bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of the Catalytic Domain of
Clostridium novyi
Alpha-Toxin
Authors: Busch Christian , Schömig Kathrin , Hofmann Fred , Aktories Klaus 
Published: July 2002

ABSTRACT

Clostridium novyi
alpha-toxin belongs to the family of large clostridial cytotoxins which act on cells through the modification of small GTP-binding proteins. We present here an analysis of the catalytic domain of alpha-toxin. A NH
2
-terminal 551-amino-acid fragment, α551, was found to contain the full enzyme activity of the holotoxin, whereas a slightly shortened fragment encompassing 509 amino acids showed no detectable enzyme activity. Further characterization of the enzymatically active fragment α551 revealed a substrate specificity for both UDP–
N
-acetylglucosamine and UDP-glucose. A Michaelis-Menten constant of 17 μM was determined for the substrate UDP–
N
-acetylglucosamine, while that for UDP-glucose was about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within α551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells. Inhibition of enzyme activity of the DXD mutant could be partially overcome by increased concentrations of manganese ions, suggesting the involvement of these aspartic acids in Mn
2+
binding. By construction of chimeras of enzymatically active fragments of
C. sordellii
lethal toxin and
C. novyi
alpha-toxin, we located the region involved in nucleotide-sugar specificity to amino acids 133 through 517.
Key Role for DsbA in Cell-to-Cell Spread of
Shigella flexneri
, Permitting Secretion of Ipa Proteins into Interepithelial Protrusions
Authors: Yu Jun , Edwards-Jones Bryn , Neyrolles Olivier , Kroll J. Simon 
Published: July 2002

ABSTRACT

DsbA, a disulfide bond catalyst, is necessary for realization of the pathogenic potential of
Shigella flexneri
. Sh42, a mutant strain differing from wild-type M90TS solely because it expresses nonfunctional DsbA33G (substitution for 33C at the active site), secreted less IpaB and IpaC than M90TS in response to various stimuli in vitro. A kinetic study demonstrated that Sh42 responded more slowly to Congo red than M90TS. By modulating relative concentrations of functional and nonfunctional DsbA within bacteria, functional enzyme has been shown to be necessary for intercellular spread. By confocal microscopy, M90TS dividing in protrusions was shown to secrete Ipa proteins from the septation furrow, anticipating lysis of protrusions, while Sh42 showed minimal Ipa secretion in this location. In the light of a previous demonstration that DsbA is not necessary for entry of epithelial cells, we conclude that a role in virulence of this disulfide bond catalyst lies in facilitating secretion of Ipa proteins specifically within epithelial protrusions, in turn allowing cell-to-cell spread of
S. flexneri.
Evidence for Vaccine Synergy between
Borrelia burgdorferi
Decorin Binding Protein A and Outer Surface Protein A in the Mouse Model of Lyme Borreliosis
Authors: Hanson Mark S. , Patel Nita K. , Cassatt David R. , Ulbrandt Nancy D. 
Published: July 2002

ABSTRACT

Mice immunized with either the predominantly vector-stage lipoprotein outer surface protein A (OspA) or the in vivo-expressed lipoprotein decorin binding protein A (DbpA) are protected against
Borrelia burgdorferi
challenge. DbpA-OspA combinations protected against 100-fold-higher challenge doses than did either single-antigen vaccine and conferred significant protection against heterologous
B. burgdorferi
,
B. garinii
, and
B. afzelii
isolates, suggesting that there is synergy between these two immunogens.
Latency-Associated Peptide of Transforming Growth Factor β Enhances Mycobacteriocidal Immunity in the Lung duringMycobacterium bovisBCG Infection in C57BL/6 Mice
Authors: Wilkinson K. A. , Martin T. D. , Reba S. M. , Aung H. , Redline R. W. , Boom W. H. , Toossi Z. , Fulton S. A. 
Published: July 2002

ABSTRACTLatency-associated peptide of transforming growth factor β (TGF-β) (LAP) was used to determine whether in vivo modulation of TGF-β bioactivity enhanced pulmonary immunity toMycobacterium bovisBCG infection in C57BL/6 mice. LAP decreased BCG growth in the lung and enhanced antigen-specific T-cell proliferation and gamma interferon mRNA expression. Thus, susceptibility of the lung to primary BCG infection may be partially mediated by the immunosuppressive effects of TGF-β.
Binding and Activation of Human Plasminogen by
Mycobacterium tuberculosis
Authors: Monroy Verónica , Amador Angelica , Ruiz Blanca , Espinoza-Cueto Patricia , Xolalpa Wendy , Mancilla Raul , Espitia Clara 
Published: July 2002

