bluuebunny/crossref_metadata_2025 huggingface dataset:
One of Two Copies of the Gene for the Activatable Shiga Toxin Type 2d in
Escherichia coli
O91:H21 Strain B2F1 Is Associated with an Inducible Bacteriophage
Authors: Teel Louise D. , Melton-Celsa Angela R. , Schmitt Clare K. , O'Brien Alison D.
Published: July 2002

Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing
Escherichia coli
(STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only
stx
2e
from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an
stx
2d1
-containing lysogen was isolated from plaques on strain DH5α that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the
stx
2d1
- and
stx
2d2
-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.
Identification of Amino Acid Residues of Anthrax Protective Antigen Involved in Binding with Lethal Factor
Authors: Chauhan Vibha , Bhatnagar Rakesh
Published: July 2002

Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for the translocation of LF to the cytosol. The binding of LF to cell surface receptor-bound PA is a prerequisite for the formation of lethal toxin. It has been hypothesized that hydrophobic residues P184, L187, F202, L203, P205, I207, I210, W226, and F236 of domain 1b of PA play an important role in the binding of PA to LF. These residues are normally buried in the 83-kDA version of PA, PA83, as determined by the crystal structure of PA. However, they become exposed due to the conformational change brought about by the cleavage of PA83 to PA63 by a cell surface protease. Mutation of the above-mentioned residues to alanine resulted in mutant proteins that were able to bind to the cell surface receptors and also to be specifically cleaved by the cellular proteases. All the mutant proteins except the F202A, L203A, P205A, and I207A mutants were able to bind to LF and were also toxic to macrophage cells in combination with LF. It was concluded that residues 202, 203, 205, and 207 of PA are essential for the binding of LF to PA.
Patterns of Epithelial Cell Invasion by Different Species of the
Burkholderia cepacia
Complex in Well-Differentiated Human Airway Epithelia
Authors: Schwab Ute , Leigh Margaret , Ribeiro Carla , Yankaskas James , Burns Kimberly , Gilligan Peter , Sokol Pamela , Boucher Richard
Published: July 2002

Burkholderia cepacia
has emerged as a serious respiratory pathogen in cystic fibrosis (CF) patients. The clinical course of
B. cepacia
infections is variable, but ∼20% of patients eventually succumb to the cepacia syndrome, which is characterized as a fatal necrotizing pneumonia with bacteremia. The mechanisms that permit
B. cepacia
to cause bacteremia are not yet known but probably involve sequential penetration of airway barriers. This study evaluated the abilities of different species of the
B. cepacia
complex, including a strain from the ET12 lineage (BC-7, genomovar III,
cblA
+
), which is associated with most cepacia syndrome fatalities among CF populations, a genomovar IV strain (HI2258), and a genomovar II strain (J-1) to penetrate polarized, well-differentiated human airway epithelial cell cultures. As revealed by light and electron microscopy, all three
B. cepacia
strains tested circumvented the mechanical barriers of mucus and ciliary transport to penetrate the airway epithelium but they used different routes. The BC-7 strain (genomovar III) formed biofilms in close proximity to the apical cell surface, followed by invasion and destruction of epithelial cells. This process involved disruption of the glycocalyx and rearrangements of the actin cytoskeleton. The HI2258 strain (genomovar IV) did not form biofilms, and the majority of bacteria that penetrated the epithelium were located between epithelial cells, suggesting paracytosis. Strain J-1 penetrated the epithelium both by cell destruction and paracytosis. These studies suggest that the distinct invasion pathways employed by
B. cepacia
may account for differences in virulence between
B. cepacia
genomovars.
CD40 Ligand (CD154) Does Not Contribute to Lymphocyte-Mediated Inhibition of VirulentMycobacterium tuberculosiswithin Human Monocytes
Authors: Larkin Rhonda , Benjamin Christopher D. , Hsu Yen-Ming , Li Qing , Zukowski Lynn , Silver Richard F.
Published: July 2002

