Search PubMed⌕ Search

Biomedical subjects

S Kajigaya

Publications and source records attributed to S Kajigaya.

At least 37 records · Page 2Linked to original sources
PubMed1994

Parvovirus particles as platforms for protein presentation.

Empty capsids of the human pathogenic parvovirus B19 can be produced in a baculovirus system. B19 capsids are composed mainly of major capsid protein (VP2) and a small amount of minor capsid protein (VP1); VP1 is identical to VP2 but contains an additional 227-aa N-terminal region ("unique" region). A portion of that region of VP1 is external to the capsid, and VP1 is not required for capsid formation. We substituted the unique region with a sequence encoding the 147 aa of hen egg white lysozyme (HEL) and constructed recombinant baculoviruses with variable amounts of retained VP1 sequence joined to the VP2 backbone. After cotransfection with VP2 baculovirus and expression in insect cells, capsids were purified by density sedimentation. Purified recombinant capsids contained HEL. External presentation of HEL was demonstrated by immunoprecipitation, ELISA, and immune electron microscopy using anti-lysozyme monoclonal antibodies or specific rabbit antisera. Empty particles showed enzymatic activity in a micrococcal cell wall digestion assay. Rabbits inoculated with capsids made antibodies to HEL. Intact heterologous protein can be incorporated in B19 particles and presented on the capsid surface, properties that may be useful in vaccine development, cell targeting, and gene therapy.

Baculoviridae↗
PubMed1994

The structure of human parvovirus B19 at 8 A resolution.

Empty capsids of the human parvovirus B19, self-assembled in a baculovirus expression system, have been crystallized in a cubic space group P2(1)3 with a = 362 A. In spite of extensive purifications, the crystals diffract X-rays to only 8.0 A resolution. Diffraction data were collected using oscillation photography with synchrotron radiation. The orientations of the particles in the unit cell were determined with a self-rotation function and their positions were obtained with an R-factor search using the known homologous canine parvovirus (CPV) structure. The resultant phases were improved by electron density averaging and solvent flattening to include all the terms between 23.0 and 8.0 A resolution. The central eight-stranded antiparallel beta-barrel, common to many viruses, is situated similarly in B19 with respect to the icosahedral symmetry axes to that observed for feline panleukopenia virus (FPV) and CPV. However, the surface structure of B19 is significantly different from the other known parvoviruses. The most striking difference is that B19 lacks the prominent spikes on the threefold icosahedral axes observed in FPV and CPV. This spike region contains residues involved in host recognition and antigenicity for the latter viruses, showing that there are major differences between subgroups of autonomous parvoviruses.

Amino Acid Sequence↗
PubMed1994

The transcriptional regulator YY1 binds to the 5'-terminal region of B19 parvovirus and regulates P6 promoter activity.

We performed a systematic study to identify cellular factors that bound to the terminal repeat region of B19 parvovirus. Using electrophoretic mobility shift assays, we detected one cellular factor which prominently bound to the repeat region. The factor was purified from K562 nuclear extract by specific DNA affinity column chromatography and identified as YY1, a multifunctional transcription factor. Of multiple possible YY1 binding sites in the upstream region of the P6 promoter, three showed specific strong binding. Transcription enhancement by YY1 was demonstrated in vitro by transient transfection assays. In studies using truncated and mutated versions of this region, YY1 activity was diminished by the alteration of any two of these three sites and abolished by the alteration of all three sites. Our results suggest that YY1 is a positive regulator of B19 parvovirus transcription.

Base Sequence↗
PubMed1994

A putative nucleoside triphosphate-binding domain in the nonstructural protein of B19 parvovirus is required for cytotoxicity.

Cytotoxicity secondary to B19 parvovirus infection is due to expression of the viral nonstructural protein. Nonstructural proteins of many parvoviruses contain a well-conserved nucleoside triphosphate (NTP)-binding motif, which has been shown to be essential for a variety of protein functions. We show here that cytotoxicity of the B19 parvovirus nonstructural protein was abolished by single mutations of amino acids within the NTP-binding domain, especially within the A motif, implicating NTP-binding in virus-induced cell death.

