bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of Intermediate Transcription Initiation Complexes at p
fliD
and p
flgM
by ς
28
RNA Polymerase
Authors: Givens Jennifer R. , McGovern Colleen L. , Dombroski Alicia J.
Published: July 2002

The ς subunit of prokaryotic RNA polymerase is an important factor in the control of transcription initiation. Primary ς factors are essential for growth, while alternative ς factors are activated in response to various stimuli. Expression of class 3 genes during flagellum biosynthesis in
Salmonella enterica
serovar Typhimurium is dependent on the alternative ς factor ς
28
. Previously, a novel mechanism of transcription initiation at the
fliC
promoter by ς
28
holoenzyme was proposed. Here, we have characterized the mechanism of transcription initiation by a holoenzyme carrying ς
28
at the
fliD
and
flgM
promoters to determine if the mechanism of initiation observed at p
fliC
is a general phenomenon for all ς
28
-dependent promoters. Temperature-dependent footprinting demonstrated that promoter binding properties and low-temperature open complex formation are similar for p
fliC
, p
fliD
, and p
flgM
. However, certain aspects of DNA strand separation and complex stability are promoter dependent. Open complexes form in a concerted manner at p
flgM
, while a sequential pattern of open complex formation occurs at p
fliD
. Open and initiated complexes formed by holoenzyme carrying ς
28
are generally unstable to heparin challenge, with the exception of initiated complexes at p
flgM
, which are stable in the presence of nucleoside triphosphates.
Regulation of rRNA Transcription Correlates with Nucleoside Triphosphate Sensing
Authors: Barker Melanie M. , Gourse Richard L.
Published: July 2002

We have previously shown that the activity of the
Escherichia coli
rRNA promoter
rrnB
P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 different
rrnB
P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The seven
rrnB
P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the
fis
gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the
rrnB
P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which
rrnB
P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).
Molecular Characterization of Global Regulatory RNA Species That Control Pathogenicity Factors in
Erwinia amylovora
and
Erwinia herbicola
pv. gypsophilae
Authors: Ma Weilei , Cui Yaya , Liu Yang , Dumenyo C. Korsi , Mukherjee Asita , Chatterjee Arun K.
Published: July 2002

rsmB
Ecc

specifies a nontranslatable RNA regulator that controls exoprotein production and pathogenicity in soft rot-causing
Erwinia carotovora
subsp.
carotovora
. This effect of

rsmB
Ecc

RNA is mediated mostly by neutralizing the function of RsmA
Ecc
, an RNA-binding protein of
E. carotovora
subsp.
carotovora
, which acts as a global negative regulator. To determine the occurrence of functional homologs of

rsmB
Ecc

in non-soft-rot-causing
Erwinia
species, we cloned the
rsmB
genes of
E. amylovora
(

rsmB
Ea

) and
E. herbicola
pv. gypsophilae (

rsmB
Ehg

). We show that

rsmB
Ea

in
E. amylovora
positively regulates extracellular polysaccharide (EPS) production, motility, and pathogenicity. In
E. herbicola
pv. gypsophilae,

rsmB
Ehg

elevates the levels of transcripts of a cytokinin (
etz
) gene and stimulates the production of EPS and yellow pigment as well as motility. RsmA
Ea
and RsmA
Ehg
have more than 93% identity to RsmA
Ecc
and, like the latter, function as negative regulators by affecting the transcript stability of the target gene. The
rsmB
genes reverse the negative effects of RsmA
Ea
, RsmA
Ehg
, and RsmA
Ecc
, but the extent of reversal is highest with homologous combinations of
rsm
genes. These observations and findings that

rsmB
Ea

and

rsmB
Ehg

RNA bind RsmA
Ecc
indicate that the
rsmB
effect is channeled via RsmA. Additional support for this conclusion comes from the observation that the
rsmB
genes are much more effective as positive regulators in a RsmA
+
strain of
E. carotovora
subsp.
carotovora
than in its RsmA
−
derivative.
E. herbicola
pv. gypsophilae produces a 290-base
rsmB
transcript that is not subject to processing. By contrast,
E. amylovora
produces 430- and 300-base
rsmB
transcripts, the latter presumably derived by processing of the primary transcript as previously noted with the transcripts of

rsmB
Ecc

. Southern blot hybridizations revealed the presence of
rsmB
homologs in
E. carotovora, E. chrysanthemi, E. amylovora, E. herbicola, E. stewartii
and
E. rhapontici
, as well as in other enterobacteria such as
Escherichia coli, Salmonella enterica
serovar Typhimurium,
Serratia marcescens, Shigella flexneri, Enterobacter aerogenes, Klebsiella pneumoniae, Yersinia enterocolitica
, and
Y. pseudotuberculosis
. A comparison of
rsmB
sequences from several of these enterobacterial species revealed a highly conserved 34-mer region which is predicted to play a role in positive regulation by
rsmB
RNA.
Genetic Evidence that the α5 Helix of the Receiver Domain of PhoB Is Involved in Interdomain Interactions
Authors: Allen Mindy P. , Zumbrennen Kimberly B. , McCleary William R.
Published: July 2002