Human monocytes displayed increased expression of CD40 following infection with virulentMycobacterium tuberculosis. Nevertheless, soluble CD40 ligand (CD40L; also designated CD154) had no effect on the intracellular growth of the organism. Restriction of the intracellular growth ofM. tuberculosisby peripheral blood lymphocytes and antigen-specific CD4+T-cell lines likewise was not reduced by blocking anti-CD40L monoclonal antibody 5c8.
Analysis of DNA Regulatory Elements Required for Expression of theLegionella pneumophilaicmanddotVirulence Genes
Authors: Gal-Mor Ohad , Zusman Tal , Segal Gil
Published: July 2002

To investigate the regulation of theLegionella pneumophila icmanddotgenes required for intracellular growth, a series of nineicm::lacZfusions were constructed. Theseicm::lacZfusions were found to have different levels of expression inL. pneumophila, and five of them were more highly expressed at stationary phase than at exponential phase. When the expression of these fusions inEscherichia coliwas tested, all of them were found to be expressed but three of them had dramatic changes in their levels of expression in comparison to those inL. pneumophila. Site-directed and PCR random mutagenesis with theseicm::lacZfusions was used to identify DNA regulatory elements oficmgenes. Fouricmgenes (icmT,icmP,icmQ, andicmM) that had low levels of expression inL. pneumophilawere found to contain a 6-bp sequence (TATACT) essential for their expression. This sequence was shown by primer extension to serve as their −10 promoter elements. A similar sequence, which constitutes the −10 promoter elements of theicmV,icmW, andicmRgenes which had high levels of expression inL. pneumophila, was also identified. In addition, regulatory elements that probably serve as binding sites for transcription regulators were found in these genes. Altogether, 12 regulatory elements, 7 of which constitute the −10 promoter elements of theicmgenes, were found. Even though all theicmanddotgenes are part of one system required forL. pneumophilaintracellular growth and even though their promoters are probably recognized by the vegetative sigma factor, it seems that they are subjected to different regulation mediated by several regulatory factors.
Mutations in the CCGTTCACA DnaA Box of
Mycobacterium tuberculosis oriC
That Abolish Replication of
oriC
Plasmids Are Tolerated on the Chromosome
Authors: Dziadek Jaroslaw , Rajagopalan Malini , Parish Tanya , Kurepina Natalia , Greendyke Rebecca , Kreiswirth Barry N. , Madiraju Murty V. V. S.
Published: July 2002

The origin of replication (
oriC
) region in some clinical strains of
Mycobacterium tuberculosis
is a hot spot for IS
6110
elements. To understand how clinical strains with insertions in
oriC
can replicate their DNA, we characterized the
oriC
regions of some clinical strains. Using a plasmid-based
oriC
-dependent replication assay, we showed that IS
6110
insertions that disrupted the DnaA box sequence CCGTTCACA abolished
oriC
activity in
M. tuberculosis
. Furthermore, by using a surface plasmon resonance technique we showed that purified
M. tuberculosis
DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of
oriC
plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain
M. tuberculosis
H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that
M. tuberculosis
strains have evolved mechanisms to tolerate mutations in the
oriC
region and that functional requirements for
M. tuberculosis oriC
replication are different for chromosomes and plasmids.
Identification of a New Class of Cytochrome P450 from a
Rhodococcus
sp
Authors: Roberts Gareth A. , Grogan Gideon , Greter Andy , Flitsch Sabine L. , Turner Nicholas J.
Published: July 2002

A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from
Rhodococcus
sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in
Escherichia coli
, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of
E. coli
was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.
An AraC/XylS Family Member at a High Level in a Hierarchy of Regulators for Phenol-Metabolizing Enzymes inComamonas testosteroniR5
Authors: Teramoto Maki , Ohnishi Kouhei , Harayama Shigeaki , Watanabe Kazuya
Published: July 2002