Amino Acid Sequence↗
PubMed1994

Formation of empty B19 parvovirus capsids by the truncated minor capsid protein.

We previously reported that empty capsids of B19 parvovirus were formed by the major capsid protein (VP2) alone expressed in a baculovirus system, but the minor capsid protein (VP1), longer by 227 amino acids, alone did not form empty capsids. We report here further investigations of the constraints on capsid formation by truncated versions of VP1. Studies were performed with recombinant baculoviruses expressed in Sf9 cells. Severely shortened VP1, extended beyond the VP2 core sequence by about 70 amino acids of the unique region, formed capsids normal in appearance; longer versions of VP1 also formed capsids but did so progressively less efficiently and produced capsids of more markedly dysmorphic appearance as the VP1-unique region was lengthened.

Animals↗
PubMed1993

Early and late gene expression in UT-7 cells infected with B19 parvovirus.

UT-7, a human megakaryocytoblastoid cell line, can be persistently infected with B19 parvovirus. We performed detailed serial analysis of parvovirus DNA replication and RNA transcription of synchronized cells. RNA transcription appeared as an early event following infection, with viral RNA detected about 6 hr after infection. In contrast, dimer-replicative intermediate forms of parvovirus DNA did not appear until more than 16 hr after infection. Northern analysis of specific transcripts showed an earlier appearance of nonstructural protein RNA (6 hr) compared to capsid protein RNA (24 hr). The addition of an inhibitor of protein synthesis to block synthesis of nonstructural protein abolished capsid protein RNA transcription as well as DNA replication. Primer extension analysis confirmed the initiation of all transcription from the single P6 promoter. RNA transcription precedes DNA replication of B19 parvovirus in these cells, and RNA processing may have a major role in regulating gene expression.

Base Sequence↗
PubMed1993

Antibody capture haemadherence tests for parvovirus B19-specific IgM and IgG.

In the absence of serum inhibitor human parvovirus B19 agglutinates primate red cells. Agglutination of baboon erythrocytes by inhibitor free baculovirus expressed VP1/VP2 proteins of B19 virus was enhanced by low pH. To avoid interference from inhibitor(s) present in serum specimens microtitre tests with an antibody capture configuration were designed and optimised. Antigen binding was demonstrated by the adherence of baboon erythrocytes. These tests (MACHAT, GACHAT) detected IgM or IgG anti-B19 in 1032 serum specimens with an overall sensitivity of 99%, specificity of 98.2% and positive predictive value of 96.8% with reference to the radioimmunoassays currently in diagnostic use. MACHAT and GACHAT are straightforward, and well suited to large scale research and seroepidemiological investigations. They are without the risks from infectious antigen and radiation associated with existing radioimmunoassays and allow the phenomenon of B19 virus haemagglutination to be put to practical advantage.

Adolescent↗
PubMed1993

Candidate recombinant vaccine for human B19 parvovirus.

Recombinant baculoviruses were used to produce human B19 parvovirus empty capsids composed of only VP2 and VP2 capsids containing 4%, 25%, 35%, or 41% VP1 protein. Immunogenicity of the purified capsids, formulated with or without adjuvant, was evaluated in mice, guinea pigs, and rabbits. Sera were analyzed for total anti-B19 parvovirus antibodies, antibodies specific to the region unique to the VP1 capsid protein, and virus neutralizing antibodies. A relationship was observed between the development of antibodies specific to sequences unique to the VP1 protein and virus neutralization. The polypeptide composition of the empty capsid immunogens appeared to be important for elicitation of potent virus neutralizing activity. VP2 capsid immunogens devoid of VP1 protein, or consisting of only 4% VP1, the composition of naturally occurring virions, were generally poor at eliciting high levels of virus neutralizing activity. Capsids consisting of > or = 25% VP1 protein efficiently and consistently provoked vigorous B19 virus neutralizing responses. Recombinant empty capsids enriched for the VP1 protein should serve as the basis for a human B19 parvovirus vaccine.

