bluuebunny/crossref_metadata_2025 huggingface dataset:
The Bacteriophage T4 Transcription Activator MotA Interacts with the Far-C-Terminal Region of the σ70Subunit ofEscherichia coliRNA Polymerase
Authors: Pande Suchira , Makela Anna , Dove Simon L. , Nickels Bryce E. , Hochschild Ann , Hinton Deborah M.
Published: July 2002

Transcription from bacteriophage T4 middle promoters usesEscherichia coliRNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the σ70subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at −30, and also interacts with σ70. We show here that the N-terminal half of MotA (MotANTD), which is thought to include the activation domain, interacts with the C-terminal region of σ70in anE. colitwo-hybrid assay. Replacement of the C-terminal 17 residues of σ70with comparable σ38residues abolishes the interaction with MotANTDin this assay, as does the introduction of the amino acid substitution R608C. Furthermore, in vitro transcription experiments indicate that a polymerase reconstituted with a σ70that lacks C-terminal amino acids 604 to 613 or 608 to 613 is defective for MotA-dependent activation. We also show that a proteolyzed fragment of MotA that contains the C-terminal half (MotACTD) binds DNA with aKD(app)that is similar to that of full-length MotA. Our results support a model for MotA-dependent activation in which protein-protein contact between DNA-bound MotA and the far-C-terminal region of σ70helps to substitute functionally for an interaction between σ70and a promoter −35 element.
Properties of 2-Oxoglutarate:Ferredoxin Oxidoreductase from
Thauera aromatica
and Its Role in Enzymatic Reduction of the Aromatic Ring
Authors: Dörner Edith , Boll Matthias
Published: July 2002

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from
Thauera aromatica
uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in
T
.
aromatica
cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 μmol min
−1
mg
−1
. The native enzyme had a molecular mass of 200 ± 20 kDa (mean ± standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (αβ)
2
composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 ± 0.5 mol of Fe, 7.2 ± 0.5 mol of acid-labile sulfur, and 1.6 ± 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low

K
m

for ferredoxin (∼10 μM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between
14
CO
2
and C
1
of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from
T
.
aromatica
belongs to the
Halobacterium
type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S]
1+/2+
clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.
Rhizobium leguminosarumHas a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family
Authors: Hosie A. H. F. , Allaway D. , Galloway C. S. , Dunsby H. A. , Poole P. S.
Published: July 2002

Amino acid uptake byRhizobium leguminosarumis dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRlshows it to be the second general amino acid permease ofR. leguminosarum. Although BraRlhas high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate,l-arginine, andl-histidine), in addition to neutral amino acids (l-alanine andl-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRlis the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRldoes not appear to be stereospecific asdamino acids cause significant inhibition of uptake ofl-glutamate andl-leucine. Unlike all other solutes tested,l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRlmembrane complex duringl-alanine uptake. Overall, the data indicate that BraRlis a general amino acid permease of the HAAT family. Furthermore, BraRlhas the broadest solute specificity of any characterized bacterial amino acid transporter.
The Putative Response Regulator BaeR Stimulates Multidrug Resistance of
Escherichia coli
via a Novel Multidrug Exporter System, MdtABC
Authors: Nagakubo Satoshi , Nishino Kunihiko , Hirata Takahiro , Yamaguchi Akihito
Published: July 2002

Overproduction of the response regulator BaeR confers resistance to novobiocin and bile salts in a Δ
acrAB
mutant by stimulating drug exporter gene expression. The
mdtABC
(multidrug transporter ABC, formerly known as
yegMNO
) genes, which encode a resistance-nodulation-cell division (RND) drug efflux system, are responsible for resistance. The MdtABC system comprises the transmembrane MdtB/MdtC heteromultimer and MdtA membrane fusion protein. MdtAC also confers bile salt, but not novobiocin, resistance. This indicates that the evolution from an MdtC homomultimer to an MdtBC heteromultimer contributed to extend the drug resistance spectrum. A BLAST search suggested that such a heteromultimer-type RND exporter constitutes a unique family among gram-negative organisms.
Structural and Genetic Analysis of the BldB Protein of
Streptomyces coelicolor
Authors: Eccleston Marcus , Ali Reem Ahmed , Seyler Richard , Westpheling Janet , Nodwell Justin
Published: July 2002