Comamonas testosteronistrain R5 expresses a higher level of phenol-oxygenating activity than any other bacterial strain so far characterized. The expression of the operon encoding multicomponent phenol hydroxylase (mPH), which is responsible for the phenol-oxygenating activity, is controlled by two transcriptional regulators, PhcS and PhcR, in strain R5. In this study, we identified a third transcriptional regulator for the mPH operon (PhcT) that belongs to the AraC/XylS family. While the disruption ofphcTin strain R5 significantly reduced the expression of the mPH operon, it did not eliminate the expression. However, the disruption ofphcTin strain R5 increased the expression ofphcR. The phenol-oxygenating activity was abolished by the disruption ofphcR, indicating that PhcT alone was not sufficient to activate the expression of the mPH operon. The disruption ofphcShas been shown in our previous study to confer the ability of strain R5 to express the mPH operon in the absence of the genuine substrate for mPH. PhcT was not involved in the gratuitous expression. Strain R5 thus possesses a more elaborate mechanism for regulating the mPH operon expression than has been found in other bacteria.
Enterococcus faecalis
3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis
Authors: Sutherlin Autumn , Hedl Matija , Sanchez-Neri Barbara , Burgner John W. , Stauffacher Cynthia V. , Rodwell Victor W.
Published: July 2002

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and
Enterococcus faecalis
genomic DNA to isolate the
mvaS
gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway.
mvaS
was expressed in
Escherichia coli
from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni
2+
-agarose to apparent homogeneity and a specific activity of 10 μmol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa;
s

20,
w

, 5.3). Optimal activity occurred in 2.0 mM MgCl
2
at 37
o
C. The ΔH
a
was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK
a
of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 ± 0.2 and that of covalent acetylation was 0.60 ± 0.02. The

K
m

for the hydrolysis of acetyl-CoA was 10 μM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from
E. faecalis
.
The RuvABC Resolvase Is Indispensable for Recombinational Repair in
sbcB15
Mutants of
Escherichia coli
Authors: Zahradka Davor , Zahradka Ksenija , Petranović Mirjana , Đermić Damir , Brčić-Kostić Krunoslav
Published: July 2002

The RuvABC proteins of
Escherichia coli
play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the
ruv
genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a
recBC sbcBC
background. Genetic analysis presented in this work revealed that the Δ
ruvABC
mutation causes an identical DNA repair defect in UV-irradiated
recBC sbcBC
,
sbcBC
, and
sbcB
strains, indicating that the
sbcB
mutation alone is responsible for the extreme UV sensitivity of
recBC sbcBC ruv
derivatives. In experiments with gamma irradiation and in conjugational crosses, however,
sbcBC
Δ
ruvABC
and
sbcB
Δ
ruvABC
mutants displayed higher recombination proficiency than the
recBC sbcBC
Δ
ruvABC
strain. The frequency of conjugational recombination observed with the
sbcB
Δ
ruvABC
strain was quite similar to that of the Δ
ruvABC
single mutant, indicating that the
sbcB
mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.
The Unique
tuf2
Gene from the Kirromycin Producer
Streptomyces ramocissimus
Encodes a Minor and Kirromycin-Sensitive Elongation Factor Tu
Authors: Olsthoorn-Tieleman Lian N. , Fischer Sylvia E. J. , Kraal Barend
Published: July 2002

