Biomedical subjects

T Daikoku

Publications and source records attributed to T Daikoku.

At least 37 records · Page 2Linked to original sources

PubMed1998

Characterization of the herpes simplex virus type 2 (HSV-2) US2 gene product and a US2-deficient HSV-2 mutant.

The herpes simplex virus type 2 (HSV-2) US2 gene product was identified by using a rabbit polyclonal antiserum raised against a recombinant 6 x His-US2 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 39 kDa protein in HSV-2 strain 186-infected cell lysates. The protein was not detectable in the presence of the virus DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the US2 protein in the cytoplasm and as discrete granules at late times post-infection within and at the periphery of the nucleus, and nuclear fractionation studies showed that the protein was partially associated with the nuclear matrix of infected cells. The protein was easily detected in purified virions. Also, a US2 insertion mutant was constructed which contained an ICP6-lacZ insertion in the US2 gene. This mutant was as virulent as wild-type virus in mice when inoculated by the footpad route. The importance of the US2 protein of HSV-2 in the virus life-cycle may be apparent only in the natural human host.

Animals↗

PubMed1998

Identification and characterization of the herpes simplex virus type 1 UL51 gene product.

The products of the herpes simplex virus type 1 UL51 gene were identified as phosphoproteins with apparent molecular masses of 27, 29 and 30 kDa. The proteins were produced at the late stage of infection, in a manner highly dependent on viral DNA synthesis, and were associated with extracellular virions. Immunofluorescence studies localized the UL51 proteins mainly to the cytoplasm, both in infected Vero cells and in transfected COS-1 cells singly expressing the UL51 gene.

Animals↗

PubMed1998

Overexpression, purification and helix-destabilizing properties of Epstein-Barr virus ssDNA-binding protein.

The Epstein-Barr virus (EBV) ssDNA-binding protein (SSB) encoded by the BALF2 gene is one of the essential replication proteins in the lytic phase of EBV DNA replication. In order to obtain the amount of EBV SSB required for characterization, a recombinant baculovirus containing the complete sequence of the BALF2 open reading frame under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 130 kDa, recognized by anti-BALF2 protein-specific polyclonal antibody. The overexpressed EBV SSB was purified homogeneously from the cytosolic fraction of the recombinant virus-infected cells. The purified protein displaced short DNA strands from their complementary sequences in the single-stranded form of M13. The helix-destabilizing activity was neutralized by the anti-BALF2 protein-specific antibody. Maximum unwinding occurred at EBV SSB concentrations exceeding saturation level of the DNA substrate. The DNA unwinding reaction mediated by the EBV SSB was highly cooperative and extremely rapid. The reaction displayed no directionality and required neither ATP nor MgCl2, two essential cofactors for DNA helicase activity. The helix-destabilizing property of the EBV SSB may function to melt out secondary structures on the ssDNA template, thereby facilitating the movement of the EBV DNA polymerase.

Animals↗

PubMed1998

Characterization of the UL55 gene product of herpes simplex virus type 2.

We have identified the herpes simplex virus type 2 (HSV-2) UL55 gene product using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL55 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 23 kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the UL55 protein within and at the periphery of the nucleus as discrete granules at late times post-infection, and nuclear fractionation studies showed that the protein was associated with the nuclear matrix of infected cells. Moreover, these discrete regions containing the UL55 protein were found to be adjacent to compartments, designated assemblons, containing the capsid protein ICP35. However, the UL55 protein was not detected in purified virions. These results suggest that the UL55 protein of HSV-2 may play an accessory role in virion assembly or maturation.

Animals↗

PubMed1998

Mode of action of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl) cycloprop-1'-yl]methyl]guanine (A-5021) against herpes simplex virus type 1 and type 2 and varicella-zoster virus.

The mode of action of (1'S,2'R)-9-([1', 2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl)guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2' site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.

Antiviral Agents↗

PubMed1997

Strand displacement associated DNA synthesis catalyzed by the Epstein-Barr virus DNA polymerase.

