Biomedical subjects

Y Isegawa

Publications and source records attributed to Y Isegawa.

At least 19 recordsLinked to original sources

PubMed2003

Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded vMIP-I and vMIP-II induce signal transduction and chemotaxis in monocytic cells.

Kaposi's sarcoma-associated herpesvirus (KSHV)/ Human herpesvirus 8 encodes three chemokines, which are called viral macrophage inflammatory protein (vMIP)-I, -II, and -III. Here, we expressed the KSHV vMIP-I and vMIP-II proteins and analyzed their biological functions. Both vMIP-I and vMIP-II had an apparent molecular mass of 7.8 kDa and were localized to the cytoplasm in a body cavity-based lymphoma cell line BC-3, stimulated with phorbol ester. We next treated a human monocytic leukemia cell line, THP-1, with purified recombinant vMIP-I and vMIP-II, or vMIP-I and vMIP-II fused with alkaline phosphatase to study Ca(2+) signalling and in vitro chemotaxis in response to these proteins. Calcium mobilization was induced by both vMIP-I and vMIP-II. Furthermore, vMIP-I and vMIP-II induced Ca(2+) mobilization in K562 cells expressing the CC chemokine receptor 5 (CCR5), suggesting that both may be agonistic for CCR5. Additionally, vMIP-I induced Ca(2+) mobilization through the intermediary of CCR8. These viral MIPs were also capable of chemotactically activating the THP-1 cells. These results imply that vMIP-I and vMIP-II may play important roles in the propagation of KS and primary effusion lymphoma by inducing the chemotaxis of CCR5-expressing monocytes.

Calcium↗

PubMed2001

The R3 region, one of three major repetitive regions of human herpesvirus 6, is a strong enhancer of immediate-early gene U95.

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-kappaB, 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 IkappaBalpha(S32A/S36A) repressed it to almost the same level. Electrophoretic mobility shift assays showed that NF-kappaB family members p50 and c-Rel bound to NF-kappaB sites derived from the R3 region. These results demonstrate that R3 strongly enhances the U95 promoter activity and that NF-kappaB and binding sites for NF-kappaB 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-kappaB site, the repetitive organization of R3 is important for regulating U95 transcription.

Base Sequence↗

PubMed2000

Structure of transcripts and proteins encoded by U79-80 of human herpesvirus 6 and its subcellular localization in infected cells.

We analyzed the U79-80 gene of human herpesvirus 6 (HHV-6), which is predicted to be a positional homolog of the UL112-113 gene of human cytomegalovirus (HCMV). The U79-80 gene encoded a family of nuclear proteins of 36, 41, 44, and 59 kDa. These proteins had common amino termini and were generated by complex alternative splicing. Transcripts from U79-80 appeared as early as 3 h postinfection and could be detected in the presence of phosphonoformate. U79-80 proteins were seen as early as 8 h postinfection and could be detected in the presence of phosphonoformate but not in the presence of cycloheximide combined with actinomycin D treatment. The U79-80 proteins were localized to the nucleus of infected cells, where they were detected as a speckled or punctuate pattern. Moreover, the U79-80 proteins colocalized with the components of the viral DNA replication machinery and appeared to distribute adjacent to or touching nuclear domain 10, where viral DNA replication occurs. From the sequence analysis of genomic DNA, the predicted amino acid similarity between U79-80 and UL112-113 was lower than between other genes, but the characteristics of the transcripts and proteins encoded by U79-80 were similar to those of UL112-113. These results suggest that the U79-80 proteins have a role in viral DNA replication and are functional homologues of the UL112-113 proteins.

Amino Acid Sequence↗

PubMed1999

Comparison of the complete DNA sequences of human herpesvirus 6 variants A and B.