Two-component signaling proteins are involved in transducing environmental stimuli into intracellular signals. Information is transmitted through a phosphorylation cascade that consists of a histidine protein kinase and a response regulator protein. Generally, response regulators are made up of a receiver domain and an output domain. Phosphorylation of the receiver domain modulates the activity of the output domain. The mechanisms by which receiver domains control the activities of their respective output domains are unknown. To address this question for the PhoB protein from
Escherichia coli
, we have employed two separate genetic approaches, deletion analysis and domain swapping. In-frame deletions were generated within the
phoB
gene, and the phenotypes of the mutants were analyzed. The output domain, by itself, retained significant ability to activate transcription of the
phoA
gene. However, another deletion mutant that contained the C-terminal α-helix of the receiver domain (α5) in addition to the entire output domain was unable to activate transcription of
phoA
. This result suggests that the α5 helix of the receiver domain interacts with and inhibits the output domain. We also constructed two chimeric proteins that join various parts of the chemotaxis response regulator, CheY, to PhoB. A chimera that joins the N-terminal ∼85% of CheY's receiver domain to the β5-α5 loop of PhoB's receiver domain displayed phosphorylation-dependent activity. The results from both sets of experiments suggest that the regulation of PhoB involves the phosphorylation-mediated modulation of inhibitory contacts between the α5 helix of its unphosphorylated receiver domain and its output domain.
Engineered Fatty Acid Biosynthesis in
Streptomyces
by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III
Authors: Smirnova Natalya , Reynolds Kevin A.
Published: July 2002

The
Streptomyces glaucescens
β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in
S. glaucescens
resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments with perdeuterated acetic acid demonstrated that 9% of the palmitate pool of the wild-type strain was generated from an intact D
3
acetyl-CoA starter unit, compared to 3% in a strain expressing the Cys122Gln KASIII. These observations support the intermediacy of malonyl-ACP in generating the SCFA precursor in a strain expressing this mutant. To study malonyl-ACP decarboxylase activity in vitro, the KASIII mutants were expressed and purified as His-tagged proteins in
Escherichia coli
and assayed. In the absence of the acyl-CoA substrate the Cys122Gln mutant and wild-type KASIII were shown to have comparable decarboxylase activities in vitro. The Cys122Ala mutant exhibited higher activity. This activity was inhibited for all enzymes by the presence of high concentrations of isobutyryl-CoA (>100 μM), a branched-chain fatty acid biosynthetic precursor. Under these conditions the mutant enzymes had no activity, while the wild-type enzyme functioned as a ketoacyl synthase. These observations indicate the likely upper and lower limits of isobutyryl-CoA and related acyl-CoA concentrations within
S. glaucescens
.
Two Regions of GerE Required for Promoter Activation in
Bacillus subtilis
Authors: Crater Dinene L. , Moran Charles P.
Published: July 2002

GerE from
Bacillus subtilis
is the smallest member of the LuxR-FixJ family of transcription activators. Its 74-amino-acid sequence is similar over its entire length to the DNA binding domain of this protein family, including a putative helix-turn-helix (HTH) motif. In this report, we sought to define regions of GerE involved in promoter activation. We examined the effects of single alanine substitutions at 19 positions that were predicted by the crystal structure of GerE to be located on its surface. A single substitution of alanine for the phenylalanine at position 6 of GerE (F6A) resulted in decreased transcription in vivo and in vitro from the GerE-dependent
cotC
promoter. However, the F6A substitution had little effect on transcription from the GerE-dependent
cotX
promoter. In contrast, a single alanine substitution for the leucine at position 67 (L67A) reduced transcription from the
cotX
promoter, but not from the
cotC
promoter. The results of DNase I protection assays and in vitro transcription reactions lead us to suggest that the F6A and L67A substitutions define two regions of GerE, activation region 1 (AR1) and AR2, that are required for activation of the
cotC
and
cotX
promoters, respectively. A comparison of our results with those from studies of MalT and BvgA indicated that other members of the LuxR-FixJ family may use more than one surface to interact with RNA polymerase during promoter activation.
The Membrane-Associated Protein-Serine/Threonine Kinase fromSulfolobus solfataricusIs a Glycoprotein
Authors: Lower Brian H. , Kennelly Peter J.
Published: July 2002