The Epstein-Barr virus (EBV) DNA polymerase (Pol) holoenzyme is an essential enzyme required for ori-Lyt dependent EBV DNA replication. Using singly primed M13ssDNA circles as template, the EBV DNA Pol holoenzyme synthesized DNA chains greater than the unit length of M13 ssDNA in addition to full length products even at a low ratio of polymerase molecule per templates. The long replication products consisted of circular double-stranded DNA with single-stranded tails that were sensitive to mung bean nuclease. Reconstitution of the EBV Pol holoenzyme by preincubation of BALF5 Pol catalytic subunit and BMRF1 Pol accessory subunit in vitro resulted in reproduction of the strand displacement DNA synthesis. Thus, the EBV DNA Pol holoenzyme by itself is able to produce strand displacement coupled to the polymerization process in a highly processive way in the absence of any other protein.

Adenosine Triphosphate↗

PubMed1997

Purification and characterization of the protein kinase encoded by the UL13 gene of herpes simplex virus type 2.

The proteins encoded by the UL13 genes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been predicted to be protein kinases. To identify the UL13 gene product, we have raised a rabbit polyclonal antiserum against a His.Tag-HSV-1 UL13 fusion protein. The antibody specifically reacted with the 60-kDa UL13 fusion protein expressed in Escherichia coli and also recognized 56- to 57-kDa late proteins in nuclear fractions of HSV-1- and HSV-2-infected cells. On the other hand, novel casein kinase activity was induced at the late stage of infection when Vero cells were infected with HSV-1 and HSV-2. The induction of the activity was most prominent in the nuclear fractions of HSV-2-infected cells and therefore we purified the protein kinase (PK) from the nuclear extracts by successive column chromatography (phosphocellulose, DEAE-cellulose, and hydroxyapatite) using casein as an exogenous substrate. The final preparation of the enzyme contained a single major protein with an apparent molecular weight of 56 kDa which was specifically reacted with the UL13 antiserum. The PK activity was optimal in the absence of NaCl and at relatively high pH. Acidic proteins such as casein and phosvitin were efficiently phosphorylated by the PK. A basic protein, protamine, which is the best substrate for the HSV-2 US3 PK, was not detectably phosphorylated but histone was a relatively good substrate for the UL13 PK. Phosphoamino acid analysis revealed that the PK phosphorylated serine and threonine but not tyrosine. Moreover the enzyme was found to be highly resistant to heparin, a potent inhibitor of casein kinase II (CK II) and also resistant to CK I-7, a synthetic inhibitor of CK I, but very sensitive to a bioflavonoid quercetin. These results indicate that the HSV-2 UL13 PK had unique catalytic properties different from those of cellular CK I, CK II, and the viral PK encoded by the US3 gene. We have also determined the complete nucleotide sequence of the HSV-2 UL13 gene. The overall amino acid homology between the HSV-2 and HSV-1 UL13 PKs was 85.9% and the homology was highly conserved in the C-terminal region.

Amino Acid Sequence↗

PubMed1997

Dramatic enhancement of the specific expression of the heart-type fatty acid binding protein in rat brown adipose tissue by cold exposure.

To understand the difference in energy metabolisms in brown (BAT) and white (WAT) adipose tissues, we examined the steady-state transcript levels of the heart-type and adipose-type fatty acid binding proteins (H-FABP and A-FABP, respectively) by Northern blot analysis. The transcript of H-FABP in rat BAT was increased about 100-fold by cold exposure, whereas that in WAT was negligible, and was increased only slightly by cold exposure. The transcript of A-FABP was observed in both BAT and WAT, the level being slightly greater in WAT. However, its transcript level was not affected by cold exposure in either adipose tissue. In addition, on treatment with norepinephrine (NE), transcript level of H-FABP was elevated markedly but that of A-FABP was not changed in rat brown adipocytes. Therefore, the stimulatory effect of cold exposure on the transcript of H-FABP in BAT was concluded to be mediated by NE, like that of the uncoupling protein (UCP). Thus, the expressions of H-FABP and UCP may be controlled by the same mechanism.