Human herpesvirus 6 (HHV-6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. Two variants of HHV-6 have been distinguished on the basis of differences in several properties. We have determined the complete DNA sequence of HHV-6 variant B (HHV-6B) strain HST, the causative agent of exanthem subitum, and compared the sequence with that of variant A strain U1102. A total of 115 potential open reading frames (ORFs) were identified within the 161,573-bp contiguous sequence of the entire HHV-6 genome, including some genes with remarkable differences in amino acid identity. All genes with <70% identity between the two variants were found to contain deleted regions when ORFs that could not be expressed were excluded from the comparison. Except in the case of U47, these differences were found in immediate-early/regulatory genes, DR2, DR7, U86/90, U89/90, and U95, which may represent characteristic differences of variants A and B. Also, we have successfully typed 14 different strains belonging to variant A or B by PCR using variant-specific primers; the results suggest that the remarkable differences observed were conserved evolutionarily as variant-specific divergence.

DNA, Viral↗

PubMed1999

Human herpesvirus 6 open reading frame U83 encodes a functional chemokine.

Some viruses including herpesviruses have undergone evolution to benefit viral infection and propagation by pirating and modifying host genes such as chemokine genes. Human herpesvirus 6 (HHV-6), acutely or persistently infects mononuclear cells in vitro. DNA sequence analysis of HHV-6 has revealed that the putative protein encoded by an open reading frame (ORF) of the U83 gene in HHV-6 variant B resembled a human chemokine. We have cloned the U83 gene and analyzed the biological function of this gene. The U83 gene contained an ORF encoding a 113-amino-acid peptide, starting at the first methionine and containing a possible signal peptide and the typical cysteine residues characteristic of the chemokines. Reverse transcription-PCR analysis of mRNA and immunofluorescent-antibody testing of infected cells both indicated that the encoded protein was a late protein. The ORF U83 gene fused to the Fc gene was expressed as a fusion protein in COS-7 cells by transfection, and the fusion protein was purified from the supernatant of transfected cells to test its biological function. The purified protein was capable of inducing transient calcium mobilization in THP-1 cells and of chemotactically activating THP-1 cells. These findings suggested that the U83 protein might play an important role in HHV-6 propagation in vivo by activating and trafficking mononuclear cells to sites of viral replication, thus aiding the development of superbly efficient virus production mechanisms.

Amino Acid Sequence↗

PubMed1998

Analysis of human herpesvirus 6 U3 gene, which is a positional homolog of human cytomegalovirus UL 24 gene.

The US22 gene family was first discovered in human cytomegalovirus (HCMV) and contains several conserved amino acid motifs. Human herpesvirus 6 (HHV-6) also encodes several genes belonging to the US22 family, including the U3 gene (a positional homolog of HCMV UL24). Because the gene products of the US22 gene family function as gene regulators in general, we analyzed the HHV-6A U3 gene. Six transcripts with different molecular weights of 7.5-1.8 kb were detected by Northern blot analysis using a U3-specific probe. Sequence analyses of the respective cDNA clones and primer extension experiments revealed that the U3 gene encoded the 2.0- and 3.5-kb transcripts and had no splicing within the U3 gene region. By immunofluorescence testing using antibodies raised to a fusion protein of MBP (maltose-binding protein) and U3, the U3 viral antigen was detected as early as 24 h p.i. in HHV-6A-infected U373 cells. The antigens were found in cytoplasmic granules, preferentially in the endoplasmic reticulum. Moreover, cotransfection assay using a luciferase gene expression system revealed that the U3 gene product was capable of activating the human immunodeficiency virus long terminal repeat promoter in CV1 cells.

Cytomegalovirus↗

PubMed1998

Characterization of neutralizing monoclonal antibody escape mutants of Hantaan virus 76118.