Aluminum Hydroxide↗
PubMed1993

Neutralizing linear epitopes of B19 parvovirus cluster in the VP1 unique and VP1-VP2 junction regions.

Presentation of linear epitopes of the B19 parvovirus capsid proteins as peptides might be a useful vaccine strategy. We produced overlapping fusion proteins to span the viral capsid sequence, inoculated rabbits, and determined whether the resulting antisera contained antibodies that neutralized the ability of the virus to infect human erythroid progenitor cells. Antibodies that bound to virus in an enzyme-linked immunosorbent assay were present in antisera raised against 10 of 11 peptides; strongest activity was found for antisera against the carboxyl-terminal half of the major capsid protein. However, strong neutralizing activity was elicited in animals immunized with peptides from the amino-terminal portion of the unique region of the minor capsid protein and peptides containing the sequence of the junction region between the minor and major capsid proteins. The development of neutralizing activity in animals was elicited most rapidly with the fusion peptide from the first quarter of the unique region. A 20-amino-acid region of the unique region of the minor capsid protein was shown to contain a neutralizing epitope. Multiple antigenic peptides, based on the sequence of the unique region and produced by covalent linkage through a polylysine backbone, elicited strong neutralizing antibody responses. Synthetic peptides and fusion proteins containing small regions of the unique portion of the minor capsid protein might be useful as immunogens in a human vaccine against B19 parvovirus.

Antibodies, Viral↗
PubMed1992

Herbimycin A, an inhibitor of tyrosine kinase, prolongs survival of mice inoculated with myeloid leukemia C1 cells with high expression of v-abl tyrosine kinase.

Herbimycin A, a benzoquinonoid ansamycin antibiotic, reduces intracellular phosphorylation by some tyrosine kinases, including v-abl. The mouse megakaryoblastic cell line C1 expresses v-abl protein at high levels. Herbimycin A at about 20 ng/ml caused 50% inhibition of growth of C1 cells but at 100 ng/ml scarcely affected the growth of another mouse leukemia cell line, M1 cells, or of normal bone marrow cells. Injection of 10(6) C1 cells into nude mice resulted in death of all the mice within 30 days. Administration of herbimycin A significantly enhanced the survival of mice inoculated with C1 cells but scarcely affected the survival of mice inoculated with M1 cells. These results suggest that herbimycin A and/or related compounds may be useful for treatment of some types of leukemia in which tyrosine kinase activity is implicated as a determinant of the oncogenic state.

Animals↗
PubMed1992

First continuous propagation of B19 parvovirus in a cell line.

The pathogenic human parvovirus B19 has extreme tropism for human erythroid progenitor cells and has resisted cultivation in conventional cell lines. We report first propagation of this virus in an erythropoietin-dependent strain of a megakaryoblastic leukemia cell line called UT-7. Virus protein was present in about 5% of cells after 1 week of culture. Appropriate ratios of major and minor capsid proteins were determined by immunoblot, and newly synthesized capsid protein was detected by immunoprecipitation of radioactively labeled cell lysates. High molecular weight monomer and dimer intermediates were detected by Southern analysis, indicating active viral replication. Approximately 1,000 genome copies were present per infected cell, and at the optimal multiplicity of infection 20- to 50-fold more virus was produced than inoculated. Virus propagation only occurred in UT-7 cells that were adapted to growth in erythropoietin; virus signal was not detected in UT-7 cells adapted for growth in granulocyte-macrophage colony-stimulating factor or interleukin-3, even with exposure to erythropoietin for several days. Infectious virus was detected in cultures as long as 3 months after inoculation. Despite persistence, there was no evidence of viral integration on Southern analysis. This cell line may prove useful for the production of infectious virus and in the analysis of B19 parvovirus persistence, cytotoxicity, and permissivity.