Adipocytes↗

PubMed1997

The product of the US10 gene of herpes simplex virus type 1 is a capsid/tegument-associated phosphoprotein which copurifies with the nuclear matrix.

We have identified the herpes simplex virus type 1 (HSV-1) US10 gene product using rabbit polyclonal antisera raised against a recombinant 6xHis-US10 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with 34 and 36 kDa proteins in HSV-1 KOS-infected cells as shown by Western blotting and immunoprecipitation experiments. The 36 kDa protein was immunoprecipitated with the US10 antiserum from 32P-labelled lysates of Vero cells infected with HSV-1 KOS, demonstrating that the US10 protein was phosphorylated. Indirect immunofluorescence studies localized the US10 protein mainly to nuclei as large discrete particles at later times post-infection (p.i.), and nuclear fractionation studies revealed that the protein was tightly associated with the nuclear matrix. Moreover, analysis of isolated intracellular capsids showed that both phosphorylated and unphosphorylated forms of the US10 product were also associated with the capsid/tegument. These results indicate that the US10 gene of HSV-1 encodes a capsid/tegument-associated phosphoprotein which copurifies with the nuclear matrix.

Animals↗

PubMed1996

Calnexin associates with the precursors of glycoproteins B, C, and D of herpes simplex virus type 1.

By a pulse-chase approach combined with immunoprecipitation, we showed that the newly synthesized precursors of glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) were associated with calnexin, a membrane-bound chaperone in the endoplasmic reticulum. Kinetics of association and dissociation was quite different among the precursors. For the precursors of gC and gD of HSV-1, the maximal association was observed immediately after the synthesis and they dissociated rapidly with half-times of 25 min for gC and 30 min for gD, respectively. In contrast, the precursor of gB showed prolonged association with calnexin. Their maximum association was observed 30 min after the synthesis, and thereafter, they dissociated slowly with a half-time of 70 min. The results suggest that, while glycoproteins that have a rapid processing rate, such as gC and gD of HSV-1, rapidly associate with calnexin and their association is quite short, those that take much time to be processed to a mature form, such as gB of HSV-1, have prolonged association kinetics with calnexin. Complete inhibition of the binding of these glycoprotein precursors to calnexin by tunicamycin or castanospermine indicates the importance of partially trimmed N-linked oligosaccharides for their association.

Calcium-Binding Proteins↗

PubMed1996

Epstein-Barr virus single-stranded DNA-binding protein: purification, characterization, and action on DNA synthesis by the viral DNA polymerase.

Epstein-Barr virus (EBV) BALF2 gene product is one of the essential components in the lytic phase of the EBV DNA replication. The BALF2 protein was purified to near homogeneity from the nuclear extract of B95-8 cells with virus productive cycle induced by chemical agents. SDS-polyacrylamide gel electrophoresis showed the presence of a single polypeptide with a molecular weight of 130 K, which was identified as BALF2 protein by Western immunoblot analysis. On Superose 6 HR 10/30 gel filtration the BALF2 protein eluted at a position corresponding to an apparent molecular mass of approximately 128 K, indicating that the BALF2 protein behaves as a monomer in solution. The purified BALF2 protein bound to single-stranded DNA preferentially over double-stranded DNA or single-stranded RNA. Replication of singly primed M13 single-stranded DNA by the EBV DNA polymerase complex in the absence of the BALF2 protein exhibited a highly processive mode of replication and generated full length products in addition to some bands of pausing sites. Although the addition of the BALF2 protein did not affect the replication rate, the average chain length of the replication products was slightly increased with eliminating bands of pausing sites. Similar effects were observed with the reconstituted polymerase complex composed of the BALF5 and BMRF1 Pol subunits. On the other hand, in the absence of the BALF2 protein, the BALF5 Pol catalytic subunit alone extended the primer slightly and paused at specific sites on M13 ssDNA template where stable secondary structure is predicted. However, addition of the BALF2 protein, in contrast to the case of herpes simplex virus ICP8 which does not affect the overall distribution of length of the replication products synthesized by the HSV Pol catalytic subunit (Gottlieb et al., 1990, J. Virol. 64, 5976-5987), stimulated DNA synthesis and yielded a distribution of replication products with long lengths in addition to full length products. Although the BALF2 protein behaved as if it converts a low processive enzyme of the EBV Pol catalytic subunit to a highly processive form like the BMRF1 Pol accessory subunit, challenger DNA experiments revealed that the EBV Pol catalytic subunit is transferred to challenger DNA even in the presence of the BALF2 protein. It is therefore likely that the EBV BALF2 protein functions to melt out the regions of secondary structure on the single-stranded DNA template, thereby reducing and eliminating pausing of the EBV DNA polymerase at specific sites. These properties indicate that the EBV BALF2 protein acts as a single-stranded DNA-binding protein during lytic phase of EBV DNA replication.