Treatment of a sodium dodecyl sulfate-polyacrylamide gel with periodic acid-Schiff (PAS) stain or blotting withGalanthus nivalisagglutinin revealed the presence of several glycosylated polypeptides in a partially purified detergent extract of the membrane fraction ofSulfolobus solfataricus.One of the glycoproteins comigrated with the membrane-associated protein-serine/threonine kinase fromS. solfataricus, which had been radiolabeled by autophosphorylation with [32P]ATP in vitro. Treatment with a chemical deglycosylating agent, trifluoromethanesulfonic acid, abolished PAS staining and reduced theMrof the protein kinase from ∼67,000 to ∼62,000. Protein kinase activity also adhered to, and could be eluted from, agarose beads containing boundG. nivalisagglutinin. Glycosylation of the protein kinase implies that at least a portion of this integral membrane protein resides on the external surface of the cell membrane.
Assembly of Colicin A in the Outer Membrane of Producing
Escherichia coli
Cells Requires both Phospholipase A and One Porin, but Phospholipase A Is Sufficient for Secretion
Authors: Cavard Daniele
Published: July 2002

Three oligomeric forms of colicin A with apparent molecular masses of about 95 to 98 kDa were detected on sodium dodecyl sulfate (SDS)-polyacrylamide gels loaded with unheated samples from colicin A-producing cells of
Escherichia coli
. These heat-labile forms, called colicins Au, were visualized both on immunoblots probed with monoclonal antibodies against colicin A and by radiolabeling. Cell fractionation studies show that these forms of colicin A were localized in the outer membrane whether or not the producing cells contained the
cal
gene, which encodes the colicin A lysis protein responsible for colicin A release in the medium. Pulse-chase experiments indicated that their assembly into the outer membrane, as measured by their heat modifiable migration in SDS gels, was an efficient process. Colicins Au were produced in various null mutant strains, each devoid of one major outer membrane protein, except in a mutant devoid of both OmpC and OmpF porins. In cells devoid of outer membrane phospholipase A (OMPLA), colicin A was not expressed. Colicins Au were detected on immunoblots of induced cells probed with either polyclonal antibodies to OmpF or monoclonal antibodies to OMPLA, indicating that they were associated with both OmpF and OMPLA. Similar heat-labile forms were obtained with various colicin A derivatives, demonstrating that the C-terminal domain of colicin A, but not the hydrophobic hairpin present in this domain, was involved in their formation.
PII T-Loop Mutations Affecting Signal Transduction to NtrB Also Abolish Yeast Two-Hybrid Interactions
Authors: Martínez-Argudo Isabel , Contreras Asunción
Published: July 2002

Mutations A49P and Δ47-53 at the T loop of the
Escherichia coli
GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P. Jiang, P. Zucker, M. R. Atkinson, E. S. Kamberov, W. Tirasophon, P. Chandran, B. R. Schepke, and A. J. Ninfa, J. Bacteriol. 179: 4342-4353, 1997). We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment. The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins.
Roles of the C-Terminal End of SecY in Protein Translocation and Viability of
Escherichia coli
Authors: Chiba Kazuhiko , Mori Hiroyuki , Ito Koreaki
Published: July 2002

SecY, a central component of the membrane-embedded sector of protein translocase, contains six cytosolic domains. Here, we examined the importance of the C-terminal cytosolic region of SecY by systematically shortening the C-terminal end and examining the functional consequences of these mutations in vivo and in vitro. It was indicated that the C-terminal five residues are dispensable without any appreciable functional defects in SecY. Mutants missing the C-terminal six to seven residues were partially compromised, especially at low temperature or in the absence of SecG. In vitro analyses indicated that the initial phase of the translocation reaction, in which the signal sequence region of the preprotein is inserted into the membrane, was affected by the lack of the C-terminal residues. SecA binding was normal, but SecA insertion in response to ATP and a preprotein was impaired. It is suggested that the C-terminal SecY residues are required for SecA-dependent translocation initiation.
Genetic Organization of the
Vibrio harveyi dnaA
Gene Region and Analysis of the Function of the
V. harveyi
DnaA Protein in
Escherichia coli
Authors: Berenstein Dvora , Olesen Kirsten , Speck Christian , Skovgaard Ole
Published: July 2002

The
Vibrionaceae
family is distantly related to
Enterobacteriaceae
within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the
dnaA
gene region of
Vibrio harveyi
and found that although the organization of the
V. harveyi dnaA
region differs from that of
Escherichia coli
, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the
dnaA
promoter region. The DnaA proteins of
V. harveyi
and
E. coli
are interchangeable and function nearly identically in controlling
dnaA
transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria.
Direct Quantification of Human Cytomegalovirus Immediate-Early and Late mRNA Levels in Blood of Lung Transplant Recipients by Competitive Nucleic Acid Sequence-Based Amplification
Authors: Greijer Astrid E. , Verschuuren Erik A. M. , Harmsen Martin C. , Dekkers Chantal A. J. , Adriaanse Henriëtte M. A. , The T. Hauw , Middeldorp Jaap M.
Published: July 2002