Neutralizing monoclonal antibody (MAb) escape mutants of Hantaan virus were generated using MAbs to envelope protein G1 (16D2) and G2 (11E10). The mutant viruses (mu16D2 and mu11E10), lacked reactivity only to the selecting MAb, or a MAb belonging to the same antigenic site. Both mutants had a single amino acid (a.a.) substitution. The a.a. substitution, found in mu16D2, was different from that found in another mutant selected with the same MAb (16D2). Although MAb 11E10 immunoprecipitated G2 protein, a deduced a.a. substitution was located in the G1 region. These results suggest that antigenic sites defined by neutralizing MAbs are composed of discontinuous epitopes over the G1 and G2 proteins. Mutant 11E10 showed a significant decrease in virulence in suckling mice. A virulence revertant of mu11E10, selected through passages in suckling mice brain, showed exactly the same deduced a.a. sequence as mu11E10 and still was not neutralized by MAb 11E10. Since mutant 16D2 was virulent for suckling mice, neutralization related epitopes found with MAbs 11E10 and 16D2 were independent of pathogenicity in BALB/c mice.

Amino Acid Substitution↗

PubMed1998

Human herpesvirus 6 open reading frame U12 encodes a functional beta-chemokine receptor.

Human herpesvirus 6 (HHV- 6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. HHV- 6 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors (GCR), while three other betaherpesviruses, human cytomegalovirus, murine cytomegalovirus, and human herpesvirus 7, have three, one, and two GCR-homologous genes, respectively. The U12 gene is expressed late in infection from a spliced mRNA. The U12 gene was cloned, and the protein was expressed in cells and analyzed for its biological characteristics. U12 functionally encoded a calcium-mobilizing receptor for beta-chemokines such as regulated upon activation, normal T expressed and secreted (RANTES), macrophage inflammatory proteins 1alpha and 1beta (MIP-1alpha and MIP-1beta) and monocyte chemoattractant protein 1 but not for the alpha-chemokine interleukin-8, suggesting that the chemokine selectivity of the U12 product was distinct from that of the known mammalian chemokine receptors. These findings suggested that the product of U12 may play an important role in the pathogenesis of HHV- 6 through transmembrane signaling by binding with beta-chemokines.

Amino Acid Sequence↗

PubMed1997

Nucleotide sequence analysis of a 30-kilobase-pair region of human herpesvirus-6B (HHV-6B) genome and strain-specific variations in major immediate-early genes.

Human herpesvirus 6 (HHV-6) is now classified into two distinct variants such as HHV-6 variant A(HHV-6A) and B(HHV-6B) (Ablashi et al., Arch. Virol. 129, 1993, 1-4) and the DNA of HHV-6A strain U1102 was completely sequenced (Gompels et al., Virology 209, 1995, 29-51). We have sequenced a 30-kilobase pair (kbp) (genomic positions around 111-141 kb) of HHV-6B strain HST, and a sequence of this region was compared with that of HHV-6A strain U1102. Dodecameric repeats, G/T and Kpn repeat elements, putative major immediate early 1 (MIE1) and major immediate early 2 (MIE2) genes were found in this region. The DNA sequences of HHV-6A (U1102) and HHV-6B (HSI) were markedly different in the MIE1 region, Kpn repeat elements and the putative MIE2 region. Dodecameric repeat element was located in the putative MIE2 locus of HHV-6. When primers covering dodecameric repeat region were used to amplify HHV-6 DNA of clinical isolates from patients with exanthem subitum (ES) by polymerase chain reaction (PCR), variations in size of PCR products in each isolate were found, indicating strain-specific features. Furthermore, the results of molecular biological analysis by PCR using DNA samples in a family suggest that HHV-6 infects within a family.

Base Composition↗

PubMed1997

Isolation of an avirulent mutant of Sendai virus with two amino acid mutations from a highly virulent field strain through adaptation to LLC-MK2 cells.

