Kazuo Yanagi

Nuclear Import of Epstein-Barr Virus Nuclear Antigen 1 Mediated by NPI-1 (Importin α5) Is Up- and Down-Regulated by Phosphorylation of the Nuclear Localization Signal for Which Lys379 and Arg380 Are Essential

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is essential for replication of episomal EBV DNAs and maintenance of latency. Multifunctional EBNA-1 is phosphorylated, but the significance of EBNA-1 phosphorylation is not known. Here, we examined the effects on nuclear translocation of Ser phosphorylation of the EBNA-1 nuclear localization signal (NLS) sequence, 379Lys-Arg-Pro-Arg-Ser-Pro-Ser-Ser386. We found that Lys379Ala and Arg380Ala substitutions greatly reduced nuclear transport and steady-state levels of green fluorescent protein (GFP)-EBNA1, whereas Pro381Ala, Arg382Ala, Pro384Ala, and Glu378Ala substitutions did not. Microinjection of modified EBNA-1 NLS peptide-inserted proteins and NLS peptides cross-linked to bovine serum albumin (BSA) showed that Ala substitution for three NLS Ser residues reduced the efficiency of nuclear import. Similar microinjection analyses demonstrated that phosphorylation of Ser385 accelerated the rate of nuclear import, but phosphorylation of Ser383 and Ser386 reduced it. However, transfection analyses of GFP-EBNA1 mutants with the Ser-to-Ala substitution causing reduced nuclear import efficiency did not result in a decrease in the nuclear accumulation level of EBNA-1. The results suggest dynamic nuclear transport control of phosphorylated EBNA-1 proteins, although the nuclear localization level of EBNA-1 that binds to cellular chromosomes and chromatin seems unchanged. The karyopherin α NPI-1 (importin α5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin α1) bound only weakly and Qip1 (importin α3) did not bind to the Ser385-phosphorylated NLS. These findings suggest that the amino-terminal 379Lys-Arg380 is essential for the EBNA-1 NLS and that Ser385 phosphorylation up-regulates nuclear transport efficiency of EBNA-1 by increasing its binding affinity to NPI-1, while phosphorylation of Ser386 and Ser383 down-regulates it.

Elevated immunoglobulin G antibodies to the proline-rich amino-terminal region of Epstein-Barr virus nuclear antigen-2 in sera from patients with systemic connective tissue diseases and from a subgroup of Sjögren's syndrome patients with pulmonary involvements.

Associations of Epstein–Barr virus (EBV) and autoimmune diseases have been hypothesized. We have analysed IgG antibodies to EBV nuclear antigen (EBNA)‐2 in sera from Japanese patients with autoimmune systemic connective tissue diseases (CTD), exemplified by systemic lupus erythematosus (SLE), primary Sjögrens syndrome (SS), rheumatoid arthritis (RA), systemic sclerosis  (SSc)  and  secondary  SS  (classical  CTDs  complicated  with  SS). An  enzyme‐linked  immunosorbent  assay  (ELISA)  which  uses  glutathione‐S‐transferase polypeptides fused to EBV nuclear antigen (EBNA)‐2 and EBNA‐1 was developed. Ratios of IgG antibody reactivity to whole IgG concentrations of sera were calculated to normalize EBNA‐2 and EBNA‐1 antibody levels to the hypergammaglobulinaemia that occurs in CTD. The ELISA optical density OD450 readings of IgG antibodies to both the amino‐terminal aa 1–116 of EBNA‐2 and carboxyl‐terminal aa 451–641 of EBNA‐1 were elevated significantly in patients with SLE, primary SS, RA, SSc and secondary SS when compared to EBNA‐1. The OD readings were divided by serum IgG concentrations to normalize for the hypergammaglobulinaemia. The specific levels of IgG antibodies to the amino‐terminal region of EBNA‐2 were elevated in patients with SLE, primary SS or RA, as well as those with secondary SS complicated with SLE or RA. The EBNA‐2 amino‐terminal region contains a polyproline tract and a proline‐rich sequence and has considerable amino acid sequence homology with many cellular proline‐rich proteins. High ratios of EBNA‐2 aa 1–116 to EBNA‐1 aa 451–641 IgG antibody levels which probably suggest reactivation of EBV latent infection were associated significantly with pulmonary involvement in SS patients. These results are consistent with the hypothesis that the sequence similarity between the amino‐terminal region of EBNA‐2 and proline‐rich cellular proteins is associated with pathogenesis in a subpopulation of CTD patients, possibly by the molecular mimicry–epitope shift mechanism.

