in the cytoplasm, increased EBNALP effects, and reversed HDAC4 or 5 repression. HDAC4 reversal depended on the HDAC4 nuclear export sequence. Consistent with EBNALP coactivation being mediated by nuclear HDAC4 depletion, HDAC4 overexpression increased nuclear HDAC4 and specifically repressed EBNA2-dependent activation as well as EBNALP-dependent coactivation. Also, EBNALP, HDAC4, and 14-3-3 could be immunoprecipitated in a single complex. Thus, these data strongly support a model in which EBNALP coactivates transcription by relocalizing HDAC4 and 5 from EBNA2 activated promoters to the cytoplasm. The observed EBNALP effects are likely also in part through HDAC5, which is highly homologous to HDAC4.

Development of a Monoclonal Antibody against Epstein‐Barr Virus Nuclear Antigen Leader Protein (EBNA‐LP) That Can Detect EBNA‐LP Expressed in P3HR1 Cells

A mouse monoclonal antibody, LP4D3, was raised against purified Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) fused to glutathione-S-transferase. The antibody detected endogenous and exogenous EBNA-LP in immunoblotting, immunofluorescence and immunoprecipitation assays, and the epitope of the antibody was mapped in the W2 domain of EBNA-LP. While another monoclonal antibody to EBNA-LP, JF186, which is widely used for analyses of the viral protein, did not react with truncated forms of EBNA-LP expressed in P3HR1 cells, as reported earlier, the LP4D3 antibody did. The LP4D3 antibody will be a useful tool for further studies of EBNA-LP, especially investigations into the phenotypes of mutant EBNA-LP expressed in P3HR1 cells.

Discovery of Epstein–Barr virus (EBV)-encoded RNA signal and EBV nuclear antigen leader protein DNA sequence in pet dogs

The aim of this study was to investigate Epstein-Barr virus (EBV)-related virus infection in pet dogs. The presence of antibodies to EBV antigens and EBV-related DNA was determined by Western blot analysis and PCR, respectively. Among 36 pet dogs examined for serum antibodies, 32 (88.9%) were positive for EBV-specific thymidine kinase, 15 (41.7%) for EBV-encoded DNA-binding protein and 10 (27.8%) for EBV-specific DNA polymerase. A BamHI W fragment sequence encoding part of the EBV nuclear antigen leader protein was detected by PCR in corresponding leukocyte DNA samples. Among 21 dogs tested, 15 (71.4%) were positive for the BamHI W fragment sequence. The specificity of the amplified DNA fragments was confirmed by DNA sequencing. Within the amplified region of the BamHI W fragment (241 bp), DNA sequences detected in 10 dogs had 99.2% (two nucleotide variations), 99.6% (one nucleotide variation) or 100% identity to that of EBV. Furthermore, an EBV-encoded RNA signal was detected by in situ hybridization in dog lymphocytes, as well as in bone-marrow sections, indicating a latent infection with EBV or an EBV-like virus. In conclusion, although the sample size was small, these results showed that a widespread EBV-related gammaherpesvirus could be detected in the peripheral blood and bone marrow of pet dogs. Although no evident zoonotic transmission was detected, further studies are imperative for disclosing the biological significance of this canine EBV-like virus, which may correlate with human disorders.

Identification of protein kinases responsible for phosphorylation of Epstein-Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization. Earlier studies have shown that the major site of phosphorylation of EBNA-LP by cellular kinase(s) is a serine residue at position 35 (Ser-35) and that the phosphorylation of Ser-35 is critical for regulation of the coactivator function of EBNA-LP (Yokoyama et al., J Virol 75, 5119-5128, 2001). In the present study, we have attempted to identify protein kinase(s) responsible for the phosphorylation of EBNA-LP at Ser-35. A purified chimeric protein consisting of glutathione S-transferase (GST) fused to a domain of EBNA-LP containing Ser-35 was found to be specifically phosphorylated by purified cdc2 in vitro, while GST fused to a mutated domain of EBNA-LP in which Ser-35 was replaced with alanine was not. In addition, overexpression of cdc2 in mammalian cells caused a significant increase in the phosphorylation of EBNA-LP, while this increased phosphorylation was eliminated if Ser-35 of EBNA-LP was replaced with alanine. These results indicate that the cellular protein kinase cdc2 mediates the phosphorylation of EBNA-LP at Ser-35. Recently, we reported that cdc2 and conserved protein kinases encoded by herpesviruses phosphorylate the same amino acid residue of target proteins (Kawaguchi et al., J Virol 77, 2359-2368, 2003). Consistent with this, the EBV-encoded conserved protein kinase BGLF4 specifically mediated the phosphorylation of EBNA-LP at Ser-35. These results indicate that the coactivator function of EBNA-LP can be regulated by the activity of these cellular and viral protein kinases.