ABSTRACT

The first evidence of the interaction of
Mycobacterium tuberculosis
with the plasminogen system is herein reported. By FACScan analysis and affinity blotting, lysine-dependent binding of plasminogen to
M. tuberculosis
was demonstrated. The binding molecules were 30-, 60-, and 66-kDa proteins present in cell wall and soluble protein extracts. The activation of plasminogen, which occurred only in presence of fibrin and was not inhibited by the host serpin, α
2
-antiplasmin, was also demonstrated.
Lack of Regulation of the Modification-Dependent Restriction Enzyme McrBC in
Escherichia coli
Authors: Murphy Mark , Schmid Nuoffer Stefanie , Bickle Thomas A. 
Published: July 2002

ABSTRACT

Restriction alleviation (RA) by the type I restriction enzyme
Eco
KI is caused by treatments that damage DNA. RA is due to proteolysis of the
Eco
KI HsdR subunit by the ClpXP ATP-dependent protease. Here we show that the modification-dependent enzyme McrBC is not subject to RA, although it is moderately sensitive to ClpAP.
The Minimal Replicator of Epstein-Barr Virus
oriP
Authors: Yates John L. , Camiolo Sarah M. , Bashaw Jacqueline M. 
Published: July 2002

ABSTRACT

oriP
is a 1.7-kb region of the Epstein-Barr virus (EBV) chromosome that supports the replication and stable maintenance of plasmids in human cells.
oriP
contains two essential components, called the DS and the FR, both of which contain multiple binding sites for the EBV-encoded protein, EBNA-1. The DS appears to function as the replicator of
oriP
, while the FR acts in conjunction with EBNA-1 to prevent the loss of plasmids from proliferating cells. Because of EBNA-1's role in stabilizing plasmids through the FR, it has not been entirely clear to what extent EBNA-1 might be required for replication from
oriP
per se, and a recent study has questioned whether EBNA-1 has any direct role in replication. In the present study we found that plasmids carrying
oriP
required EBNA-1 to replicate efficiently even when assayed only 2 days after plasmids were introduced into the cell lines 143B and 293. Significantly, using 293 cells it was demonstrated that the plasmid-retention function of EBNA-1 and the FR did not contribute significantly to the accumulation of replicated plasmids, and the DS supported efficient EBNA-1-dependent replication in the absence of the FR. The DS contains two pairs of closely spaced EBNA-1 binding sites, and a previous study had shown that both sites within either pair are required for activity. However, it was unclear from previous work what additional sequences within the DS might be required. We found that each “half” of the DS, including a pair of closely spaced EBNA-1 binding sites, had significant replicator activity when the other half had been deleted. The only significant DNA sequences that the two halves of the DS share in common, other than EBNA-1 binding sites, is a 9-bp sequence that is present twice in the “left half” and once in the “right half.” These nonamer repeats, while not essential for activity, contributed significantly to the activity of each half of the DS. Two thymines occur at unique positions within EBNA-1 binding sites 1 and 4 at the DS and become sensitive to oxidation by permanganate when EBNA-1 binds, but mutation of each to the consensus base, adenine, actually improved the activity of each half of the DS slightly. In conclusion, the DS of
oriP
is an EBNA-1-dependent replicator, and its minimal active core appears to be simply two properly spaced EBNA-1 binding sites.
Identification and Analysis of a Novel Heparin-Binding Glycoprotein Encoded by Human Herpesvirus 7
Authors: Skrincosky David , Hocknell Peter , Whetter Linda , Secchiero Paola , Chandran Bala , Dewhurst Stephen 
Published: July 2002