Streptomyces ramocissimus
, the producer of elongation factor Tu (EF-Tu)-targeted antibiotic kirromycin, contains three divergent
tuf
-like genes, with
tuf1
encoding regular kirromycin-sensitive EF-Tu1; the functions of
tuf2
and
tuf3
are unknown. Analysis of the
tuf
gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of
tuf1
and sometimes of
tuf3
but that
tuf2
was found in
S. ramocissimus
only. The
tuf2
-flanking regions were sequenced, and the two
tuf2
-surrounding open reading frames were shown to be oriented in opposite directions. In vivo transcription analysis of the
tuf2
gene displayed an upstream region with bidirectional promoter activity. The transcription start site of
tuf2
was located approximately 290 nucleotides upstream of the coding sequence. Very small amounts of
tuf2
transcripts were detected in both liquid- and surface-grown cultures of
S. ramocissimus
, consistent with the apparent absence of EF-Tu2 in total protein extracts. The
tuf2
transcript level was not influenced by the addition of kirromycin to exponentially growing cultures. To assess the function of
S. ramocissimus
EF-Tu2, the protein was overexpressed in
Streptomyces coelicolor
LT2. This strain is a J1501 derivative containing His
6
-tagged EF-Tu1 as the sole EF-Tu species, which facilitated the separation of EF-Tu2 from the interfering EF-Tu1.
S. ramocissimus
EF-Tu1 and EF-Tu2 were indistinguishable in their ability to stimulate protein synthesis in vitro and exhibited the same kirromycin sensitivity, which excludes the possibility that EF-Tu2 is directly involved in the kirromycin resistance mechanism of
S. ramocissimus
.
The Chloramphenicol-Inducible
catB
Gene in
Agrobacterium tumefaciens
Is Regulated by Translation Attenuation
Authors: Rogers Elizabeth J. , Rahman M. Sayeedur , Hill Russell T. , Lovett Paul S.
Published: July 2002

Agrobacterium tumefaciens
strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the
catB
gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the
catB
gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible
catA
genes resident in gram-positive bacteria and the inducible
cmlA
gene that confers chloramphenicol resistance in
Pseudomonas
spp.
Minimizing the Workup of Blood Culture Contaminants: Implementation and Evaluation of a Laboratory-Based Algorithm
Authors: Richter S. S. , Beekmann S. E. , Croco J. L. , Diekema D. J. , Koontz F. P. , Pfaller M. A. , Doern G. V.
Published: July 2002

An algorithm was implemented in the clinical microbiology laboratory to assess the clinical significance of organisms that are often considered contaminants (coagulase-negative staphylococci, aerobic and anaerobic diphtheroids,
Micrococcus
spp.,
Bacillus
spp., and viridans group streptococci) when isolated from blood cultures. From 25 August 1999 through 30 April 2000, 12,374 blood cultures were submitted to the University of Iowa Clinical Microbiology Laboratory. Potential contaminants were recovered from 495 of 1,040 positive blood cultures. If one or more additional blood cultures were obtained within ±48 h and all were negative, the isolate was considered a contaminant. Antimicrobial susceptibility testing (AST) of these probable contaminants was not performed unless requested. If no additional blood cultures were submitted or there were additional positive blood cultures (within ±48 h), a pathology resident gathered patient clinical information and made a judgment regarding the isolate's significance. To evaluate the accuracy of these algorithm-based assignments, a nurse epidemiologist in approximately 60% of the cases performed a retrospective chart review. Agreement between the findings of the retrospective chart review and the automatic classification of the isolates with additional negative blood cultures as probable contaminants occurred among 85.8% of 225 isolates. In response to physician requests, AST had been performed on 15 of the 32 isolates with additional negative cultures considered significant by retrospective chart review. Agreement of pathology resident assignment with the retrospective chart review occurred among 74.6% of 71 isolates. The laboratory-based algorithm provided an acceptably accurate means for assessing the clinical significance of potential contaminants recovered from blood cultures.
Ralstonia pickettii
Traced in Blood Culture Bottles
Authors: Boutros Névine , Gonullu Nevriye , Casetta Anne , Guibert Michèle , Ingrand Didier , Lebrun Léa
Published: July 2002

Over a 9-month period, 14 strains of
Ralstonia pickettii
were isolated from various biological samples inoculated in a blood culture medium. Molecular epidemiological investigation confirmed the relatedness of the strains. The source of the contamination proved to be the blood culture bottle caps.
Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins
Authors: Buonocore Linda , Blight Keril J. , Rice Charles M. , Rose John K.
Published: July 2002

