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.

Monoclonal antibodies against regions topologically surrounding the homodimeric beta-barrel interface of Epstein-Barr virus nuclear antigen-1.

Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is essential for maintenance of EBV latency. Four mouse monoclonal antibodies (mAbs) against the part of the EBNA-1 sequence (amino acids 451-641) containing the domain that forms a homodimeric eight-stranded beta-barrel were generated and characterized, examined for immunocytochemical staining, immunoblotting and isoelectric focusing of EBNA-1 proteins, and used to examine interactions between EBNA-1 polypeptides by far-Western blot assays. Far-Western blot analyses using the mAbs suggest that both the beta-strand (aa 593-604) and alpha helix (aa 568-582) are essential for EBNA-1 dimerization, consistent with yeast two-hybrid studies of mutant EBNA-1 polypeptides. These mAbs should be useful for studies on the structure and function of EBNA-1 proteins.

Analysis of herpes simplex virus type 1 restriction fragment length polymorphism variants associated with herpes gladiatorum and Kaposi's varicelliform eruption in sumo wrestlers.

The geographical distribution of herpes simplex virus type 1 (HSV-1) restriction fragment length polymorphism (RFLP) variants BgK(L) and BgO(L) and the high relative frequency (RF) of BgK(L) in orolabial lesions has led to a dispersion-replacement hypothesis for these variants. The pathogenic properties of HSV-1 variants in mice and professional sumo wrestlers were examined here. The wrestlers herpes gladiatorum (HG) was caused by primary and non-primary HSV-1 infections and recurred in many wrestlers. HSV-1 neutralizing antibody titres in sera from wrestlers who did not develop HG were relatively high. HG was caused by distinct HSV-1 variants and strains from wrestlers living in the same sumo stable. The BgK(L) RF was significantly higher in HG cases, particularly in those with Kaposi's varicelliform eruption. These data indicated that reactivation and transmission of latent HSV-1 infections, especially BgK(L), occurred frequently among wrestlers and was caused by severe skin damage. These results support the BgK(L) dispersion hypothesis.

Contrasting geographic distribution profiles of the herpes simplex virus type 1 BgOL and BgKL variants in Japan suggest dispersion and replacement.

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 BgK(L) variant, which is a good marker for the BgK(L):SaCFJ(M):SaGH(M):SaD/E(L):KpM(S) variant, suggested that BgK(L) dispersed geographically. Southern hybridization analyses showed that in BgK(L) 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 (SaE(L) variation). The RFLP and geographic distribution of one more HSV-1 RFLP variant, BgO(L), were comparatively analyzed. The BglII cleavage site between the BglII O and Q/#13 fragments is lost in BgOL. BgO(L) clinical isolates were not associated with any of the SaCFJ(M), SaE(L), SaGH(M), or KpM(S) variations, whereas one-fourth of the non-BgO(L):non-BgK(L) isolates was associated with SaCFJ(M) and SaGH(M), indicating that BgK(L) and BgO(L) are distant in terms of diversification. BgO(L) 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 BgK(L). 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.

The herpes simplex virus type 1 BgKL variant, unlike the BgOL variant, shows a higher association with orolabial infection than with infections at other sites, supporting the variant-dispersion-replacement hypothesis.

The identification and geographic distribution of the herpes simplex virus type 1 (HSV-1) BglII restriction fragment length polymorphism (RFLP) variants named BgK(L) and BgO(L) in clinical isolates from orolabial and cutaneous sites were described in our previous reports, in which the dispersion and replacement of HSV-1 variants were proposed. The base substitution sites deduced from the BgK(L) multiple RFLP variations were mapped to the U(L)12 (DNase), R(L)2 (alpha0 transactivator), and latency-associated transcript genes in the present study. The results show that the relative frequencies (RFs) of BgK(L) are significantly higher in orolabial and cutaneous HSV-1 infections than in ocular infections. For the BgO(L) variant, the opposite was found; i.e., the RF of BgO(L) was significantly lower in orolabial and cutaneous infections than in ocular infections. No significant differences in the RFs of non-BgK(L):non-BgO(L) isolates were observed. The ratio of the BgK(L) RF to the BgO(L) RF was much higher for the orolabial and cutaneous infection groups than for the ocular infection group, whereas the BgK(L) RF-to-non-BgK(L):non-BgO(L) RF ratios for the former groups were slightly higher than those for the latter group. The higher efficiency of orolabial and cutaneous infections caused by BgK(L) compared to the efficiency of infections caused by BgO(L) allows BgK(L) to spread more efficiently in human populations and to displace BgO(L), because the mouth and lips are the most common HSV-1 infection sites in children. The present study supports our HSV-1 dispersion-and-replacement hypothesis and suggests that HSV-1, the latency-reactivation of which allows variants to accumulate in human populations, has evolved under competitive conditions, providing a new perspective on the polymorphism or variation of HSV-1.