ABSTRACTHuman herpesvirus 6 (HHV-6) and HHV-7 are closely related betaherpesviruses that encode a number of genes with no known counterparts in other herpesviruses. The product of one such gene is the HHV-6 glycoprotein gp82-105, which is a major virion component and a target for neutralizing antibodies. A 1.7-kb cDNA clone from HHV-7 was identified which contains a large open reading frame capable of encoding a predicted primary translational product of 468 amino acids (54 kDa) with 13 cysteine residues and 9 potential N-linked glycosylation sites. This putative protein, which we have termed gp65, was homologous to HHV-6 gp105 (30% identity) and contained a single potential membrane-spanning domain located near its amino terminus. Comparison of the cDNA sequence with that of the viral genome revealed that the gene encoding gp65 contains eight exons, spanning almost 6 kb of the viral genome at the right (3′) end of the HHV-7 genome. Northern (RNA) blot analysis with poly(A)+RNA from HHV-7-infected cells revealed that the cDNA insert hybridized to a single major RNA species of 1.7 kb. Antiserum raised against a purified, recombinant form of gp65 recognized a protein of roughly 65 kDa in sucrose density gradient-purified HHV-7 preparations; treatment with PNGase F reduced this glycoprotein to a putative precursor of approximately 50 kDa. Gp65-specific antiserum also neutralized the infectivity of HHV-7, while matched preimmune serum did not do so. Finally, analysis of the biochemical properties of recombinant gp65 revealed a specific interaction with heparin and heparan sulfate proteoglycans and not with closely related molecules such asN-acetylheparin and de-N-sulfated heparin. At least two domains of the protein were found to contribute to heparin binding. Taken together, these findings suggest that HHV-7 gp65 may contribute to viral attachment to cell surface proteoglycans.
Directional Transneuronal Infection by Pseudorabies Virus Is Dependent on an Acidic Internalization Motif in the Us9 Cytoplasmic Tail
Authors: Brideau Amy D. , Eldridge Marlies G. , Enquist Lynn W. 
Published: July 2002

ABSTRACT

The Us9 gene is conserved among most alphaherpesviruses. In pseudorabies virus (PRV), the Us9 protein is a 98-amino-acid, type II membrane protein found in the virion envelope. It localizes to the
trans
-Golgi network (TGN) region in infected and transfected cells and is maintained in this compartment by endocytosis from the plasma membrane. Viruses with Us9 deleted have no observable defects in tissue culture yet have reduced virulence and restricted spread to retinorecipient neurons in the rodent brain. In this report, we demonstrate that Us9-promoted transneuronal spread in vivo is dependent on a conserved acidic motif previously shown to be essential for the maintenance of Us9 in the TGN region and recycling from the plasma membrane. Mutant viruses with the acidic motif deleted have an anterograde spread defect indistinguishable from that of Us9 null viruses. Transneuronal spread, however, is not dependent on a dileucine endocytosis motif in the Us9 cytoplasmic tail. Through alanine scanning mutagenesis of the acidic motif, we have identified two conserved tyrosine residues that are essential for Us9-mediated spread as well as two serine residues, comprising putative consensus casein kinase II sites, that modulate the rate of PRV transneuronal spread in vivo.
Identification of a
cis
-Acting Replication Element within the Poliovirus Coding Region
Authors: Goodfellow Ian , Chaudhry Yasmin , Richardson Andrew , Meredith Janet , Almond Jeffrey W. , Barclay Wendy , Evans David J. 
Published: July 2002

ABSTRACT

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein. Covariance analysis suggested the structure was well conserved in the
Enterovirus
genus of the
Picornaviridae
. Site-directed mutagenesis, disrupting the structure without affecting the 2C product, destroyed genome viability and suggested that the structure was required in the positive sense for function. Recovery of revertant viruses suggested that integrity of the structure was critical for function, and analysis of replication demonstrated that nonviable mutants did not synthesize negative strands. Our conclusion, that this RNA secondary structure constitutes a novel poliovirus
cis
-acting replication element (CRE), is supported by the demonstration that subgenomic replicons bearing lethal mutations in the native structure can be restored to replication competence by the addition of a second copy of the 61-nt wild-type sequence at another location within the genome. This poliovirus CRE functionally resembles an element identified in rhinovirus type 14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) and the cardioviruses (P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560–11565, 1999) but differs in sequence, structure, and location. The functional role and evolutionary significance of CREs in the replication of positive-sense RNA viruses is discussed.
Influenza Virus Assembly and Lipid Raft Microdomains: a Role for the Cytoplasmic Tails of the Spike Glycoproteins
Authors: Zhang Jie , Pekosz Andrew , Lamb Robert A. 
Published: July 2002