We have demonstrated that the
bldB
gene of
Streptomyces coelicolor
is required for the formation of aerial hyphae and the synthesis of antibiotics. We also found that BldB forms a higher-order complex (most likely a dimer) and that amino acid residues 20 to 78 are important for this interaction. This region is conserved in the BldB family, suggesting that dimer formation may be a common feature of these proteins.
Transcriptome and Proteome Analysis ofBacillus subtilisGene Expression Modulated by Amino Acid Availability
Authors: Mäder Ulrike , Homuth Georg , Scharf Christian , Büttner Knut , Bode Rüdiger , Hecker Michael
Published: July 2002

A comprehensive study ofBacillus subtilisgene expression patterns in response to amino acid availability was performed by means of proteomics and transcriptomics. The methods of two-dimensional protein gel electrophoresis and DNA macroarray technology were combined to analyze cells exponentially grown in minimal medium with and without 0.2% Casamino Acids (CAA). This approach revealed about 120 genes predominantly involved in amino acid biosynthesis, sporulation, and competence, which were downregulated in CAA-containing medium. Determination of sporulation frequencies confirmed the physiological relevance of the expression data.
Clinical Evaluation of the ZstatFlu-II Test: a Chemiluminescent Rapid Diagnostic Test for Influenza Virus
Authors: Hamilton Marilyn S. , Abel David M. , Ballam Yolanda J. , Otto Mary K. , Nickell Angela F. , Pence Lisa M. , Appleman James R. , Shimasaki Craig D. , Achyuthan Komandoor E.
Published: July 2002

Exploiting the high sensitivity of the chemiluminescence phenomenon, an accurate and sensitive point-of-care test, called the ZstatFlu-II test (ZymeTx, Inc., Oklahoma City, Okla.), was developed to detect influenza virus infections. The ZstatFlu-II test takes 20 min and requires approximately 2 min of “hands-on” time for operational steps. The ZstatFlu-II test does not distinguish between infections with influenza virus types A and B. ZstatFlu-II test results are printed on Polaroid High-Speed Detector Film, allowing test results to be archived. A prototype version of the ZstatFlu-II test was evaluated during the 2000-to-2001 flu season with 300 nasal aspirate specimens from children at a pediatric hospital. Compared to culture, the ZstatFlu-II test had 88% sensitivity and 92% specificity. The Directigen test had a sensitivity of 75% and a specificity of 93%. The sensitivity of the ZstatFlu-II test was significantly higher than that of the Directigen test (
P
< 0.0574).
Comparison of an Industry-Derived LCx
Chlamydia pneumoniae
PCR Research Kit to In-House Assays Performed in Five Laboratories
Authors: Chernesky Max , Smieja Marek , Schachter Julius , Summersgill James , Schindler Laura , Solomon Natalie , Campbell Karen , Campbell LeeAnn , Cappuccio Alison , Gaydos Charlotte , Chong Sylvia , Moncada Jeanne , Phillips Jack , Jang Dan , Wood Billie Jo , Petrich Astrid , Hammerschlag Margaret , Cerney Mike , Mahony James
Published: July 2002

In a multicenter comparison of PCR assays utilizing 120 quantitated samples of 16
Chlamydia pneumoniae
isolates, an LCx research-use-only (RUO) PCR developed by Abbott Laboratories demonstrated 100% sensitivity on 48 samples with >1 copy of DNA per μl of specimen. The sensitivities of five in-house PCR assays ranged from 54 to 94% for the same samples. All six assays showed decreased sensitivities as the DNA copy numbers of the samples decreased. Overall, sensitivities ranged from 68% for the LCx PCR assay to 29% for one of the in-house tests. The LCx RUO PCR and three of the five in-house PCR tests reported no false positives with the 24 negative samples. Increasing the number of replicates tested increased the sensitivities of all of the assays, including the LCx PCR. The LCx RUO assay showed high reproducibility for a single technologist and between technologists, with a kappa agreement of 0.77. The within-center agreements of the five in-house PCR tests varied from 0.19 to 0.74 on two challenges of 60 specimens 1 month apart. The LCx
C. pneumoniae
RUO PCR shows excellent potential for use in clinical studies, which could enable standardization of results in the field.
Evaluation of the C6 Peptide Enzyme-Linked Immunosorbent Assay for Individuals Vaccinated with the Recombinant OspA Vaccine
Authors: Marques Adriana R. , Martin Dale S. , Philipp Mario T.
Published: July 2002