Animals↗

PubMed1996

Calnexin acts as a molecular chaperone during the folding of glycoprotein B of human cytomegalovirus.

Human cytomegalovirus glycoprotein B (gB) is synthesized as a 105-kDa nonglycosylated polypeptide and cotranslationally modified by addition of N-linked oligosaccharides to a 160-kDa precursor in the endoplasmic reticulum (ER). It is then transported to the Golgi complex, where it is endoproteolytically cleaved to form the disulfide-linked mature gp55-116 complex. Pulse-chase experiments demonstrate that the 160-kDa gB precursor was transiently associated with calnexin, a membrane-bound chaperone, in the ER. The association was maximal immediately after synthesis, and they dissociated with a half-time of 15 min. Complete inhibition of binding by tunicamycin or castanospermine indicates the importance of N-linked oligosaccharides for it. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that during an initial stage in the biogenesis, the 160-kDa gB precursor was first synthesized as a fully reduced form and rapidly converted to an oxidized form, with a half-time of 18 min. Both forms of the gB precursor could bind to calnexin. The kinetics of the conversion from the fully reduced to the oxidized form coincided with that of dissociation of the 160-kDa gB precursor from calnexin, suggesting that the two steps are closely related.

1-Deoxynojirimycin↗

PubMed1995

The US3 protein kinase of herpes simplex virus type 2 is associated with phosphorylation of the UL12 alkaline nuclease in vitro.

Herpes simplex virus type 2 (HSV-2) gene US3 encodes a serine-threonine protein kinase. We previously described the isolation of a US3-inactivated mutant which is able to replicate in Vero cells but not in murine macrophages. To learn more about the biological role of the US3 protein kinase, we have sought to identify the target proteins of the enzyme. Studies of in vitro phosphorylation with extracts of infected cells demonstrate that the US3 protein kinase is involved in phosphorylation of the UL12 alkaline nuclease in vitro, suggesting that the nuclease is a possible target of the protein kinase.

Animals↗

PubMed1995

Growth and cytopathogenicity of herpes simplex virus in a macrophage cell line, RAW264: A good indicator of intraperitoneal pathogenicity.

Macrophages are known to play a critical role in host resistance to herpes simplex virus (HSV). In this study, we investigate the interaction between various HSV strains with different virulence and a murine macrophage cell line, RAW264. Highly attenuated strains replicated poorly in RAW264 cells and were cleared from the cultures. For the eleven viruses tested, there was good correlation between intraperitoneal pathogenicity for adult mice and replication in RAW264 cells. It was also shown that interferon alpha/beta was involved in restricted replication of some strains.

Animals↗

PubMed1994

Identification of a target protein of US3 protein kinase of herpes simplex virus type 2.