ABSTRACTInfluenza viruses encoding hemagglutinin (HA) and neuraminidase (NA) glycoproteins with deletions in one or both cytoplasmic tails (HAt− or NAt−) have a reduced association with detergent-insoluble glycolipids (DIGs). Mutations which eliminated various combinations of the three palmitoylation sites in HA exhibited reduced amounts of DIG-associated HA in virus-infected cells. The influenza virus matrix (M1) protein was also found to be associated with DIGs, but this association was decreased in cells infected with HAt− or NAt− virus. Regardless of the amount of DIG-associated protein, the HA and NA glycoproteins were targeted primarily to the apical surface of virus-infected, polarized cells. The uncoupling of DIG association and apical transport was augmented by the observation that the influenza A virus M2protein as well as the influenza C virus HA-esterase-fusion glycoprotein were not associated with DIGs but were apically targeted. The reduced DIG association of HAt− and NAt− is an intrinsic property of the glycoproteins, as similar reductions in DIG association were observed when the proteins were expressed from cDNA. Examination of purified virions indicated reduced amounts of DIG-associated lipids in the envelope of HAt− and NAt− viruses. The data indicate that deletion of both the HA and NA cytoplasmic tails results in reduced DIG association and changes in both virus polypeptide and lipid composition.
The Epithelial Integrin αvβ6 Is a Receptor for Foot-and-Mouth Disease Virus
Authors: Jackson Terry , Sheppard Dean , Denyer Michael , Blakemore Wendy , King Andrew M. Q. 
Published: July 2002

ABSTRACTField isolates of foot-and-mouth disease virus (FMDV) have been shown to use the RGD-dependent integrin αvβ3 as a cellular receptor on cultured cells. However, several other RGD-dependent integrins may have the potential to act as receptors for FMDV in vivo. Of these, αvβ6 is a likely candidate for use as a receptor by FMDV as it is expressed on epithelial cells, which correlates with the tissue tropism of the virus. In this report, we show that human colon carcinoma cells (SW480) that are normally nonpermissive for FMDV become susceptible to infection as a result of transfection with the integrin β6 subunit and expression of αvβ6 at the cell surface. Integrin αvβ6 is the major site for virus attachment on the β6-transfected cells, and binding to αvβ6 serves to increase the rate of virus entry into these cells. In addition, we show that virus binding and infection of the β6-transfected cells is mediated through an RGD-dependent interaction that is specifically inhibited by a monoclonal antibody (10D5) that recognizes αvβ6. These studies establish a role for αvβ6 as a cellular receptor for FMDV.
Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter
Authors: Aurrekoetxea-Hernández Koldo , Buetti Elena 
Published: July 2002

ABSTRACTB lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position −139 to −146 from the cap site) and fp2 (at −157 to −164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways.
Host Protein Interactions with the 3′ End of Bovine Coronavirus RNA and the Requirement of the Poly(A) Tail for Coronavirus Defective Genome Replication
Authors: Spagnolo Jeannie F. , Hogue Brenda G. 
Published: July 2002

ABSTRACT

RNA viruses have 5′ and 3′ untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5′ end and has a 3′ UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3′ UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3′ UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important
cis
-acting signal for coronavirus replication.
Cell Fusion Activity of Hepatitis C Virus Envelope Proteins
Authors: Takikawa Shingo , Ishii Koji , Aizaki Hideki , Suzuki Tetsuro , Asakura Hitoshi , Matsuura Yoshiharu , Miyamura Tatsuo 
Published: July 2002

ABSTRACT
To examine the cell fusion activity of hepatitis C virus (HCV) envelope proteins (E1 and E2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68:5411–5422, 1994). The chimeric HCV E1 and E2 proteins, each consisting of the ectodomain of the E1 and E2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G glycoprotein, were expressed on the cell surface. Cells expressing the chimeric envelope proteins and T7 RNA polymerase were cocultured with the various target cell lines transfected with a reporter plasmid encoding the luciferase gene under the control of the T7 promoter. After cocultivation, the cell fusion activity was determined by the expression of luciferase in the cocultured cells. The induction of cell fusion requires both the chimeric E1 and E2 proteins and occurs in a low-pH-dependent manner. Although it has been shown that HCV E2 protein binds human CD81 (P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Science 282:938–941, 1998), the expression of human CD81 alone is not sufficient to confer susceptibility to cell fusion in the mouse cell line. Treatment of the target cells with pronase, heparinase, or heparitinase reduced the cell fusion activity induced by the chimeric envelope proteins. These results suggest (i) that both HCV E1 and E2 proteins are responsible for fusion with the endosomal membrane after endocytosis and (ii) that certain protein molecules other than human CD81 and some glycosaminoglycans on the cell surface are also involved in the cell fusion induced by HCV.