We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These ΔG viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.
Analysis of a Temperature-Sensitive Mutant Rotavirus Indicates that NSP2 Octamers Are the Functional Form of the Protein
Authors: Taraporewala Zenobia F. , Schuck Peter , Ramig Robert F. , Silvestri Lynn , Patton John T.
Published: July 2002

Evidence that NSP2 plays a role in packaging and replication comes from studies on
tsE
(
1400
), a rotavirus mutant with a temperature-sensitive (
ts
) lesion in the NSP2 gene. Cells infected with
tsE
and maintained at nonpermissive temperature contain few replication-assembly factories (viroplasms) or replication intermediates and produce virus particles that are mostly empty. Sequence analysis has indicated that an A152V mutation in NSP2 is responsible for the
ts
phenotype of
tsE
. To gain insight into the effect of the mutation on the octameric structure and biochemical activities of
tsE
NSP2, the protein was expressed in bacteria and purified to homogeneity. Analytical ultracentrifugation showed that
tsE
NSP2 formed octamers which, like those formed by wild-type (
wt
) NSP2, undergo conformational change into more compact structures upon binding of nucleotides. However, exposure to Mg
2+
and the nonpermissive temperature caused disruption of the
tsE
octamers and yielded the formation of polydisperse NSP2 aggregates, events not observed with
wt
octamers. Biochemical analysis showed that the RNA-binding, helix-destabilizing and NTPase activities of
tsE
NSP2 were significantly less at the nonpermissive temperature than at the permissive temperature. In contrast, these activities for
wt
NSP2 were higher at the nonpermissive temperature. Our results indicate that the octamer is the fully functional form of NSP2 and the form required for productive virus replication. The propensity of
tsE
NSP2 to form large aggregates provides a possible explanation for the inability of the protein to support packaging and/or replication in the infected cell at the nonpermissive temperature.
Herpes Simplex Virus Type 1/Adeno-Associated Virus
rep
+
Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration
Authors: Wang Y. , Camp S. M. , Niwano M. , Shen X. , Bakowska J. C. , Breakefield X. O. , Allen P. D.
Published: July 2002

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A
rep
−
HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a
rep
+
HSV/AAV hybrid amplicon was made by inserting
rep68
/
78
outside the
rep
−
vector 3′ AAV ITR sequence. Both vectors also had a pair of
loxP
sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The
rep
+
, but not the
rep
−
, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the
rep
+
hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-
loxP
recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.
RNA 3′ Readthrough of Oncoretrovirus and Lentivirus: Implications for Vector Safety and Efficacy
Authors: Zaiss Anne-Kathrin , Son Sodany , Chang Lung-Ji
Published: July 2002

The expression of reporter genes driven by the same human elongation factor 1α (EF1α) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1α intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1α transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1α intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3′-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3′ polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.
Characterization of Varicella-Zoster Virus Gene 21 and 29 Proteins in Infected Cells
Authors: Cohrs Randall J. , Wischer Jeanne , Essman Carrie , Gilden Donald H.
Published: July 2002

Varicella-zoster virus (VZV) transcription is limited in latently infected human ganglia. Note that much of the transcriptional capacity of the virus genome has not been analyzed in detail; to date, only VZV genes mapping to open reading frames (ORFs) 4, 21, 29, 62, and 63 have been detected. ORF 62 encodes the major immediate-early virus transcription transactivator IE62, ORF 29 encodes the major virus DNA binding protein, and ORF 21 encodes a protein associated with the developing virus nucleocapsid. We analyzed the cellular location of proteins encoded by ORF 21 (21p) and ORF 29 (29p), their phosphorylation state during productive infection, and their ability form a protein-protein complex. The locations of both 21p and 29p within infected cells mimic those of their herpes simplex virus type 1 (HSV-1) homologues (UL37 and ICP8); however, unlike these homologues, 21p is not phosphorylated and neither 21p nor 29p exhibits a protein-protein interaction. Transient transfection assays to determine the effect of 21p and 29p on transcription from VZV gene 20, 21, 28, and 29 promoters revealed no significant activation of transcription by 21p or 29p from any of the VZV gene promoters tested, and 21p did not significantly modulate the ability of IE62 to activate gene transcription. A modest increase in IE62-induced activation of gene 28 and 29 promoters was seen in the presence of 29p; however, IE62-induced activation of gene 28 and 29 promoters was reduced in the presence of 21p. A
Saccharomyces cerevisiae
two-hybrid analysis of 21p indicated that the protein can activate transcription when tethered within a responsive promoter. Together, the data reveal that while VZV gene 21 and HSV-1 UL37 share homology at the nucleic acid level, these proteins differ functionally.
Human Immunodeficiency Virus-Specific CD8+T Cells in Human Breast Milk
Authors: Sabbaj Steffanie , Edwards Bradley H. , Ghosh Mrinal K. , Semrau Katherine , Cheelo Sanford , Thea Donald M. , Kuhn Louise , Ritter G. Douglas , Mulligan Mark J. , Goepfert Paul A. , Aldrovandi Grace M.
Published: July 2002