A field strain of Sendai virus (SeV) Ohita-M1 (M1) was isolated from an epidemic in an animal laboratory by passaging in mice. A mutant strain, Ohita-MVC11 (MVC11), was then obtained by passaging M1 in rhesus monkey (LLC-MK2) cells. MVC11 was adapted to LLC-MK2 cells and produced 20 times higher levels of infectious virus than M1. This increased production of infectious virus in LLC-MK2 cells was associated with enhanced viral gene expression. However, MVC11 could not replicate efficiently in mouse lung and was not lethal to mice even when inoculated at a titre of 8 x 10(5) cell-infecting units (CIU) per mouse. On the other hand, with an inoculum of only 4 x 10(1) CIU per mouse, corresponding to 1 LD50, M1 replicated well in mouse lung and was highly virulent to mice. Nucleotide and deduced amino acid sequence analyses of the entire genomes of M1 and MVC11 revealed that adaptation to LLC-MK2 cells and the attenuation of mouse pathogenicity of MVC11 were associated with only two amino acid substitutions; one on the C protein (Phe substituted by Ser at position 170) and the other on the RNA polymerase, the L protein (Glu substituted by Ala at position 2050).

Amino Acids↗

PubMed1997

Identification of a variant B-specific neutralizing epitope on glycoprotein H of human herpesvirus-6.

We have identified the human herpesvirus-6 variant B (HHV-6B)-specific neutralizing epitope on glycoprotein H (gH) which is recognized by monoclonal antibody (MAb) OHV3, with complement-independent neutralizing activity. HHV-6 gHs from HHV-6A (strain U1102) and HHV-6B (strain HST) were expressed in a T7-vaccinia virus transient expression system. OHV3 reacted with HST gH, but not with U1102 gH, in an immunoprecipitation assay and an indirect immunofluorescence assay. In addition, OHV3 reacted with chimeric gHs, formed between U1102 gH and HST gH, containing amino acids 272 to 422 of HST gH. Sequence comparison between U1102 and HST showed seven amino acid differences in this region. Site-specific mutations were introduced into these positions and then reactivity against OHV3 was investigated. The arginine at position 389 of HST gH was shown to be a determinant of the HHV-6B-specific reactivity of OHV3.

Antibodies, Monoclonal↗

PubMed1997

Characterization of glycoprotein H and L of human herpesvirus 7.

The genes encoding the glycoproteins H (gH) and L (gL) of human herpesvirus 7 (HHV-7) have been identified. The gH open reading frame (ORF) was 2,070 base pairs in length and encoded a predicted 690 amino-acid protein. The gH contained characteristics of a transmembrane glycoprotein including 10 consensus N-linked glycosylation sites, 12 cysteine residues, a potential amino-terminal signal sequence and a predicted transmembrane segment located near the carboxyl terminus. The gL ORF was 738 base pairs in length and encoded a predicted 246 amino-acid protein. Four possible N-glycosylation sites and 6 cysteine residues existed within gL. The predicted amino-acid sequences of the HHV-7 gH and human herpesvirus 6 variant A (HHV-6A) gH gene products exhibited 23.6% identity to each other; and those of the gL gene products had 26.0% identity. Upon in vitro translation of the gL gene, the addition of microsomal membranes resulted in two modified products with molecular weights of 32 kDa and 35 kDa from the unmodified initial translation product of 26 kDa. An amino-terminal portion of gH and the full length of gL were expressed as glutathione S-transferase fusion proteins, and these proteins were used to raise immune sera in mice. Lysates of cells infected with HHV-7 were subjected to immunoprecipitation analysis. Approximate molecular weights of 33, 37, 80 and 90 kDa polypeptides were immunoprecipitated with antibodies against the gH protein. Antibodies against the gL protein polypeptides with the same molecular weights were also precipitated, and were observed with the antibodies against the gH protein. These results suggest that HHV-7 gH and gL may form a heterodimeric complex with each other in HHV-7 infected cells, as has been reported for other herpesviruses.

Animals↗

PubMed1996

Identification of a variant A-specific neutralizing epitope on glycoprotein B (gB) of human herpesvirus-6 (HHV-6).