The C6 enzyme-linked immunosorbent assay (ELISA), based on a peptide (C6) that reproduces the sequence of invariable region 6 of VlsE, the antigenic variation protein ofBorrelia burgdorferi, has been shown to be a sensitive and specific test for the serologic diagnosis of Lyme disease. We now report that none of 29 uninfected individuals vaccinated with the recombinant OspA vaccine had an antibody response to the C6 peptide. The C6 peptide ELISA can be used to diagnose Lyme disease in patients who have received the OspA vaccine.
Three Cases of Infection with Hepatitis C Virus Genotype 5 among Brazilian Hepatitis Patients
Authors: Levi José Eduardo , Takaoka Deise Tihe , Garrini Regina Helena , Fachini Roberta Maria , Focaccia Roberto , de Bortholi Santos Edgar , Mitre Heloisa Pedrosa , de Mendonça João Silva , de Paula Cavalheiro Norma , Barone Antonio Alci , Wendel Silvano
Published: July 2002

During the course of routine genotyping of hepatitis C virus isolates by 5′ noncoding region sequencing, three samples were found to bear genotype 5-specific nucleotides. A serotyping method was subsequently applied and confirmed the finding. This is the first report of the occurrence of genotype 5 in Brazil.
Nationwide Survey of In Vitro Activities of Itraconazole and Voriconazole against Clinical
Aspergillus fumigatus
Isolates Cultured between 1945 and 1998
Authors: Verweij Paul E. , Te Dorsthorst Debbie T. A. , Rijs Anthonius J. M. M. , De Vries-Hospers Hilly G. , Meis Jacques F. G. M. ,   the Dutch Interuniversity Working Party for Invasive Mycoses
Published: July 2002

The isolates in a collection of 170
Aspergillus fumigatus
isolates recovered from 114 patients and 21 different medical centers in The Netherlands over a period of 53 years were tested for the presence of resistance to itraconazole and voriconazole according to the guidelines of NCCLS document M38-P and by the E-test. Three isolates were highly resistant to itraconazole, and voriconazole MICs were low for all isolates.
Rapid Identification of
Staphylococcus aureus
and the
mecA
Gene from BacT/ALERT Blood Culture Bottles by Using the LightCycler System
Authors: Shrestha Nabin K. , Tuohy Marion J. , Hall Gerri S. , Isada Carlos M. , Procop Gary W.
Published: July 2002

One hundred BacT/ALERT blood culture bottles growing gram-positive cocci in clusters were cultured and studied by LightCycler PCR for the
sa442
and
mecA
genes. PCR was 100% sensitive and specific for detecting
Staphylococcus aureus
and methicillin resistance in
S. aureus
but was less accurate for methicillin resistance in coagulase-negative staphylococci.
PCR for Detection of
cdt-III
and the Relative Frequencies of Cytolethal Distending Toxin Variant-Producing
Escherichia coli
Isolates from Humans and Cattle
Authors: Clark Clifford G. , Johnson Shelley T. , Easy Russell H. , Campbell Jennifer L. , Rodgers Frank G.
Published: July 2002

A PCR assay that uses primers whose sequences were obtained from the published sequence of the
cdt-III
gene was developed to determine the frequencies of the
cdt-I
,
cdt-II
, and
cdt-III
genes in
Escherichia coli
isolates from humans and animals.
E. coli
isolates producing cytolethal distending toxin (CDT) were infrequently detected. The
cdt-I
gene was preferentially detected in strains with the
cnf1
gene, while the
cdt-III
gene was found in strains carrying the
cnf2
gene. The
cdt-III
genotype was more prevalent in animal isolates, while the
cdt-I
and
cdt-II
genotypes were more evident in human isolates. The presence of further
cdt
gene variants was indicated by the presence of toxin activity in cell culture in the absence of PCR amplification of the
cdt-I
,
cdt-II
, or
cdt-III
gene.
Characterization of Low- and Very-Low-Density Hepatitis C Virus RNA-Containing Particles
Authors: André P. , Komurian-Pradel F. , Deforges S. , Perret M. , Berland J. L. , Sodoyer M. , Pol S. , Bréchot C. , Paranhos-Baccalà G. , Lotteau V.
Published: July 2002

