. Here, we show that the HHV-6 U47 gene, which is a positional homolog of the human cytomegalovirus glycoprotein O (gO) gene, encodes a third component of the HHV-6 gH-gLcontaining envelope complex. A monoclonal antibody (MAb) against the amino terminus of HHV-6 gO reacted in immunoblots with protein species migrating at 120 to 130 kDa and 74 to 80 kDa in lysates of HHV-6-infected cells and with a 74-to 80-kDa protein species in purified virions. The 80-kDa form of gO was coimmunoprecipitated with an anti-gH MAb, but an anti-gQ MAb, which coimmunoprecipitated gH, did not coprecipitate gO. Furthermore, the gH-gL-gO complex did not bind to human CD46, indicating that the complex was not a ligand for CD46. These findings suggested that the viral envelope contains at least two kinds of tripartite complexes, gH-gL-gQ and gH-gL-gO, and that the gH-gL-gO complex may play a role different from that of gH-gL-gQ during viral infection. This is the first report of two kinds of gH-gL complexes on the viral envelope in a member of the herpesvirus family.Human herpesvirus 6 (HHV-6) was first isolated from the peripheral blood of patients with AIDS and lymphoproliferative disorders (8, 28). HHV-6 isolates can be classified into two variants, HHV-6A and HHV-6B. HHV-6B is the causative agent of exanthem subitum (40). The classification of the two variants is based on nucleotide sequence differences, as well as their immunological and biological characteristics (1-3, 6, 37).In the herpesvirus family, the envelope glycoproteins play critical roles in viral infection, including attachment, penetration, cell-to-cell spread, and the envelopment and maturation of nascent viral particles. In all of the human and animal herpesviruses studied to date, homologs of glycoprotein H (gH) and glycoprotein L (gL) have been found (9, 14-17, 20, 21, 26, 31-33, 38, 39, 41, 42). These two envelope glycoproteins, which associate to form a gH-gL complex, have been implicated as key participants in fusion events that are critical to herpesvirus infection. Studies of the HHV-6 gH (14, 27) and gL proteins have shown them to be representative gH and gL homologs (19,20).Santoro et al. showed that human CD46 is a cellular receptor for HHV-6 (30). Recently, we showed that HHV-6A, but not HHV-6B, mediates fusion from without in a variety of cells expressing human CD46 (23) and that anti-gH and anti-gB monoclonal antibodies (MAbs) inhibit the cell-cell fusion induced by HHV-6A. Furthermore, we found that the HHV-6A gH-gL complex interacts with one form of the U100 gene products, which we designated glycoprotein Q (gQ) (22), and we identified the gH-gL-gQ complex of HHV-6A as the viral ligand for human CD46 (25). Santoro et al. have also reported that HHV-6 gH associates with CD46 by a coimmunoprecipitation study (29).In the case of Epstein-Barr virus (EBV), a third viral glycoprotein, gp42, associates with the gH-gL complex (18,35,36). Recently, a third viral gene product of human cytomegalovirus (HCMV), glycoprotein O (gO), was identified as a member of the...

In an aim to create a "sharp" molecular knife, we have studied site-specific fragmentation caused by Si:2p core photoionization of bridged trihalosilyltrimethylsilyl molecules in the vapor phase. Highly site-specific bond dissociation has been found to occur around the core-ionized Si site in some of the molecules studied. The site specificity in fragmentation and the 2p binding energy difference between the two Si sites depend in similar ways on the intersite bridge and the electronegativities of the included halogen atoms. The present experimental and computational results show that for efficient "cutting" the following conditions for the two atomic sites to be separated by the knife should be satisfied. First, the sites should be located far from each other and connected by a chain of saturated bonds so that intersite electron migration can be reduced. Second, the chemical environments of the atomic sites should be as different as possible.

The UV protection and singlet oxygen quenching of aloesaponarin I have been studied by means of laser spectroscopy. The excited-state intramolecular proton transfer that provides the UV protection takes place along only one of the molecule's two intramolecular hydrogen bonds, and this can be understood by considering the nodal pattern of the wave function. The functional groups participating in the excited-state intramolecular proton transfer also play important roles in the singlet oxygen quenching. Aloesaponarin I has a quenching rate constant larger than that of vitamin E and has a long duration of action due to its resistance to UV degradation and chemical attacks by singlet oxygen and free radicals.

An immediate-early (IE) gene of human herpesvirus 6 (HHV-6), U95, has similarity at the amino acid level to the murine cytomegalovirus (MCMV) IE2 gene and is related to the human cytomegalovirus (HCMV) US22 gene family. Sequence analyses of U95 cDNA clones revealed that the transcription start site was located about 1.6 kbp upstream of the putative initiating ATG and that the transcript consisted of two exons. A single intron extended from nucleotides 142589 to 144229, which contained ORF U94. A protein with a molecular mass of about 120 kDa was translated from this cDNA clone in an in vitro transcription-translation assay. The transcription start site was found to be 220 bp downstream of the R3 region by primer extension analysis. HHV-6 has three repetitive elements, R1, R2, and R3, in or near the IE-A locus. R3 is composed of 24 copies of a 104-to 107-bp sequence element, which contains multiple putative binding sites for cellular transcription factors such as AP2 and NF-B, and its biological significance has yet to be elucidated. The region between ؊710 and ؉46 relative to the transcription start site of U95 was analyzed in this study. Deletion from ؊710 to ؊396, corresponding to three copies of an R3 unit, decreased the promoter activity by 15-fold, and coexpression of IB␣(S32A/S36A) repressed it to almost the same level. Electrophoretic mobility shift assays showed that NF-B family members p50 and c-Rel bound to NF-B sites derived from the R3 region. These results demonstrate that R3 strongly enhances the U95 promoter activity and that NF-B and binding sites for NF-B in the R3 region play an important role in its activation. Because U95 promoter activity correlated with the number of R3 units, which each contained an NF-B site, the repetitive organization of R3 is important for regulating U95 transcription.

A mass spectroscopic analysis of proteins from human herpesvirus 6 (HHV-6)-infected cells showed that the HHV-6 U14 protein coimmunoprecipitated with the tumor suppressor p53. The binding of U14 to p53 was verified by coimmunoprecipitation experiments in both Molt-3 cells infected with HHV-6 and 293 cells cotransfected with U14 and p53 expression vectors. Indirect immunofluorescence assays (IFAs) showed that by 18 h postinfection (hpi) U14 localized to the dot-like structures observed in both the nucleus and cytoplasm where p53 was partly accumulated. Despite Northern blotting evidence that U14 follows late kinetics, the U14 protein was detected immediately after infection (at 3 hpi) by IFA. In addition, by Western blotting, U14 was detected at 0 hpi or in the presence of cycloheximide which completely abolished the expression of IE1 protein.In addition to U14, p53 was detected at 0 hpi although it was not detected in mock-infected cells. Furthermore, both U14 and p53 were clearly detected in the viral particles by Western blotting and immunoelectron microscopy, supporting the idea that U14 and p53 are incorporated into virions. Our study provides the first evidence of the incorporation of cellular p53 into viral particles and suggests that p53 may play an important role in viral infection.