Breast-feeding infants of human immunodeficiency virus (HIV)-infected women ingest large amounts of HIV, but most escape infection. While the factors affecting transmission risk are poorly understood, HIV-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in controlling HIV levels in blood. We therefore investigated the ability of breast milk cells (BMC) from HIV-infected women from the United States and Zambia to respond to HIV-1 peptides in a gamma interferon enzyme-linked immunospot assay. All (n= 11) HIV-infected women had responses to pools of Gag peptide (range, 105 to 1,400 spot-forming cells/million; mean = 718), 8 of 11 reacted to Pol, 7 reacted to Nef, and 2 of 5 reacted to Env. Conversely, of four HIV-negative women, none responded to any of the tested HIV peptide pools. Depletion and tetramer staining studies demonstrated that CD8+T cells mediated these responses, and a chromium-release assay showed that these BMC were capable of lysing target cells in an HIV-specific manner. These data demonstrate the presence of HIV-specific major histocompatibility complex class I-restricted CD8+CTLs in breast milk. Their presence suggests a role in limiting transmission and provides a rationale for vaccine strategies to enhance these responses.
Addition of Exogenous Protease Facilitates Reovirus Infection in Many Restrictive Cells
Authors: Golden Joseph W. , Linke Jessica , Schmechel Stephen , Thoemke Kara , Schiff Leslie A.
Published: July 2002

Virion uncoating is a critical step in the life cycle of mammalian orthoreoviruses. In cell culture, and probably in extraintestinal tissues in vivo, reovirus virions undergo partial proteolysis within endosomal or/or lysosomal compartments. This process converts the virion into a form referred to as an intermediate subvirion particle (ISVP). In natural enteric reovirus infections, proteolytic uncoating takes place extracellularly within the intestinal lumen. The resultant proteolyzed particles, unlike intact virions, have the capacity to penetrate cell membranes and thereby gain access to cytoplasmic components required for viral gene expression. We hypothesized that the capacity of reovirus outer capsid proteins to be proteolyzed is a determinant of cellular host range. To investigate this hypothesis, we asked if the addition of protease to cell culture medium would expand the range of cultured mammalian cell lines that can be productively infected by reoviruses. We identified many transformed and nontransformed cell lines, as well as primary cells, that restrict viral infection. In several of these restrictive cells, virion uncoating is inefficient or blocked. Addition of proteases to the cell culture medium generates ISVP-like particles and promotes viral growth in nearly all cell lines tested. Interestingly, we found that some cell lines that restrict reovirus uncoating still express mature cathepsin L, a lysosomal protease required for virion disassembly in murine L929 cells. This finding suggests that factors in addition to cathepsin L are required for efficient intracellular proteolysis of reovirus virions. Our results demonstrate that virion uncoating is a critical determinant of reovirus cellular host range and that many cells which otherwise support productive reovirus infection cannot efficiently mediate this essential early step in the virus life cycle.
Intersubtype Human Immunodeficiency Virus Type 1 Superinfection following Seroconversion to Primary Infection in Two Injection Drug Users
Authors: Ramos Artur , Hu Dale J. , Nguyen Lily , Phan Kim-Oanh , Vanichseni Suphak , Promadej Nattawan , Choopanya Kachit , Callahan Margaret , Young Nancy L. , McNicholl Janet , Mastro Timothy D. , Folks Thomas M. , Subbarao Shambavi
Published: July 2002