Based on genetic, antigenic, and growth properties, human herpesvirus-6 (HHV-6) can be classified into two groups, variant A (HHV-6A) and variant B (HHV-6B). We have mapped the HHV-6A-specific epitope on glycoprotein B (gB), which was recognized by a monoclonal antibody (MAb), 87-y-13, with a complement-independent neutralizing activity. Plasmids carrying various chimeric gB sequences formed between strains U1102 (HHV-6A) and HST (HHV-6B) and carrying sequences for a series of carboxy-terminal deletions of U1102 gB were constructed. By using the plasmids, in vitro transcription and subsequent in vitro translation were carried out. Immunoprecipitation assay of the translated products with MAb 87-y-13 revealed that MAb 87-y-13 was able to react only with in vitro translation products containing the sequence between amino acid residues 335 and 395 of U1102 gB. Amino acid sequence comparison between HHV-6A and HHV-6B in this region showed that amino acid residues 347, 387, and 393-395 were HHV-6A-specific. To determine which amino acid residue(s) was involved in recognition by MAb 87-y-13 as well as in the neutralizing activity, point mutations were introduced at those amino acid positions. Immunoprecipitation assay of the mutagenized gB with point mutations suggested that the neutralizing MAb-y-13 was involved in the recognition of the amino acid Asn at residue 347 of U1102 gB (HHV-6A). This site may play an important role in viral infection.

Amino Acid Sequence↗

PubMed1996

Human herpesvirus 6 induces IL-8 gene expression in human hepatoma cell line, Hep G2.

The infectivity of human herpesvirus 6 (HHV-6) in a human hepatoma cell line, Hep G2 cells, and the effect of HHV-6 on production of inflammatory cytokines in these cells were examined to analyze pathogenesis of HHV-6 in the liver. We demonstrated that Hep G2 cells were susceptible to infection with HHV-6, and produced infectious virus. Moreover, infection of Hep G2 cells by HHV-6 induced the expression of IL-8 mRNA, but not IL-1 beta. The effect on induction of IL-8 gene expression was observed only in Hep G2 cells infected with infectious virus, whereas both heat-inactivated HHV-6 and UV-irradiated HHV-6 did not change the IL-8 mRNA level in these cells. These data suggest that HHV-6 may induce the cytokine-mediated inflammatory response by infecting liver cells, which could result in liver dysfunction in vivo.

Antigens, Viral↗

PubMed1996

Analysis of T cell receptor V beta expression in rabbit T lymphocytes induced to proliferate by an HTLV-I-transformed leukemogenic T cell line.

Peripheral blood lymphocytes (PBL) from rabbits of the Chbb:HM strain proliferated in coculture with an X-ray-irradiated HTLV-I-transformed leukemogenic cell line of (B/J x Chbb:HM) F1 origin, whereas PBL from rabbits of the B/J and F1 strains hardly proliferated at all in co-culture with the same cell line. A proviral HTLV-I genome was detected in high-molecular-mass DNA from these proliferating cells. An analysis of T cell receptor V beta expression revealed that these lymphocytes were of restricted V beta subfamilies, suggesting that the preferential stimulation and transformation of lymphocytes occurred in this co-culture. Staphylococcal enterotoxins similarly stimulated lymphocytes and the proliferated lymphocytes were mostly of distinct V beta subfamilies depending on stimulator enterotoxins. These results suggested that the leukemogenic cell line possesses an antigen that preferentially stimulates lymphocytes of restricted V beta subfamilies.

Animals↗

PubMed1996

Prokaryotic expression of an immediate-early gene of human herpesvirus 6 and analysis of its viral antigen expression in human cells.