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.
Correlation of Major Histocompatibility Complex Class I Downregulation with Resistance of Human T-Cell Leukemia Virus Type 1-Infected T Cells to Cytotoxic T-Lymphocyte Killing in a Rat Model
Authors: Ohashi Takashi , Hanabuchi Shino , Suzuki Reiko , Kato Hirotomo , Masuda Takao , Kannagi Mari
Published: July 2002

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. Despite the apparent transforming ability of HTLV-1 under experimental conditions, most HTLV-1 carriers are asymptomatic. These facts suggest that HTLV-1 is controlled by host immunity in most carriers. To understand the interplay between host immunity and HTLV-1-infected cells, in this study, we isolated several HTLV-1 Tax-specific cytotoxic T-lymphocyte (CTL) lines from rats inoculated with Tax-coding DNA and investigated the long-term effects of the CTL on syngeneic HTLV-1-infected T cells. Our results demonstrated that long-term mixed culture of these CTL and the virus-infected T cells led to the emergence of CTL-resistant HTLV-1-infected cells. Although the Tax expression level in these resistant cells was equivalent to that in the parental cells, expression of surface major histocompatibility complex class I (MHC-I) was significantly downregulated in the resistant cells. Downregulation of MHC-I was more apparent in RT1.Al, which presents a Tax epitope recognized by the CTL established in this study. Moreover, peptide pulsing resulted in killing of the resistant cells by CTL, indicating that resistance was caused by a decreased epitope density on the infected cell surface. This may be one of the mechanisms for persistence of HTLV-1-infected cells that evade CTL lysis and potentially develop ATL.
Aura Virus Structure Suggests that the T=4 Organization Is a Fundamental Property of Viral Structural Proteins
Authors: Zhang Wei , Fisher Bonnie R. , Olson Norman H. , Strauss James H. , Kuhn Richard J. , Baker Timothy S.
Published: July 2002

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 Å, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.
Sequence Requirements for Viral RNA Replication and VPg Uridylylation Directed by the Internal
cis
-Acting Replication Element (
cre
) of Human Rhinovirus Type 14
Authors: Yang Yan , Rijnbrand Rene , McKnight Kevin L. , Wimmer Eckard , Paul Aniko , Martin Annette , Lemon Stanley M.
Published: July 2002

Until recently, the
cis
-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5′ and 3′ noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA
4:
1569-1584, 1998). The nucleotide sequence of the apical loop of this internal
cis
-acting replication element (
cre
) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar
cre
s have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol.
74:
10359-10370, 2000). Here, we show that the minimal functional HRV-14
cre
resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5′ half of this loop are critically important for
cre
function and suggest that a common RNNNAARNNNNNNR loop motif exists among the
cre
sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated
cre
s to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14
cre
and provide additional support for a model in which the role of the
cre
in RNA replication is to act as template for VPg uridylylation.
Transgenic Expression of Theiler's Murine Encephalomyelitis Virus Genes inH-2bMice Inhibits Resistance to Virus-Induced Demyelination
Authors: Lin Xiaoqi , Njenga M. Kariuki , Johnson Aaron J. , Pavelko Kevin D. , David Chella S. , Pease Larry R. , Rodriguez Moses
Published: July 2002

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2b) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8+T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8+T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.
Identification of Rotavirus VP6 Residues Located at the Interface with VP2 That Are Essential for Capsid Assembly and Transcriptase Activity
Authors: Charpilienne Annie , Lepault Jean , Rey Felix , Cohen Jean
Published: July 2002

Rotavirus
has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.
Resistance to Infection by Subgroups B, D, and E Avian Sarcoma and Leukosis Viruses Is Explained by a Premature Stop Codon within a Resistance Allele of the
tvb
Receptor Gene
Authors: Klucking Sara , Adkins Heather B. , Young John A. T.
Published: July 2002