Blotting, Southern↗
PubMed1992

Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface.

Capsids of the B19 parvovirus are composed of major (VP2; 58 kD) and minor (VP1; 83 kD) structural proteins. These proteins are identical except for a unique 226 amino acid region at the amino terminus of VP1. Previous immunization studies with recombinant empty capsids have demonstrated that the presence of VP1 was required to elicit virus-neutralizing antibody activity. However, to date, neutralizing epitopes have been identified only on VP2. Crystallographic studies of a related parvovirus (canine parvovirus) suggested the unique amino-terminal portion of VP1 assumed an internal position within the viral capsid. To determine the position of VP1 in both empty capsids and virions, we expressed a fusion protein containing the unique region of VP1. Antisera raised to this protein recognized recombinant empty capsids containing VP1 and VP2, but not those containing VP2 alone, in an enzyme-linked immunosorbent assay. The antisera immunoprecipitated both recombinant empty capsids and human plasma-derived virions, and agglutinated the latter as shown by immune electron microscopy. The sera contained potent neutralizing activity for virus infectivity in vitro. These data indicate that a portion of the amino terminus of VP1 is located on the virion surface, and that this region contains intrinsic neutralizing determinants. The external location of the VP1-specific tail may provide a site for engineered heterologous epitope presentation in novel recombinant vaccines.

Animals↗
PubMed1991

Induction by some protein kinase inhibitors of differentiation of a mouse megakaryoblastic cell line established by coinfection with Abelson murine leukemia virus and recombinant SV40 retrovirus.

Mouse C1 line cells are megakaryoblastic cells established by coinfection of Abelson murine leukemia virus and recombinant simian virus 40. We examined the effects of various compounds on growth and differentiation of these cells. Megakaryocytic differentiation of C1 cells was not induced by cytokines that stimulate megakaryocytic maturation of normal progenitor cells, such as interleukin 3 and 6 and granulocyte-macrophage colony-stimulating factor. However, the cells were induced to differentiate into megakaryocytes by treatment with some protein kinase inhibitors. The inhibition of v-abl tyrosine kinase activity preceded induction of differentiation of the cells treated with tyrosine kinase inhibitors such as genistein, herbimycin A, and erbstatin. Treatment of C1 cells with a v-abl antisense oligomer inhibited their proliferation and induced acetylcholinesterase activity, a typical marker of megakaryocytic differentiation. These results suggest that inhibition of v-abl function is associated with induction of megakaryocytic differentiation of C1 cells. Among the compounds tested, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of cyclic nucleotide-dependent and Ca(2+)-phospholipid-dependent (protein kinase C) protein kinases, was the most potent inducer of differentiation of C1 cells. However, the differentiation-inducing effect of H-7 was unlikely to be mediated through inhibition of protein kinase C or cyclic nucleotide-dependent kinases, because other types of inhibitors of these kinases were not effective, and a protein kinase activator (phorbol ester) induced differentiation of C1 cells. Moreover, neither v-abl mRNA expression nor v-abl kinase activity in C1 cells was affected by treatment with H-7. These findings indicate that induction of megakaryocytic differentiation by H-7 is not related to inhibition of v-abl kinase, but rather to some novel function of H-7.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Self-assembled B19 parvovirus capsids, produced in a baculovirus system, are antigenically and immunogenically similar to native virions.