Segments of an immediate-early (1E) protein (1E03; 958 amino acids (aa)), encoded by clone pSTY03, of human herpesvirus 6 (HHV-6) variant B strain HST were expressed as beta-galactosidase fusion proteins in Escherichia coli. Using Western blot analysis, and the serum of a patient having high titer anti-HHV-6 antibodies, an antigenic region of the IE03 protein was mapped between residues 340 and 505 (pUE03IE-M). The fusion protein expressed in E. coli harboring plasmid pUE03IE-M was purified after electrophoresis in SDS-PAGE, and then immunized in mice to obtain a monospecific antibody. Monospecific antibody raised against the fusion protein reacted with IE03 protein species with apparent molecular weights of 155 and 170 kDa, and was detected as granular fluorescence in nuclei of infected cells by an immunofluorescence antibody test. Furthermore, this antibody reacted only with HHV-6 variant B, but did not react with HHV-6 variant A. The IE03 protein was confirmed to be an IE protein, since the synthesis of this protein was observed in infected cells that were first treated with cycloheximide, which was then replaced with actinomycin D. Further, it was also detected as early as 4 h after infection.

Animals↗

PubMed1996

Identification and analyses of glycoprotein B of human herpesvirus 7.

The gene for the human herpes virus 7 (HHV-7) glycoprotein B (gB) has been identified by sequencing a molecularly cloned HHV-7 DNA fragment. A 2.5-kb open reading frame (ORF) encoded a protein of 822 amino acids with characteristics of a transmembrane glycoprotein, and showed the strongest similarity (56.5%) with the human herpesvirus 6 (HHV-6) gB. The genes for the transport/capsid assembly protein (tp/cap) and the DNA polymerase (pol) existed upstream and downstream of the gB gene, respectively. This arrangement was the same as that of HHV-6. Antisera were generated by immunizing mice with a glutathione S-transferase-carboxy terminal gB fusion protein. Immunofluorescent tests demonstrated that the antisera reacted specifically with HHV-7 antigens in cytoplasm of infected cells. The antisera immunoprecipitated proteins with apparent molecular masses of 51, 63 and 112 kDa from HHV-7 infected cells by pulse-chase analysis. In the presence of tunicamycin, the protein with a molecular mass of 112 kDa was replaced by a protein with a molecular mass of 88 kDa, and this size was consistent with the predicted size of the primary translation product of the HHV-7 gB gene. These results suggested that the protein with a molecular mass of 112 kDa was a glycoprotein synthesized by addition of N-linked oligosaccharides to a non-glycosylated precursor of the protein with a molecular mass of 88 kDa and then cleaved into the proteins with molecular masses of 51 and 63 kDa in HHV-7 infected cells.

Amino Acid Sequence↗

PubMed1996

Mitochondrial NADH- or NADPH-linked aquacobalamin reductase activity is low in human skin fibroblasts with defects in synthesis of cobalamin coenzymes.

Mammalian livers have been reported to contain NADH- and NADPH-linked aquacobalamin reductases, which are distributed in both mitochondria and microsomes. The four aquacobalamin reductase isozymes have been purified and characterized from rat liver. It is unclear which aquacobalamin reductase among the four reductase isozymes participates in the synthesis of cobalamin coenzymes. To clarify the physiological roles of the aquacobalamin reductase isozymes, human mutant fibroblasts (cblC and cblA cells) with defects in cobalamin reductases involved in the coenzyme synthesis were used. In the cblC cells, the activity of the mitochondrial NADH-linked aquacobalamin reductase was reduced significantly, compared with normal human fibroblasts but the mitochondrial NADPH-linked enzyme was not. The reduced specific activity of the NADH-linked enzyme was not due to reduction in levels of the enzyme, but in its affinity for NADH. Although there was not a significant difference in the mitochondrial NADH-linked enzyme activity between normal and cblA cells, the activity of the mitochondrial NADPH-linked enzyme was not detectable in the mutant cells. These results indicate that the defects in the mitochondrial NADH- and NADPH-linked aquacobalamin reductases underlie cblC and cblA disorders, respectively.

Cells, Cultured↗