Here we present the first molecular characterization of the defect associated with an avian sarcoma and leukosis virus (ASLV) receptor resistance allele,
tvb
r
. We show that resistance to infection by subgroups B, D, and E ASLV is explained by the presence of a single base pair mutation that distinguishes this allele from
tvb
s1
, an allele which encodes a receptor for all three viral subgroups. This mutation generates an in-frame stop codon that is predicted to lead to the production of a severely truncated protein.
Genetic and Biochemical Analyses of Receptor and Cofactor Determinants for T-Cell-Tropic Feline Leukemia Virus Infection
Authors: Lauring Adam S. , Cheng Heather H. , Eiden Maribeth V. , Overbaugh Julie
Published: July 2002

Entry by retroviruses is mediated through interactions between the viral envelope glycoprotein and the host cell receptor(s). We recently identified two host cell proteins, FeLIX and Pit1, that are necessary for infection by cytopathic, T-cell-tropic feline leukemia viruses (FeLV-T). Pit1 is a classic multiple transmembrane protein used as a receptor by several other simple retroviruses, including subgroup B FeLV (FeLV-B), and FeLIX is a secreted cellular protein expressed from endogenous FeLV-related sequences (enFeLV). FeLIX is nearly identical to FeLV-B
envelope
sequences that encode the N-terminal half of the viral surface unit (SU), because these FeLV-B sequences are acquired by recombination with enFeLV. FeLV-B SUs can functionally substitute for FeLIX in mediating FeLV-T infection. Both of these enFeLV-derived cofactors can efficiently facilitate FeLV-T infection only of cells expressing Pit1, not of cells expressing the related transport protein Pit2. We therefore have used chimeric Pit1/Pit2 receptors to map the determinants for cofactor binding and FeLV-T infection. Three distinct determinants appear to be required for cofactor-dependent infection by FeLV-T. We also found that Pit1 sequences within these same domains were required for binding by FeLIX to the Pit receptor. In contrast, these determinants were not all required for receptor binding by the FeLV-B SU cofactors used in this study. These data indicate that cofactor binding is not sufficient for FeLV-T infection and suggest that there may be a direct interaction between FeLV-T and the Pit1 receptor.
Heterologous Protection Induced by the Inner Capsid Proteins of Rotavirus Requires Transcytosis of Mucosal Immunoglobulins
Authors: Schwartz-Cornil Isabelle , Benureau Yann , Greenberg Harry , Hendrickson Barbara A. , Cohen Jean
Published: July 2002

Protective immunization against rotavirus (RV) can be achieved with heterologous RV, i.e., virus isolated from another species, and with heterologous inner core VP2 and VP6 proteins assembled as virus-like particles (VLP). Although the antigenically conserved VP6 protein does not induce in vitro-neutralizing antibodies, it may, however, elicit immunoglobulins (Ig) involved in heterologous protection, as some IgA against VP6 prevent RV infection in a backpack mouse model. The protective role of Ig directed to the RV inner core proteins VP2 and VP6 was investigated in J-chain-deficient mice (J chain−/−), which have a defect in the polymeric Ig receptor (pIgR)-mediated transcytosis of IgA and IgM. J chain−/−mice and wild-type (WT) mice were intranasally vaccinated with bovine RV-derived VLP2/6 and then challenged with highly infectious murine ECw RV. Whereas WT mice were totally protected, immunized J chain−/−mice shed RV for several days. In addition, naïve J chain−/−mice exhibited a 2-day delay in clearing RV compared with WT mice. The immunized J chain−/−mice displayed unaltered VLP2/6-specific B-cell numbers in spleen and in mesenteric nodes and similar levels of serum anti-VLP2/6 Ig, confirming that the adaptive B-cell response is preserved in J chain−/−mice. These results indicate that J-chain-mediated transcytosis of Ig participates in the clearance of RV and that epithelial pIgR-mediated transport of Ig is involved in the heterologous protection induced by VLP2/6.
Effects of Prostratin on T-Cell Activation and Human Immunodeficiency Virus Latency
Authors: Korin Yael D. , Brooks David G. , Brown Stephen , Korotzer Andrew , Zack Jerome A.
Published: July 2002