Geographical distribution of the herpes simplex virus type 1 BgKL variant in Japan suggests gradual dispersion of the virus from Shikoku Island to the other Islands.

Restriction endonuclease fragment length polymorphism (RFLP) is useful for the epidemiological study of herpes simplex virus type 1 (HSV-1). We report here the identification of a major BglII RFLP variant of HSV-1, designated BgKL, found in 27.0% of 636 HSV-1 clinical isolates. We have also established its geographic distribution in Japan. BgKL has an unusually large BglII K fragment. SalI cleavage analyses showed that 97% of BgKL variant isolates lack both the SalI C-J and the F-J cleavage sites and have an unusually large SalI D or E fragment, and 91% of the BgKL variants lack both SalI G and H fragments. Furthermore, 96% of BgKL isolates have an unusually small KpnI M fragment. Therefore, BgKL is a marker for these five mutations in most HSV-1 isolates and is a useful HSV-1 RFLP marker. The BgKL variant was found in 59% of HSV-1 isolates from Shikoku Island, 44% of HSV-1 isolates from the Chugoku region of Honshu Island, 31% of HSV-1 isolates from Kyushu Island, 0% of HSV-1 isolates from Okinawa Island, 49% of HSV-1 isolates from Osaka, 27% of HSV-1 isolates from Shiga, 13% of HSV-1 isolates from the Chubu Region, and 9% of HSV-1 isolates from the Tohoku Region of Honshu Island. Differences in the frequency of BgKL between the Shikoku-Chugoku-Osaka area (49%) and Kyushu, between Kyushu and Okinawa, between the Shikoku-Chugoku-Osaka area and Shiga, and between Shiga and Tohoku are all statistically significant. The BgKL frequency decreases in a geographical gradient suggest that this HSV-1 variant was dispersed from Shikoku to the surrounding regions and then to more distant regions. The BgKL frequency in Tokyo was similar to the nationwide average. These are the first data to suggest a geographic and demographic dispersion pattern of HSV-1. Implications for the epidemiology and diversification of HSV-1 are discussed.

Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up- and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential.

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 alpha NPI-1 (importin alpha5), a nuclear import adaptor, bound more strongly to Ser385-phosphorylated NLS than to any other phosphorylated or nonphosphorylated forms. Rch1 (importin alpha1) bound only weakly and Qip1 (importin alpha3) 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.

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids.

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.

Epstein-Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular.

Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition of oriP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP-EBNA-1) is examined by confocal microscopy combined with a 'premature chromosome condensation' (PCC) procedure. Analyses show that GFP-EBNA-1 expressed in living cells that lack oriP plasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP-EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.

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.

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 ( approximately 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.

Enhancement of immunity against VZV by giving live varicella vaccine to the elderly assessed by VZV skin test and IAHA, gpELISA antibody assay.

The enhancement of immunity against varicella-zoster vaccine (VZV) by subcutaneous injection of a live varicella vaccine was assessed by the VZV skin test for cell-mediated immunity (CMI), and immunoadherence hemagglutination assay (IAHA) and gpELISA antibody assays in the elderly people of 50-79 years of age. A total of 127 subjects were examined: 79 aged 50-59, 25 aged 60-69, and 25 aged 70-79. All were seropositive by the gpELISA assay (one was seronegative in the IAHA antibody assay). In contrast, a notable decline was observed in the VZV skin test with increasing age. Negative reaction was observed in 16/79 (20.2%) of the subjects in their 50s, 12/25 (48.0%) in their 60s and 14/25 (56.0%) in the 70s. After the vaccination, the results of the VZV skin test changed from negative to positive in 15/16 (91.8%) of subjects in their 50s, 11/12 (91.7%) in their 60s and 12/14 (85.7%) in their 70s. The mean antibody titer in the IAHA and the gpELISA increased approximately two-fold after the vaccination in each group. Immunity to VZV in 35 elderly subjects who were vaccinated previously was followed up for 4 years. All were positive by the VZV skin test after the previous vaccination. After 4 years, 31 (88.6%) were positive by the skin test, 4 were negative and became positive after revaccination. Although this study was uncontrolled open study, the results suggest that administering live varicella vaccine to the elderly is effective for enhancing immunity, particularly CMI to VZV.