Physical interaction of Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) with human oestrogen-related receptor 1 (hERR1): hERR1 interacts with a conserved domain of EBNA-LP that is critical for EBV-induced B-cell immortalization.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and plays a critical role in EBV-induced transformation. To identify the cellular proteins associating with EBNA-LP, we performed a yeast two-hybrid screen using EBNA-LP cDNA containing a single W1W2 domain as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) A cDNA in the positive yeast colony was found to encode a cellular protein, human oestrogen-related receptor 1 (hERR1), which is a constitutive transcriptional activator of the various types of oestrogen response elements. (ii) A purified chimeric protein consisting of glutathione S-transferase (GST) fused to hERR1 specifically formed complexes with EBNA-LPs containing one (EBNA-LPR1), two (EBNA-LPR2) or four W1W2 repeats (EBNA-LPR4) transiently expressed in COS-7 cells. Reciprocally, GST fused to EBNA-LPR1 or EBNA-LPR2 pulled down hERR1 transiently expressed in COS-7 cells. (iii) Mutational analyses of EBNA-LP revealed that the Y2 domain of EBNA-LP is responsible for the interaction with hERR1 and two leucines in the Y2 domain (Leu-78 and -82), which are conserved among a subset of primate gammaherpesviruses, are interactive sites for hERR1. So far, it has been reported that the only domain of EBNA-LP critical for EBV-induced transformation is the Y1Y2 domain. Potential roles of hERR1 in EBV-induced transformation are discussed.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) forms complexes with a cellular anti-apoptosis protein Bcl-2 or its EBV counterpart BHRF1 through HS1-associated protein X-1.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in EBV-induced transformation. An earlier report (Y. Kawaguchi et al., J. Virol. 74: 10104-10111, 2000) showed that EBNA-LP interacts with a cellular protein HS1-associated protein X-1 (HAX-1). The predicted amino acid sequence of HAX-1 exhibits similarity to that of another cellular protein Nip3 which has been shown to interact with cellular and viral anti-apoptotic proteins such as Bcl-2 and BHRF1, an EBV homolog of Bcl-2. Here we investigated whether HAX-1, like Nip3, interacts with Bcl-2 proteins and report the following. (i) A purified chimeric protein consisting of gluthathione S-transferase (GST) fused to BHRF1 (GST-BHRF1) or Bcl-2 (GST-Bcl-2) specifically pulled down HAX-1 transiently expressed in COS-7 cells. (ii) GST-BHRF1 or GST-Bcl-2 was not able to pull down EBNA-LP transiently expressed in COS-7 cells, whereas each of the GST fusion proteins formed complexes with EBNA-LP in the presence of RAX-1. These results indicated that EBNA-LP interacts with the viral and cellular Bcl-2 proteins through HAX-1, suggesting that EBNA-LP possesses a potential function in the regulation of apoptosis in EBV-infected cells.

Conserved region CR2 of Epstein-Barr virus nuclear antigen leader protein is a multifunctional domain that mediates self-association as well as nuclear localization and nuclear matrix association.

Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate gamma-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401-413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

EVIDENCE THAT HAX-1 IS AN INTERLEUKIN-1α N-TERMINAL BINDING PROTEIN

During studies aimed at understanding the function of the N-terminal peptide of interleukin-1α (IL-1 NTP, amino acids 1–112), which is liberated from the remainder of IL-1α during intracellular processing, we identified by yeast two-hybrid analysis a putative interacting protein previously designated as HAX-1. In vitro binding studies and transient transfection experiments confirmed that HAX-1 can associate with the IL-1 NTP. HAX-1 was first identified as a protein that associates with HS1, a target of non-receptor protein tyrosine kinases within haematopoietic cells. Recent data have also revealed interactions between HAX-1 and three disparate proteins, polycystin-2 (derived from the PKD2 gene), a protein linked to polycystic kidney disease, cortactin, and Epstein-Barr virus nuclear antigen leader protein (EBNA-LP). Sequence analysis of different HAX-1 binding domains revealed a putative consensus binding motif that is present in various intracellular proteins. Overlapping peptides comprising the IL-1 NTP were synthesized, and binding experiments revealed that discrete peptides were capable of interacting with HAX-1. HAX-1 may serve to retain the IL-1 NTP in the cytoplasm, and complex formation between the IL-1 NTP and HAX-1 may play a role in motility and/or adhesion of cells.