Human immunodeficiency virus (HIV) replication is linked to cellular gene transcription and requires target cell activation. The latent reservoir of HIV-1 in quiescent T cells is thought to be a major obstacle to clearance of infection by highly active antiretroviral therapy (HAART). Thus, identification of agents that can induce expression of latent virus may, in the presence of HAART, allow elimination of the infected cells by the immune response. We previously used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis. Latently infected mature thymocytes can be exported into the periphery, providing an efficient primary cell model to determine cellular activation signals that induce renewed expression of latent virus. Here we characterized the effects of prostratin, a non-tumor-promoting phorbol ester, on primary human peripheral blood lymphocytes (PBLs) and assessed its ability to reactivate latent HIV infection from thymocytes and PBLs in the SCID-hu (Thy/Liv) model. Prostratin stimulation alone did not induce proliferation of quiescent PBLs; however, it could provide a secondary signal in the context of T-cell receptor stimulation or a primary activation signal in the presence of CD28 stimulation to induce T-cell proliferation. While prostratin alone was not sufficient to allow de novo HIV infection, it efficiently reactivated HIV expression from latently infected cells generated in the SCID-hu mouse. Our data indicate that prostratin alone is able to specifically reactivate latent virus in the absence of cellular proliferation, making it an attractive candidate for further study as an adjunctive therapy for the elimination of the latent HIV reservoir.
Rebound of Hepatitis B Virus Replication in HepG2 Cells after Cessation of Antiviral Treatment
Authors: Abdelhamed Ayman M. , Kelley Colleen M. , Miller Thomas G. , Furman Phillip A. , Isom Harriet C.
Published: July 2002

Treatment of patients with lamivudine (3TC) results in loss of detectable levels of hepatitis B virus (HBV) DNA from serum; however, the relapse rate, with regard to both reappearance of virus in the bloodstream and hepatic inflammation, is high when therapy is terminated. Although the rebound observed in patients has also been seen in animal hepadnavirus models, rebound has not been analyzed in an in vitro cell culture system. In this study, we used the HBV recombinant baculovirus/HepG2 system to measure the time course of antiviral agent-mediated loss of HBV replication as well as the time course and magnitude of HBV production after release from antiviral treatment. Because of the sensitivity of the system, it was possible to measure secreted virions, intracellular replicative intermediates, and nuclear non-protein-bound HBV DNA and separately analyze individual species of DNA, such as single-stranded HBV DNA compared to the double-stranded form and relaxed circular compared to covalently closed circular HBV DNA. We first determined that HBV replication in the HBV recombinant baculovirus/HepG2 system could proceed for at least 35 days, with a 30-day plateau level of replication, making it possible to study antiviral agent-mediated loss of HBV followed by rebound after cessation of drug treatment. All HBV DNA species decreased in a time-dependent fashion following antiviral treatment, but the magnitude of decline differed for each HBV DNA species, with the covalently closed circular form of HBV DNA being the most resistant to drug therapy. When drug treatment ceased, HBV DNA species reappeared with a pattern that recapitulated the initiation of replication, but with a different time course.
The Block in Assembly of Modified Vaccinia Virus Ankara in HeLa Cells Reveals New Insights into Vaccinia Virus Morphogenesis
Authors: Sancho M. Carmen , Schleich Sibylle , Griffiths Gareth , Krijnse-Locker Jacomine
Published: July 2002

It has previously been shown that upon infection of HeLa cells with modified vaccinia virus Ankara (MVA), assembly is blocked at a late stage of infection and immature virions (IVs) accumulate (G. Sutter and B. Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992). In the present study the morphogenesis of MVA in HeLa cells was studied in more detail and compared to that under two conditions that permit the production of infectious particles: infection of HeLa cells with the WR strain of vaccinia virus (VV) and infection of BHK cells with MVA. Using several quantitative and qualitative assays, we show that early in infection, MVA in HeLa cells behaves in a manner identical to that under the permissive conditions. By immunofluorescence microscopy (IF) at late times of infection, the labelings for an abundant membrane protein of the intracellular mature virus, p16/A14L, and the viral DNA colocalize under permissive conditions, whereas in HeLa cells infected with MVA these two structures do not colocalize to the same extent. In both permissive and nonpermissive infection, p16-labeled IVs first appear at 5 h postinfection. In HeLa cells infected with MVA, IVs accumulated predominantly outside the DNA regions, whereas under permissive conditions they were associated with the viral DNA. At 4 h 30 min, the earliest time at which p16 is detected, the p16 labeling was found predominantly in a small number of distinct puncta by IF, which were distinct from the sites of DNA in both permissive and nonpermissive infection. By electron microscopy, no crescents or IVs were found at this time, and the p16-labeled structures were found to consist of membrane-rich vesicles that were in continuity with the cellular endoplasmic reticulum. Over the next 30 min of infection, a large number of p16-labeled crescents and IVs appeared abruptly under both permissive and nonpermissive conditions. Under permissive conditions, these IVs were in close association with the sites of DNA, and a significant amount of these IVs engulfed the viral DNA. In contrast, under nonpermissive conditions, the IVs and DNA were mostly in separate locations and relatively few IVs acquired DNA. Our data show that in HeLa cells MVA forms normal DNA replication sites and normal viral precursor membranes but the transport between these two structures is inhibited.
Molecular Basis of the Attenuation Exhibited by Molecularly Cloned Highly Passaged Chicken Anemia Virus Isolates
Authors: Todd Daniel , Scott Alistair N. J. , Ball Neris W. , Borghmans Borghert J. , Adair Brian M.
Published: July 2002