The domain of Epstein-Barr virus nuclear antigen 1 essential for binding to oriP region has a sequence fitted for the hypothetical basic-helix-loop-helix structure

The domain of Epstein-Barr virus nuclear antigen 1 (EBNA-1) which is essential for binding to a region containing oriP, an episomal replication origin of EBV DNA, was analyzed by DNA binding assay with beta-galactosidase-EBNA-1 fusion proteins. It was revealed that a 159-amino acid (aa) domain, 460-618 aa, of EBNA-1 retained the oriP-binding activity and the domains activity was abolished by a deletion of 29 aa from its amino-terminal end and by a 38 aa deletion from its carboxyl-end as well. One of five monoclonal antibodies against EBNA-1 specifically inhibited the binding of the beta-galactosidase-EBNA-1 fusion protein to the oriP region. The epitope recognized by the monoclonal antibody was mapped in the crucial 29 aa region. An analysis of the domains putative secondary structure and a computer search of amino acid sequence homology indicated that the 159-aa domain contains the hypothetical basic-helix-loop-helix structure which is considered to be a common characteristic structure of a family of DNA binding proteins. Examinations of DNA binding activity of the other EBNA polypeptides with a series of fusion proteins and similar structural analyses of their amino acid sequences were also performed. This study suggests that EBNA-1 is a constituent of the family of DNA binding proteins which are involved in transcriptional regulation critical for cell differentiation or cell-type determination.

Maintenance of Serum Immunoglobulin G Antibodies to Epstein-Barr Virus (EBV) Nuclear Antigen 2 in Healthy Individuals from Different Age Groups in a Japanese Population with a High Childhood Incidence of Asymptomatic Primary EBV Infection

ABSTRACT Immunoglobulin G (IgG) antibodies to Epstein-Barr virus (EBV) nuclear antigens 2 and 1 (EBNA-2 and EBNA-1, respectively) were studied using sera from healthy individuals of a population with a high incidence of asymptomatic primary EBV infections during infancy or childhood in Japan. Two CHO-K1 cell lines expressing EBNA-2 and EBNA-1 were used for anticomplement and indirect immunofluorescence assays. The positivity rate for EBNA-2 IgG rose in the 1- to 2-year age group, increased and remained at a plateau (∼45%) between 3 and 29 years of age (3- to 4-, 5- to 9-, 10- to 14-, and 15- to 29-year age groups), and then reached 98% by age 40 (≥40-year age group). Both seropositivity for EBNA-1 and seropositivity for EBNAs in Raji cells (EBNA/Raji) were detected in the 1- to 2-year age group, remained high, and finally reached 100% by age 40. The geometric mean titer (GMT) of EBNA-2 IgG reached a plateau in the 5- to 9- and 10- to 14-year-old groups and remained elevated in the older age groups (15 to 29 and ≥40 years). The GMT of EBNA-1 IgGs increased to a plateau in the 1- to 2-year-old group and remained unchanged in the older age groups. The GMT of EBNA/Raji IgGs also reached a plateau in the 1- to 2-year-old group, remained level throughout the 3- to 14-year age groups, and decreased in the 15- to 29-year-olds. EBNA-2 IgGs emerged earlier than EBNA-1 IgGs in 8 of 10 patients with infectious mononucleosis, who were between 1 and 27 years old, and declined with time in three of eight cases. These results suggest that EBNA-2 IgG antibodies evoked in young children by asymptomatic primary EBV infections remain elevated throughout life, probably because of reactivation of latent and/or exogenous EBV superinfection.

Telomere size and telomerase activity in Epstein-Barr virus (EBV)-positive and EBV-negative Burkitt's lymphoma cell lines.

Summary.The telomere repeat lengths of BL cell lines were quantified by measuring terminal restriction fragment (TRF). Epstein-Barr virus (EBV)-positive Namalwa, Raji, and EB-3 cell lines have long telomeres, i.e. TRFs 10–19 kbp, whereas the Daudi cell line, producing a transformation-defective EBV mutant, has TRFs ∼2.2 kbp. EBV-negative BJAB and DG75 cell lines have short TRFs 3.9–5.4 kbp, shorter than the ∼12 kbp TRFs in PBLs. Telomerase activities of these BL cell lines are similar. TRFs of non-BL lymphoma cell lines are 2.3–5.5 kbp. Fluorescent in situ hybridization (FISH) studies of these cell lines showed remarkable heterogeneity of telomere size in chromosomes in the same BL cell. These results suggest that EBV-positive and EBV-negative BL cell lines have experienced various telomere dynamics.