The dynamics of active human cytomegalovirus (HCMV) infection was monitored by competitive nucleic acid sequence-based amplification (NASBA) assays for quantification of IE1 (UL123) and pp67 (UL65) mRNA expression levels in the blood of patients after lung transplantation. RNA was isolated from 339 samples of 13 lung transplant recipients and analyzed by the quantitative IE1 and pp67 NASBA in parallel with pp65 antigenemia and serology. Rapid increases in IE1 RNA exceeding 10
4
copies per 100 μl of blood were associated with active infection, whereas lower levels were suggestive for abortive, subclinical viral activity. Any positive value for pp67 RNA was indicative for active infection, and quantification of pp67 mRNA did not give additional diagnostic information. The onset of IE1-positive NASBA preceded pp67 NASBA and was earlier than the pp65 antigenemia assay, confirming previous studies with qualitative NASBA. Effective antiviral treatment was reflected by a rapid disappearance of pp67 mRNA, whereas IE1 mRNA remained detectable for longer periods. Quantification of IE1 might be relevant to monitor progression of HCMV infection but should be validated in prospective studies.
Serum Immunoglobulin G Immune Response to
Helicobacter pylori
Antigens in Mongolian Gerbils
Authors: Kumagai Toshiko , Yan Jing , Graham David Y. , Tozuka Minoru , Okimura Yukie , Ikeno Tatsuo , Sugiyama Atsushi , Katsuyama Tsutomu , Ota Hiroyoshi
Published: July 2002

The Mongolian gerbil model for
Helicobacter pylori
infection is an animal model that mimics human disease. We examined the serum immune response to
H. pylori
infection in gerbils by enzyme-linked immunosorbent assay (ELISA) and Western blotting, both with whole-cell (
H. pylori
) extracts. A total of 66 7-week-old specific-pathogen-free male gerbils were inoculated orogastrically with
H. pylori
strain ATCC 43504. Sera were collected 1, 2, 4, 8, 12, 26, 38, and 52 weeks after
H. pylori
inoculation. Sixty-nine noninfected gerbils and their sera were used as controls. The specificity of the ELISA was 95.7%. The frequency of seropositivity increased over time: 2 of 10 (20%), 7 of 10 (70%), and 7 of 7 (100%) samples of sera from inoculated gerbils were positive for
H. pylori
at 2, 4, and 8 weeks postinoculation, respectively. Western blot assays showed that the primary immunoglobulin G (IgG) response against low-molecular-mass (25-, 30-, and 20-kDa) proteins appeared after a lag period of 2 to 8 weeks after inoculation. Antibodies against 160-, 150-, 110-, 120-, 80-, 66-, and 63-kDa proteins were observed 12 weeks after inoculation. The early reactive 30-kDa protein was identified as a urease α subunit by N-terminal amino acid sequencing. After 26 weeks, two groups of animals could be distinguished: one group developed ulcers (
n
= 5), and the other developed hyperplastic polyps without ulcers (
n
= 19). Gerbils in the gastric ulcer group showed significantly higher serum anti-
H. pylori
IgG levels than did gerbils in the hyperplastic group (
P
= 0.001) as measured by ELISA. Furthermore, a higher proportion of animals developed antibodies to
H. pylori
proteins of 26, 25, and 20 kDa in the ulcer group than those animals with hyperplastic polyps (75 to 100% versus 17 to 50%) in Western blot assays. These results highlight the importance of the immune response of the host in the development of
H. pylori
-related gastric lesions.
Prevalence of Human Immunodeficiency Virus Type 1 (HIV-1) Non-B Subtypes in Foreigners Living in Madrid, Spain, and Comparison of the Performances of the AMPLICOR HIV-1 MONITOR Version 1.0 and the New Automated Version 1.5
Authors: Holguı́n Africa , Aracil Belén , Álvarez Amparo , Barros Carlos , Soriano Vincent
Published: July 2002