Chimeric virus experiments indicated that the pathogenicity and monoclonal antibody reactivity differences between two molecularly cloned, highly passaged chicken anemia virus isolates could be attributed to the VP1 amino acid change at residue 89. The introduction of this change into a pathogenic cloned low-passage isolate was not sufficient to cause attenuation.
Inserting a Nuclear Targeting Signal into a Replication-Competent Moloney Murine Leukemia Virus Affects Viral Export and Is Not Sufficient for Cell Cycle-Independent Infection
Authors: Seamon Jennifer A. , Jones Kathryn S. , Miller Christina , Roth Monica J.
Published: July 2002

The effects of inserting reported nuclear localization signals (NLSs) into the Moloney murine leukemia virus (Mo-MuLV) integrase (IN) protein, within a replication-competent viral construct, were studied. In contrast to the virus harboring IN fused to the simian virus 40 (SV40) large T antigen NLS (SV40 NLS) (J. A. Seamon, M. Adams, S. Sengupta, and M. J. Roth, Virology
274:
412-419, 2000), a codon-modified SV40 NLS was stably expressed during viral propagation. Incorporation of the codon-modified SV40 NLS into IN, however, altered the packaging of the Gag-Pol precursor in the virus; viral particles contained decreased levels of reverse transcriptase (RT) and IN. In addition, the virus showed delayed kinetics of viral DNA synthesis upon infection. A panel of infectious MuLVs containing alternative IN-NLS fusions was generated and assayed for cell cycle-independent infection. Viral infection with the NLS-tagged proteins, however, remained dependent on passage of the cells through mitosis. This finding has direct implications for engineering murine-based retroviral vectors for gene therapy.
Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function
Authors: Wang Huating , Norris Kendra M. , Mansky Louis M.
Published: July 2002

Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the
Deltaretrovirus
genus of the
Retroviridae
family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain).
Chemistry of closo-Dodecaborate Anion [B12H12]2-: A Review
Authors: Sivaev Igor B. , Bregadze Vladimir I. , Sjöberg Stefan
Published: July 2002

Synthesis and chemical properties of the closo-dodecaborate anion [B12H12]2- and its derivatives are reviewed. Attention is also paid to potential applications of the closo-dodecaborate derivatives with emphasis on medical applications. A review with 325 references.
Strained Configurations in Three-Dimensional Analogues of Kekulé-Type Structures for Deltahedral Boranes
Authors: King R. Bruce
Published: July 2002

Localized structures analogous to the Kekulé structures for benzenoid hydrocarbons can be constructed for the deltahedral boranes BnHn2-. These localized structures contain exactly three two-center two-electron (2c-2e) B-B bonds and n - 2 three-center two-electron (3c-2e) B-B-B bonds. The number of equivalent such Kekulé-type structures corresponds to the index of the symmetry group of the Kekulé structure, K, in the symmetry group, D, of the deltahedron. Three-dimensional Kekulé-type structures with the following configurations exhibit excessive strain and are therefore unfavorable: (i) structures having one or more pairs of boron atoms connected simultaneously by a 2c-2e B-B bond and a 3c-2e B-B-B bond (violation of the O'Neill-Wade restrictions); (ii) structures in which the three 2c-2e B-B bonds are excessively concentrated occupying only three or four vertices (the undesirable ∆, U, and Y configurations). Computations by Lipscomb and coworkers with partial retention of diatomic differential overlap (PRDDO) suggest that wide distribution of the three 2c-2e B-B bonds throughout the deltahedron and a minimum number of empty faces are more important than maximum symmetry in leading to the most favorable Kekulé-type structure.
Two Forgotten Ten-Vertex arachno Triheteroboranes: arachno-5,6,9-C2SB7H11 and arachno-5,6,9-C3B7H13, Their Molecular Structure Determination by ab initio/NMR Approach and Synthesis of the Thiacarbaborane
Authors: Hnyk Drahomír , Holub Josef
Published: July 2002