In this study, we describe two cases of human immunodeficiency virus type 1 (HIV-1) intersubtype superinfection with CRF01_AE and subtype B strains, which occurred in two injection drug users participating in a prospective cohort study in Bangkok, Thailand. In both cases, the superinfecting strain was detected by molecular and serologic analyses several weeks after complete seroconversion to the primary infection with a strain belonging to a different subtype. Superinfection occurred despite specific T-cell and humoral antibody responses to the primary virus. In both cases, cross-subtype immune responses were limited or absent prior to the second infection. These data show that, in some individuals, the quality and quantity of the immune response elicited by primary HIV-1 infection may not protect against superinfection. This finding has important implications for vaccine design. HIV-1 vaccines, at a minimum, will need to include potent, broadly protective, conserved immunogens derived from several group M subtypes.
Identification of an Upstream Sequence Element Required for Vesicular Stomatitis Virus mRNA Transcription
Authors: Hinzman Edward E. , Barr John N. , Wertz Gail W.
Published: July 2002

Vesicular stomatitis virus (VSV), the prototypic rhabdovirus, has a nonsegmented negative-sense RNA genome with five genes flanked by 3′ leader and 5′ trailer sequences. Transcription of VSV mRNAs is obligatorily sequential, starting from a single 3′ polymerase entry site, and termination of an upstream mRNA is essential for transcription of a downstream gene.
cis
-acting signals for transcription of VSV mRNAs are present within the leader region, at the leader-N junction, and at the internal gene junctions. The gene junctions of VSV consist of a conserved 23-nucleotide region that includes the gene end sequence of the upstream gene, 3′-AUACU7-5′, a nontranscribed intergenic dinucleotide, 3′-G/CA-5′, and the gene start sequence, 3′-UUGUCNNUAG-5′, at the beginning of the gene immediately downstream. Previous work has shown that the gene end sequence and intergenic region are sufficient to signal polyadenylation and termination of VSV transcripts. Mutagenesis of the gene start sequence has determined the importance of this region in the processes of initiation and 5′-end modification of mRNAs. However, because the gene end sequence is positioned directly upstream of the gene start sequence in the gene junction, and because of the requirement for termination of the upstream gene prior to transcription of the downstream gene, it has not been possible to investigate whether the gene end sequence contributes to transcription of the downstream gene. In this study, we inserted an additional gene end sequence upstream of the gene junction in a subgenomic replicon of VSV, which extended the intergenic region from 2 to 88 nucleotides. This duplication of termination signals allowed us to separate the signals required for termination from those required for initiation. We investigated the effect that the upstream gene end sequences had on downstream mRNA transcription. Our data show that the U7 tract of the upstream gene end sequence is necessary for optimal transcription of the downstream gene, independent of its role in termination of the upstream gene. Altering the sequence or changing the length of the U tract directly upstream of the gene start sequence significantly decreased transcription of the downstream gene. These results show that the U tract is a multifunctional region that is required not only for polyadenylation and termination of the upstream mRNA but also for efficient transcription of the downstream gene.
Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein
Authors: Schülke Norbert , Vesanen Mika S. , Sanders Rogier W. , Zhu Ping , Lu Min , Anselma Deborah J. , Villa Anthony R. , Parren Paul W. H. I. , Binley James M. , Roux Kenneth H. , Maddon Paul J. , Moore John P. , Olson William C.
Published: July 2002