Herpes simplex virus type 2 (HSV-2) gene US3 has been shown to encode a serine-threonine protein kinase. In this study, we have tried to identify target proteins of the US3 protein kinase using a US3 lacZ insertion mutant of HSV-2. When permeabilized cells were labelled with [gamma-32P]ATP under the optimum conditions for the US3 enzyme, the most striking difference between wild-type HSV-2 strain 186- and mutant-infected cells was observed in the phosphorylation of proteins ranging in M(r) values from 14K to 22K. Studies of in vitro phosphorylation with purified virions and with cells infected with a US9-defective HSV-1 mutant suggested that a tegment phosphoprotein encoded by the US9 gene may be a target of HSV-2 US3 protein kinase.

Amino Acid Sequence↗

PubMed1994

Further characterization of the interaction between the Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3'-to-5' exonucleolytic activity and stability of initiation complex at primer terminus.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, possesses an intrinsic 3'-to 5' proofreading exonuclease activity in addition to 5'-to-3' DNA polymerase activity (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993). The exonuclease hydrolyzed both double-and single-stranded DNA substrates with 3'-to-5' directionality, releasing deoxyribonucleoside 5'-monophosphates. The double-strand exonucleolytic activity catalyzed by the BALF5 polymerase catalytic subunit was very sensitive to high ionic strength, whereas the single-strand exonucleolytic activity was moderately resistant. The addition of the BMRF1 polymerase accessory subunit to the reaction enhanced the double-strand exonucleolytic activity in the presence of high concentrations of ammonium sulfate (fourfold stimulation at 75 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 and higher, identical to the values required for reconstituting the optimum DNA polymerizing activity (T. Tsurumi, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:7648-7653, 1993). Furthermore, product size analyses revealed that the polymerase catalytic subunit alone excised a few nucleotides from the 3' termini of the primer hybridized to template DNA and that the addition of the BMFR1 polymerase accessory subunit stimulated the nucleotide excision several times. In contrast, the hydrolysis of single-stranded DNA by the BALF5 protein was not affected by the addition of the BMRF1 polymerase accessory subunit at all. These observations suggest that the BMRF1 polymerase accessory subunit forms a complex with the BALF5 polymerase catalytic subunit to stabilize the interaction of the holoenzyme complex with the 3'-OH end of the primer on the template DNA during exonucleolysis. On the other hand, challenger DNA experiments revealed that the BALF5 polymerase catalytic subunit alone stably binds to the primer terminus in a stationary state, whereas the reconstituted polymerase holoenzyme is unstable. The instability of the initiation complex of the EBV DNA polymerase would allow the rapid removal of the EBV DNA polymerase holoenzyme from the lagging strand after it has replicated up to the previous Okazaki fragment. This feature of the EBV DNA polymerase holoenzyme in a stationary state is in marked contrast to the moving holoenzyme complex tightly bound to the primer end during polymerization and exonucleolysis.

Antigens, Viral↗

PubMed1993

The US 9, 10, 11, and 12 genes of herpes simplex virus type 1 are of no importance for its neurovirulence and latency in mice.

We have studied the pathogenic and latency/reactivation potential of a herpes simplex virus type 1 (HSV-1) variant (N38) which has a deletion of four genes, US9, US10, US11, and US12, in the short unique region of the HSV-1 genome. N38 was as pathogenic as the parental wild-type virus following intracerebral infection of mice and replicated with wild-type kinetics in the brain. After intraperitoneal infection, some restricted replication of N38 was observed in the adrenal gland and the deletion virus was about 20-fold less virulent than the parental virus. There was no significant difference between two viruses in the frequency of reactivation or in reactivation time. The results indicate that US9, US10, US11, and US12 gene products are dispensable for its neurovirulence and latency in mice.

Animals↗

PubMed1993

Purification and biochemical characterization of the protein kinase encoded by the US3 gene of herpes simplex virus type 2.

Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to successive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mM exerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 microM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.

Adenosine Triphosphate↗