A solution of nido-5,6-C2B8H12 reacted in the presence of Et3N with sulfur to yield the first mixed triheteroborane based on the [arachno-B10H14]2- framework (C2v symmetry), i.e. arachno-5,6,9-C2SB7H11. This compound is isoelectrolobal with known arachno-5,6,9-C3B7H13. The structures of these two ten-vertex arachno triheteroboranes have been established by the ab initio/IGLO (and GIAO-SCF)/NMR method. The presence of three heteroatoms and the absence of the hydrogen atom bridging the 5 and 10 atoms are responsible for a symmetry reduction from C2v to C1 in both compounds. The carbon atoms are compressed toward the center and the sulfur atom is pushed away from the center of the respective cluster relative to the positions they would have in [arachno-B10H14]2-. These distortions from the parent compound are well described by the MP2/6-31* geometrical parameters. A good fit between the computed and measured 11B chemical shifts revealed that the MP2/6-31G* internal coordinates are good representations of the molecular geometries of these two ten-vertex arachno heteroboranes.
A clinical neurological, neurophysiological, and neuropsychological study of sheep farmers and dippers exposed to organophosphate pesticides
Authors: Jamal G A , Hansen S , Pilkington A , Buchanan D , Gillham R A , Abdel-Azis M , Julu P O O , Al-Rawas S F , Hurley F , Ballantyne J P
Published: July 2002

Objectives:To classify clinical diseases of the subjects with abnormal indices of peripheral neuropathy identified in field studies of sheep farmers and dippers exposed to organophosphate pesticides. To explore what neuropsychological profiles, if any, may be associated with neurophysiological damage in these subjects.

Methods:A case-control study (79 subjects) nested within the cross sectional study (685 subjects) of sheep farmers from the field study. Three groups with no, possible, and probable or definite neuropathy according to field studies were recruited. Investigations comprised symptoms of neuropathy, neurologial signs, motor and sensory nerve conduction, electromyography, quantitative sensory testing, and neuropsychological tests.

Results:The incidence of clinical neuropathy increased from 7% in the no neuropathy group to 52% in the probable or definite neuropathy group based on nerve conduction measurements or presence of neurological signs. Sensory abnormalities were found more often than motor deficits. Small diameter nerve fibres were also affected more than large fibres.

Conclusions:The neuropathy is predominantly sensory and is characteristic of distal, chronic neuropathy with no acute features. Small fibre populations are affected more than large fibre populations. Increasing severity of neuropathy was associated with anxiety and depression as measured in the neuropsychological tests.
Methicillin resistantStaphylococcus aureus(MRSA) in the intensive care unit
Authors: Haddadin A S , Fappiano S A , Lipsett P A
Published: July 2002

Methicillin resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that causes severe morbidity and mortality worldwide. MRSA strains are endemic in many American and European hospitals and account for 29%–35% of all clinical isolates. Recent studies have documented the increased costs associated with MRSA infection, as well as the importance of colonisation pressure. Surveillance strategies have been proposed especially in high risk areas such as the intensive care unit. Pneumonia and bacteraemia account for the majority of MRSA serious clinical infections, but intra-abdominal infections, osteomyelitis, toxic shock syndrome, food poisoning, and deep tissue infections are also important clinical diseases. The traditional antibiotic therapy for MRSA is a glycopeptide, vancomycin. New antibiotics have been recently released that add to the armamentarium for therapy against MRSA and include linezolid, and quinupristin/dalfopristin, but cost, side effects, and resistance may limit their long term usefulness.