B19 parvovirus is pathogenic in humans, causing fifth disease, transient aplastic crisis, some cases of hydrops fetalis, and acquired pure red cell aplasia. Efforts to develop serologic assays and vaccine development have been hampered by the virus's extreme tropism for human bone marrow and the absence of a convenient culture system. We constructed recombinants containing either the major (VP2) or minor (VP1) structural proteins of B19 in a baculovirus-based plasmid, from which the polyhedrin gene had been deleted; these recombinant plasmids were used to generate recombinant infectious baculovirus. Subsequent infection of insect cells in vitro resulted in high-level expression of either B19VP1 or VP2. Parvovirus capsids were obtained by self-assembly in cell cultures coinfected with either VP1- and VP2-containing baculoviruses or, surprisingly, VP2-containing baculoviruses alone. Empty B19 capsids composed of VP1 and VP2 could replace serum virus as a source of antigen in a conventional immunoassay for detection of either IgG or IgM antiparvovirus antibodies in human serum. Immunization of rabbits with capsids composed of VP1 and VP2 resulted in production of antisera that recognized serum parvovirus on immunoblot and neutralized parvovirus infectivity for human erythroid progenitor cells. Baculovirus-derived parvovirus antigen can substitute for scarce viral antigen in immunoassays and should be suitable as a human vaccine.

Amino Acid Sequence↗
PubMed1991

Human parvovirus B19 specific IgG, IgA, and IgM antibodies and DNA in serum specimens from persons with erythema infectiosum.

To determine the diagnostic use of different markers of acute parvovirus B19 infection, serum specimens obtained from 128 persons with erythema infectiosum were tested for specific immunoglobulin G (IgG), IgA, and IgM antibodies by capture enzyme immunoassay (EIA) using Chinese hamster ovary (CHO) cell-expressed B19 antigen, and tested for circulating B19 DNA by polymerase chain reaction (PCR). A significant rise in specific IgG and IgA antibodies was detected in 87% and 77%, respectively, of persons from whom acute- and convalescent-phase serum specimens were available. Specific IgA antibodies were detected in single serum specimens from 90% of cases and were present in 22 (18%) of 120 persons from a control group without a history of recent exposure to B19. Specific IgM antibodies were detected in 97% of cases and one person (1%) from the control group. B19 DNA was detected in 94% of cases and was absent in 20 persons from the control group positive for both IgG and IgA antibodies. Serum specimens obtained between 4 and 6 months after onset of illness from six additional persons were also tested. All had specific IgG antibodies, four (67%) had IgA, five (83%) had IgM, and none had detectable B19 DNA. Our data indicate that 1) specific IgA antibodies are too persistent to be a useful indicator of recent B19 infection; 2) specific IgM antibodies are the most sensitive indicator of acute B19 infection in immunologically normal persons but can persist up to 6 months; and 3) B19 DNA can often be detected up to 2 months after onset of illness even in immunologically normal hosts and might be a useful adjunct test for diagnosis of acute B19 infection.

Adolescent↗
PubMed1991

Preliminary X-ray crystallographic investigation of human parvovirus B19.

Crystals that diffract X rays to at least 8 A resolution have been grown from human B19 parvovirus empty capsids. These particles consist of VP-2 derived from a baculovirus expression system. This is possibly the first time that a self-assembled empty viral capsid, grown in other than normal host cells, has been crystallized. Partial X-ray diffraction data have been collected using synchrotron radiation. The space group is P2(1)3 with a = 362 A. The particle position in the crystal cell is given, at least roughly, from packing considerations.

Animals↗
PubMed1991

A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response.

We used 18 monoclonal antibodies against B19 parvovirus to identify neutralizing epitopes on the viral capsid. Of the 18 antibodies, 9 had in vitro neutralizing activity in a bone marrow colony culture assay. The overlapping polypeptide fragments spanning the B19 structural proteins were produced in a pMAL-c Escherichia coli expression system and used to investigate the binding sites of the neutralizing antibodies. One of the nine neutralizing antibodies reacted with both VP1 and VP2 capsid proteins and a single polypeptide fragment on an immunoblot, identifying a linear neutralizing epitope between amino acids 57 and 77 of the VP2 capsid protein. Eight of nine neutralizing antibodies failed to react with either of the capsid proteins or any polypeptide fragments, despite reactivities with intact virions in a radioimmunoassay, suggesting that additional conformationally dependent neutralizing epitopes exist.

ATP-Binding Cassette Transporters↗