Plasma specimens collected in 1999 from 32 human immunodeficiency virus type 1 (HIV-1)-infected foreigners living in Madrid, Spain, were examined for the presence of non-B subtypes. Furthermore, plasma viremia was quantified using two different AMPLICOR HIV-1 MONITOR tests, version 1.0 and the new upgraded and automated version 1.5 (COBAS). Most patients came from Africa, where they most likely had acquired HIV-1 infection through sexual contact. HIV-1 genetic subtyping was based on the phylogenetic analysis of the
protease
gene. Twenty-two subtype B, six subtype G, two subtype C, one subtype A, and one D subtype infection were found. Overall, non-B subtypes represented 31.25% of the study population. Irrespective of the HIV-1 variant, viral load values above the detection limit (200 HIV RNA copies/ml) increased from 56.2 to 71.9% for results obtained using MONITOR version 1.0 and COBAS, respectively. Moreover, significant differences in viral load values (>0.5 logs) were recognized in up to 37.5% of samples. In summary, COBAS seemed to be more reliable for testing plasma viral load in HIV-infected immigrants living in Spain, one third of whom carried non-B subtypes.
Identification of Strains of
Alcaligenes
and
Agrobacterium
by a Polyphasic Approach
Authors: Clermont Dominique , Harmant Christine , Bizet Chantal
Published: July 2002

The number of stable discriminant biochemical characters is limited in the genera
Alcaligenes
and
Agrobacterium
, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example,
Alcaligenes xylosoxidans
was transferred to the genus
Achromobacter
in 1998. Twenty-five strains of
Achromobacter xylosoxidans
, three strains of an
Agrobacterium
sp., five strains of an
Alcaligenes
sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with
Eco
RI or
Pst
I restriction endonuclease. This polyphasic approach allowed the two subspecies of
A. xylosoxidans
to be clearly separated. Relationships between five strains and the
Ralstonia paucula
type strain were demonstrated. Likewise, three strains were found to be related to the
Ochrobactrum anthropi
type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying
A. xylosoxidans
subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing.
PCR M Typing: a New Method for Rapid Typing of Group A Streptococci
Authors: Vitali Luca A. , Zampaloni Claudia , Prenna Manuela , Ripa Sandro
Published: July 2002

A new approach for the M-typing ofStreptococcus pyogenesis reported. Oligonucleotide primers were used in a PCR to amplify the N-terminal region of theemmgene. The presence of the PCR amplification product is associated with the corresponding M serotype. This technique offers potential advantages over other molecular typing methods.
Rapid Typing of
Bordetella pertussis
Pertussis Toxin Gene Variants by LightCycler Real-Time PCR and Fluorescence Resonance Energy Transfer Hybridization Probe Melting Curve Analysis
Authors: Mäkinen Johanna , Mertsola Jussi , Viljanen Matti K. , Arvilommi Heikki , He Qiushui
Published: July 2002

A LightCycler real-time PCR hybridization probe assay was developed for rapid typing of gene variants of the
Bordetella pertussis
virulence factor pertussis toxin. The assay correctly identified the
ptxS1
alleles of all strains tested, comprising 57 Finnish clinical isolates and 2 vaccine strains. The method is simple, reliable, and suitable for large-scale screening of
B. pertussis
strains.
Comparative Study of Two Methods for RNA Extraction Prior to Detection of Resistance to Human Immunodeficiency Virus Type 1 with the Line Probe Assay
Authors: Eiros Jose María , Labayru Cristina , Hernández Beatriz , Ortiz de Lejarazu Raúl , Rodríguez Torres Antonio
Published: July 2002

We evaluated two methods from Roche and Promega for RNA extraction prior to the genotypic detection of human immunodeficiency virus type 1 resistance by line probe assay (LiPA). Fifty plasma RNA extracts were processed in parallel by LiPA. Results obtained by the Roche method were superior in the proportion of amplified samples, the percentage of mutated samples, and band intensity.
Quantitative Analysis of the Antiviral Activity of CD8+T Cells from Human Immunodeficiency Virus-Positive Asymptomatic Patients with Different Rates of CD4+T-Cell Decrease
Authors: Salerno-Gonçalves Rosângela , Lu Wei , Andrieu Jean-Marie
Published: July 2002

We have measured in 22 asymptomatic human immunodeficiency virus type 1-infected patients (10 rapid progressors and 12 slow progressors) the proviral load of CD4+T cells homogeneously superinfected by the same dose of a non-syncytium-inducing virus in the presence or in the absence of autologous CD8+T cells. We demonstrated that the antiviral activity of CD8+T cells was highly predictive of the rate of peripheral CD4+T-cell decline.
Analysis of Gene Expression in a Human Cell Line Stably Transduced with Herpesvirus Saimiri
Authors: Hall Kersten T. , Giles Mathew S. , Goodwin Delyth J. , Calderwood Michael A. , Carr Ian M. , Stevenson Alex J. , Markham Alex F. , Whitehouse Adrian
Published: July 2002

Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus; it has significant homology to the human gammaherpesviruses Kaposi's sarcoma-associated virus and Epstein-Barr virus and the murine gammaherpesvirus murine herpesvirus 68. HVS causes a persistent asymptomatic infection in its natural host, the squirrel monkey. Both subgroups A and C possess the ability to immortalize common marmoset T lymphocytes to interleukin-2-independent proliferation. However, only subgroup C is capable of transforming human, rabbit, and rhesus monkey lymphocytes in vitro. In addition, HVS can stably transduce a variety of human cell lines where the virus persists as a nonintegrating circular episome. In this study, we have developed a system in which the HVS DNA is stably maintained as a nonintegrated circular episome in the human lung carcinoma cell line A549. Virus production can be reactivated using chemical inducing agents, including tetradecanoyl phorbol acetate andn-butyrate, suggesting that the infection in human A549 cells is latent. To analyze virus gene expression in these stably transduced cells, Northern blot analysis was performed using a series of probes produced from restriction fragments spanning the entire coding region of the HVS genome. This demonstrated that an adjacent set of genes containing open reading frames (ORFs) 71 to 73 are expressed in this stably transduced cell line. Moreover, these genes are transcribed as a polycistronic mRNA species produced from a common promoter upstream of ORF 73. This model may serve as a useful tool in the further analysis of the role of ORFs 71 to 73 in gamma-2 herpesvirus latency.
Determination of the Frequency of Retroviral Recombination between Two Identical Sequences within a Provirus
Authors: Li Ting , Zhang Jiayou
Published: July 2002

Retroviruses use RNA as their genetic material within viral particles and DNA (provirus) as their genetic material within cells. The rate of recombination during reverse transcription between two identical sequences within the same RNA molecule is very high. In this study, we have developed a sensitive system to study recombination occurring within the proviral sequence. This system includes a murine Moloney leukemia virus vector which contains a neomycin resistance gene (
neo
) and two mutated green fluorescent protein genes (
gfp
) in tandem positions. The 3′ end of the first
gfp
and the 5′ end of the second
gfp
gene are both mutated, so that neither of these two
gfp
genes is functional. However, if recombination occurs between the two
gfp
genes it will create a functional
gfp
protein. Cells containing such a functional recombinant
gfp
appear green under fluorescence microscopy. The rate of recombination between the two
gfp
sequences during a single round of replication is as high as 51%. Green cells appear during proliferation of a clonal clear-cell population and allow a small portion of these recombinations between sequences of proviral DNA to be detected. The frequency of recombination at the proviral DNA level is about 10
−5
events/cell division, which is very low compared with the frequency of recombination (51%) caused by reverse transcriptase and/or RNA polymerase II.
Moloney Murine Leukemia Virus-Induced Tumors Show Altered Levels of Proapoptotic and Antiapoptotic Proteins
Authors: Bonzon Christine , Fan Hung
Published: July 2002

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphomas when inoculated into neonatal mice. The tumor cells are typically derived from immature T cells. During preleukemic times, a marked decrease in thymic size is apparent in M-MuLV-inoculated mice. We previously demonstrated that this thymic regression is correlated with enhanced levels of thymocyte apoptosis (C. Bonzon and H. Fan, J. Virol. 73:2434-2441, 1999). In this study, we investigated the apoptotic state of M-MuLV-induced tumors. M-MuLV-induced tumors were screened for expression of the apoptotic proteins Fas and Bcl-2 by three-color flow cytometric analysis. Single-positive (SP; CD4+CD8−and CD4−CD8+) tumor cells generally displayed lower cell surface expression of Fas than SP thymocytes from uninoculated control mice. Double-positive (DP; CD4+CD8+) M-MuLV-induced tumor cells fell into two categories: those with normal high levels of Fas and those with low levels of Fas. Additionally, the vast majority of DP tumors showed elevated Bcl-2 levels. The DP tumor cells retaining normal/high Fas expression were capable of transducing an apoptotic signal upon anti-Fas engagement. In addition, DP and CD4+SP tumor populations displayed higher levels of Fas ligand than normal thymocytes with the same phenotypes. In contrast, CD8+SP and CD4−CD8−tumors did not show elevated Fas ligand expression. There was no significant correlation between Fas and Fas ligand expression in the DP tumors, suggesting that Fas Ligand expression was not the driving force behind Fas down-regulation. These results suggest that both the Fas death receptor and mitochondrial pathways of apoptotic death are active in M-MuLV-induced tumors and that they must be modulated to permit cell survival and tumor outgrowth.
Anti-Feline Immunodeficiency Virus (FIV) Soluble Factor(s) Produced from Antigen-Stimulated Feline CD8+T Lymphocytes Suppresses FIV Replication
Authors: Choi In-Soo , Hokanson Regina , Collisson Ellen W.
Published: July 2002