Identification of major phosphorylation sites of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP): ability of EBNA-LP to induce latent membrane protein 1 cooperatively with EBNA-2 is regulated by phosphorylation.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization of B cells. One of the potential functions of EBNA-LP is a cooperative induction with EBNA-2 of viral and cellular gene expression, including that of the genes for viral latent membrane protein 1 (LMP-1) and cellular cyclin D2. We report here that the phosphorylation of EBNA-LP by cellular kinase(s) is critical to its ability to cooperate with EBNA-2 in up-regulating the expression of LMP-1 in a B-lymphoma cell line. Our conclusion is based on the following observations. (i) Mass-spectrometric analysis of purified EBNA-LP and mutational analyses of EBNA-LP revealed that the serine residue at position 35 in the W2 repeat domain is the major phosphorylation site of EBNA-LP in vivo. (ii) Substitutions of this site in each W2 repeat domain with alanine markedly reduced the ability of the protein to induce LMP-1 expression in combination with EBNA-2 in Akata cells. (iii) Replacement at the major phosphorylation sites with glutamic acids restored the wild-type phenotype. It is well established that this substitution mimics constitutive phosphorylation. These results indicated that the coactivator function of EBNA-LP is regulated by phosphorylation.

The Conserved Domain CR2 of Epstein–Barr Virus Nuclear Antigen Leader Protein Is Responsible Not Only for Nuclear Matrix Association but Also for Nuclear Localization

There is a growing body of evidence for the importance of the nuclear matrix in various nuclear events including gene expression and DNA replication. Epstein–Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a nuclear matrix-associated protein that has been suggested to play an important role in EBV-induced transformation. To define the biological significance of the association of EBNA-LP with the nuclear matrix, we mapped the domain of EBNA-LP responsible for nuclear matrix association and investigated the functions of the EBNA-LP mutant mutagenized by substitution of alanines for the cluster of arginine residues in the mapped region. The results of the present study were as follows. (i) Transiently expressed EBNA-LP in COS-7 or BOSC23 cells was associated with the nuclear matrix, similarly to that in EBV-infected B cells. (ii) Mutational analysis of EBNA-LP revealed that a 10-amino acid segment of EBNA-LP is critical for nuclear matrix association of the protein. Interestingly, the identified region overlapped with the region CR2 of EBNA-LP conserved among a subset of primate gammaherpesviruses. The identified segment is referred to as EBNA-LP NMTS (nuclear matrix targeting signal). (iii) The EBNA-LP mutant with the arginine to alanine substitutions in NMTS was no longer localized not only to the nuclear matrix but also to the nucleus. (iv) The EBNA-LP mutant lacked its ability to coactivate EBNA-2-dependent transactivation. These results indicated that EBNA-LP needs to be localized in the nucleus and/or associated with the nuclear matrix through CR2 to elicit its function such as the coactivation of the EBNA-2-dependent transcriptional activation.

Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1. (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1.

Ultrastructural Characterization of Hearts from Transgenic Mice Expressing the Epstein-Barr Virus Nuclear Antigen-Leader Protein

Mice expressing the Epstein-Barr virus nuclear antigen-leader protein (EBNA-LP) develop congestive heart failure and atrial thrombosis as early as four months of age. Epstein-Barr virus (EBV) is a common human herpes virus with a complex life cycle involving chronic replication in epithelial tissues. The EBNA-LP, one of six EBV nuclear antigens, has an unknown biochemical function but is associated with EBV-induced growth transformation. This transgenic line was originally produced to investigate oncogenic consequences, but unexpectedly resulted in mice with progressive heart failure. Since the progression and development of the heart failure is highly predictable, this transgenic line may serve as a valuable model for studying the pathophysiologic changes associated with human dilated cardiomyopathy. We describe here the ultrastructural characteristics of the myocardium from EBNA-LP transgenic mice in order to better delineate the pathogenesis of the associated heart disease.