Epstein-Barr Virus (EBV) Nuclear Antigen 1 Colocalizes with Cellular Replication Foci in the Absence of EBV Plasmids

ABSTRACT Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.

Induced CD25 Expression in a Human B-Lymphoma Cell Line Transfected with the Epstein-Barr Virus Nuclear Antigen 2 Gene

Two EBV‐negative human B‐lymphoma cell lines, BJAB and DG75, were transfected with an Epstein‐Barr virus (EBV) nuclear antigen 2 (EBNA‐2) gene, which plays a critical role in the EBV‐induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co‐transfected with the EBNA‐2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA‐2‐expressing cell clones was analyzed by flowcytometry. None of the EBNA‐2‐expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA‐2 in a different EBV‐negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL‐2R/Tac) on cell surface, however, was induced in two of six DG75‐derived cell clones. One of the two CD25‐induced cell clones was expressing EBNA‐2 only, and the other was co‐expressing EBNA‐2 and LMP. The results suggest that EBNA‐2 has a potential to up‐regulate CD25 independently of CD23 on human B cells.

Molecular mechanism of activation of human Cdc7 kinase: bipartite interaction with Dbf4/activator of S phase kinase (ASK) activation subunit stimulates ATP binding and substrate recognition.

Cdc7 is a serine/threonine kinase conserved from yeasts to human and is known to play a key role in the regulation of initiation at each replication origin. Its catalytic function is activated via association with the activation subunit Dbf4/activator of S phase kinase (ASK). It is known that two conserved motifs of Dbf4/ASK are involved in binding to Cdc7, and both are required for maximum activation of Cdc7 kinase. Cdc7 kinases possess unique kinase insert sequences (kinase insert I–III) that are inserted at defined locations among the conserved kinase domains. However, precise mechanisms of Cdc7 kinase activation are largely unknown. We have identified two segments on Cdc7, DAM-1 (Dbf4/ASK interacting motif-1; amino acids 448–457 near the N terminus of kinase insert III) and DAM-2 (C-terminal 10-amino acid segment), that interact with motif-M and motif-C of ASK, respectively, and are essential for kinase activation by ASK. The C-terminal 143-amino acid polypeptide (432–574) containing DAM-1 and DAM-2 can interact with Dbf4/ASK. Characterization of the purified ASK-free Cdc7 and Cdc7-ASK complex shows that ATP binding of the Cdc7 catalytic subunit requires Dbf4/ASK. However, the “minimum” Cdc7, lacking the entire kinase insert II and half of kinase insert III, binds to ATP and shows autophosphorylation activity in the absence of ASK. However, ASK is still required for phosphorylation of exogenous substrates by the minimum Cdc7. These results indicate bipartite interaction between Cdc7 and Dbf4/ASK subunits facilitates ATP binding and substrate recognition by the Cdc7 kinase.

Contrasting Geographic Distribution Profiles of the Herpes Simplex Virus Type 1 BgOL and BgKL Variants in Japan Suggest Dispersion and Replacement

ABSTRACT Thelifelong latent infection-reactivation mode of infection of herpes simplex virus type 1 (HSV-1) transmitted by close contact has allowed a diversity of restriction fragment length polymorphism (RFLP) variations to accumulate in human populations. Whether and how the variants of the HSV-1 that is ubiquitous worldwide spread to different human populations is not clear. In our previous study the geographically gradient distribution of the HSV-1 BgKL variant, which is a good marker for the BgKL:SaCFJM:SaGHM:SaD/EL:KpMS variant, suggested that BgKL dispersed geographically. Southern hybridization analyses showed that in BgKL the BglII cleavage site between the BglII K and small “Q/#13” fragments is lost, the SalI cleavage sites between the SalI J and C and between SalI F and J fragments are lost, and the SalI E fragment is abnormally large (SaEL variation). The RFLP and geographic distribution of one more HSV-1 RFLP variant, BgOL, were comparatively analyzed. The BglII cleavage site between the BglII O and Q/#13 fragments is lost in BgOL. BgOL clinical isolates were not associated with any of the SaCFJM, SaEL, SaGHM, or KpMS variations, whereas one-fourth of the non-BgOL:non-BgKL isolates was associated with SaCFJM and SaGHM, indicating that BgKL and BgOL are distant in terms of diversification. BgOL is distributed highly in the northeastern region and the southwestern island of Kyushu but is rare between the two regions in Japan, in a remarkable contrast to BgKL. These are the first epidemiologic data to show contrasting geographic distribution profiles of two HSV-1 variants and suggest the gradual dispersion and replacement of HSV-1 variants.

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.