Feline immunodeficiency virus (FIV) causes AIDS-like symptoms in infected cats. Concanavalin A (ConA)-stimulated peripheral blood mononuclear cells (PBMC) from chronically FIV strain PPR-infected cats readily expressed FIV. In contrast, when PBMC from these animals were stimulated with irradiated, autologous antigen-presenting cells (APC), at least a 10-fold drop in viral production was observed. In addition to FIV-specific cytotoxic T lymphocytes, anti-FIV activity was demonstrated in the cell-free supernatants of effector T lymphocytes stimulated with APC. The FIV-suppressive activity was induced from APC-stimulated PBMC of either FIV-infected or uninfected cats but not from ConA-stimulated PBMC. Suppression of FIV strain PPR replication was observed for both autologous and heterologous feline PBMC, was dose dependent, and demonstrated cross-reactivity and cell specificity. It was also demonstrated that the anti-FIV activity originated from CD8+T lymphocytes and was mediated by a noncytolytic mechanism.
Measles Virus-Induced Disruption of the Glial-Fibrillary-Acidic Protein Cytoskeleton in an Astrocytoma Cell Line (U-251)
Authors: Duprex W. Paul , McQuaid Stephen , Rima Bert K.
Published: July 2002

A recombinant measles virus which expresses enhanced green fluorescent protein (MVeGFP) has been used to infect two astrocytoma cell lines (GCCM and U-251) to study the effect of virus infection on the cytoskeleton. Indirect immunocytochemistry was used to demonstrate the cellular localization of the cytoskeletal components. Enhanced green fluorescent protein autofluorescence was used to identify measles virus-infected cells. No alteration of the actin, tubulin, or vimentin components of the cytoskeleton was observed in either cell type, whereas a disruption of the glial-fibrillary-acidic protein filament (GFAP) network was noted in MVeGFP-infected U-251 cells. The relative amounts of GFAP present in infected and uninfected U-251 cells were quantified by image analysis of data sets obtained by confocal microscopy by using vimentin, another intermediate filament on which MVeGFP has no effect, as a control.
The Vaccinia Virus A14.5L Gene Encodes a Hydrophobic 53-Amino-Acid Virion Membrane Protein That Enhances Virulence in Mice and Is Conserved among Vertebrate Poxviruses
Authors: Betakova Tatiana , Wolffe Elizabeth J. , Moss Bernard
Published: July 2002

A short sequence, located between the A14L and A15L open reading frames (ORFs) of vaccinia virus, was predicted to encode a hydrophobic protein of 53 amino acids that is conserved in orthopoxviruses, leporipoxviruses, yatapoxiruses, and molluscipoxviruses. We constructed a recombinant vaccinia virus with a 10-codon epitope tag appended to the C terminus of the A14.5L ORF. Synthesis of the tagged protein occurred at late times and was blocked by an inhibitor of DNA replication, consistent with regulation by a predicted late promoter just upstream of the A14.5L ORF. Hydrophobicity of the protein was demonstrated by extraction into the detergent phase of Triton X-114. The protein was associated with purified vaccinia virus particles and with membranes of immature and mature virions that were visualized by electron microscopy of infected cells. Efficient release of the protein from purified virions occurred after treatment with a nonionic detergent and reducing agent. A mutant virus, in which the A14.5L ORF was largely deleted, produced normal-size plaques in several cell lines, and the yields of infectious intra- and extracellular viruses were similar to those of the parent. In contrast, with a mouse model, mutant viruses with the A14.5L ORF largely deleted were attenuated relative to that of the parental virus or a mutant virus with a restored A14.5L gene.
An Epstein-Barr Virus Protein Interacts with Notch
Authors: Kusano Shuichi , Raab-Traub Nancy
Published: July 2002

The Epstein-Barr virus (EBV)
Bam
HI A mRNAs were originally identified in cDNA libraries from nasopharyngeal carcinoma, where they are expressed at high levels. The RNAs are differentially spliced to form several open reading frames and also contain the BARF0 open reading frame at the 3′ end. One cDNA, RK-BARF0, included a potential endoplasmic reticulum-targeting signal peptide sequence. The RK-BARF0 protein is shown here to interact with the Notch4 ligand binding domain, using yeast two-hybrid screening, coimmunoprecipitation, and confocal microscopy. This interaction induces translocation of a portion of the full-length unprocessed Notch4 to the nucleus by using the Notch nuclear localization signal. These effects of RK-BARF0 on Notch intracellular location indicate that EBV possibly modulates Notch signaling. Unprocessed Notch4 was also detected in immunoprecipitated complexes from EBV-infected cells by using a rabbit antiserum raised against a BARF0-specific peptide. This finding provides additional evidence for expression of RK-BARF0 and its interaction with Notch during EBV infection. In EBV-infected, EBNA2-negative cells, RK-BARF0 induced the expression of EBV latent membrane protein 1 (LMP1), and this induction was dependent on the RK-BARF0/Notch interaction domain. The activation of LMP1 expression by RK-BARF0 may be responsible for expression of LMP1 in EBV latent infections in the absence of EBNA2.
Nonspecific Down-Regulation of CD8+T-Cell Responses in Mice Expressing Human Papillomavirus Type 16 E7 Oncoprotein from the Keratin-14 Promoter
Authors: Tindle Robert W. , Herd Karen , Doan Tracy , Bryson Greg , Leggatt Graham R. , Lambert Paul , Frazer Ian H. , Street Michael
Published: July 2002

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-14 (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8+cells is down-regulated compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter. Down-regulation did not involve deletion of CD8+T cells of high affinity or high avidity, and T-cell receptor (TCR) Vβ-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge. We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T. Doan et al., J. Virol. 73:6166–6170, 1999). The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.
Heparin Inhibits Retrovirus Binding to Fibronectin as Well as Retrovirus Gene Transfer on Fibronectin Fragments
Authors: Carstanjen D. , Dutt P. , Moritz T.
Published: July 2002

Fibronectin fragments have been shown to improve retrovirus gene transfer efficiency by binding retrovirus and target cells. Using a novel virus adhesion assay, we confirmed binding of type C oncoretrovirus vectors to the heparin II domain of fibronectin and demonstrated inhibition of viral binding and gene transfer by heparin.
Identification and Characterization of a Spliced C-Type Lectin-Like Gene Encoded by Rat Cytomegalovirus
Authors: Voigt Sebastian , Sandford Gordon R. , Ding Lijun , Burns William H.
Published: July 2002

The English isolate of rat cytomegalovirus (RCMV) encodes a 20-kDa protein with a C-type lectin-like domain that is expressed in the delayed-early and late phases of the viral replication cycle. Genomic sequence analysis of the restriction fragmentKpnR of RCMV revealed significant homology to several C-type lectin-containing molecules implicated in natural killer (NK) and T-cell interactions, as well as genes from four poxviruses and African swine fever virus. The gene is spliced into five exons and shows a splicing pattern with exon boundaries similar to those observed in the human differentiation antigen CD69. The cap site of the gene was mapped by RNase protection, 5′ rapid amplification of cDNA ends, and primer extension experiments. This analysis demonstrated that the core promoter of the RCMV lectin-like gene contains a GATA rather than a TATA box. Splicing patterns were confirmed with isolates from an infected-cell cDNA library. A unique aspect of the protein is that its translation is not initiated by the canonical methionine but rather by alanine. To study its role in virus replication and pathogenesis, a recombinant virus was constructed in which the gene is interrupted. Replication in tissue culture was similar to that of wild-type virus.
Donor- and Ligand-Dependent Differences in C-C Chemokine Receptor 5 Reexpression
Authors: Sabbe Rebecca , Picchio Gastón R. , Pastore Cristina , Chaloin Olivier , Hartley Oliver , Offord Robin , Mosier Donald E.
Published: July 2002

N-terminal modifications of the chemokine RANTES bind to C-C chemokine receptor 5 (CCR5) and block human immunodeficiency virus type 1 (HIV-1) infection with greater efficacy than native RANTES. Modified RANTES compounds induce rapid CCR5 internalization and much slower receptor reexpression than native RANTES, suggesting that receptor sequestration is one mode of anti-HIV activity. The rates of CCR5 internalization and reexpression were compared using the potentn-nonanoyl (NNY)-RANTES derivative and CD4+T cells derived from donors with different CCR5 gene polymorphisms. NNY-RANTES caused even more rapid receptor internalization and slower reexpression than aminooxypentane (AOP)-RANTES. Polymorphisms in the promoter and coding regions of CCR5 significantly affected the receptor reexpression rate after exposure of cells to NNY-RANTES. These observations may be relevant for understanding the protective effects of different CCR5 genotypes against HIV-1 disease progression.
Protective Mucosal Immunity to Ocular Herpes Simplex Virus Type 1 Infection in Mice by UsingEscherichia coliHeat-Labile Enterotoxin B Subunit as an Adjuvant
Authors: Richards C. M. , Aman A. T. , Hirst T. R. , Hill T. J. , Williams N. A.
Published: July 2002

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relativeEscherichia coliheat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 μg of rEtxB or above with 10 μg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 μg) with a trace (0.5 μg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 μg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.
Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway
Authors: Brunori Michele , Malerba Maddalena , Kashiwazaki Haruhiko , Iggo Richard
Published: July 2002

Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and β-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable β-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.