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1
JR-FL
SOS gp140, proteolytically uncleaved gp140 (gp140
UNC
), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140
UNC
, SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140
UNC
displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41
ECTO
) occludes the “nonneutralizing” face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140
UNC
comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140
UNC
were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140
UNC
) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.
Similarities in the Induction of Post-Golgi Vesicles by the Vaccinia Virus F13L Protein and Phospholipase D
Authors: Husain Matloob , Moss Bernard
Published: July 2002

Intracellular mature vaccinia virions are wrapped by cisternae, derived from virus-modified trans-Golgi or endosomal membranes, and then transported via microtubules to the cell periphery. Two viral proteins, encoded by the F13L and B5R open reading frames, are essential for the membrane-wrapping step. Previous transfection studies indicated that F13L induces the formation of post-Golgi vesicles that incorporate the B5R protein and that this activity depends on an intact F13L phospholipase motif. Here we show that the F13L protein has a general effect on the trafficking of integral membrane proteins from the Golgi apparatus, as both the vaccinia virus A36R protein and the vesicular stomatitis virus G protein also colocalized with the F13L protein in vesicles. In addition, increased expression of cellular phospholipase D, which has a similar phospholipase motif as, but little amino acid sequence identity with, F13L, induced post-Golgi vesicles that contained B5R and A36R proteins. Butanol-1, which prevents the formation of phosphatidic acid by phospholipase D and specifically inhibits phospholipase D-mediated vesicle formation, also inhibited F13L-induced vesicle formation, whereas secondary and tertiary alcohols had no effect. Moreover, inhibition of phospholipase activity by butanol-1 also reduced plaque size and decreased the formation of extracellular vaccinia virus without affecting the yield of intracellular mature virus. Phospholipase D, however, could not complement a vaccinia virus F13L deletion mutant, indicating that F13L has additional virus-specific properties. Taken together, these data support an important role for F13L in inducing the formation of vesicle precursors of the vaccinia virus membrane via phospholipase activity or activation.
A Chimeric Human T-Cell Lymphotropic Virus Type 1 with the Envelope Glycoprotein of Moloney Murine Leukemia Virus Is Infectious for Murine Cells
Authors: Delebecque Frédéric , Pramberger Karin , Prévost Marie-Christine , Brahic Michel , Tangy Frédéric
Published: July 2002

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4
+
T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4
+
T lymphocytes, infected by the ΔR chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Two Functional Lytic Origins of DNA Replication
Authors: AuCoin David P. , Colletti Kelly S. , Xu Yiyang , Cei Sylvia A. , Pari Gregory S.
Published: July 2002

We used a transient-transfection replication assay to identify two functional copies of the human herpesvirus 8 (HHV8) lytic origin of DNA replication (oriLyt). BCLB-1 cells were transfected with HHV8 subgenomic fragments containing the putative lytic origin along with a plasmid expressing viral transactivator open reading frame (ORF) 50. The HHV8 left-end oriLyt (oriLyt-L) lies between ORFs K4.2 and K5 and is composed of a region encoding various transcription factor binding sites and an A+T-rich region and a G+C repeat region. The right-end oriLyt (oriLyt-R) maps between ORF 69 and vFLIP, a region similar to the RRV oriLyt, and is an inverted duplication of oriLyt-L.
Comparative Sequence Analysis of the Largest E1A Proteins of Human and Simian Adenoviruses
Authors: Avvakumov Nikita , Wheeler Russ , D'Halluin Jean Claude , Mymryk Joe S.
Published: July 2002

The early region 1A (
E1A
) gene is the first gene expressed after infection with adenovirus and has been most extensively characterized in human adenovirus type 5 (hAd5). The E1A proteins interact with numerous cellular regulatory proteins, influencing a variety of transcriptional and cell cycle events. For this reason, these multifunctional proteins have been useful as tools for dissecting pathways regulating cell growth and gene expression. Despite the large number of studies using hAd5 E1A, relatively little is known about the function of the E1A proteins of other adenoviruses. In 1985, a comparison of E1A sequences from three human and one simian adenovirus identified three regions with higher overall levels of sequence conservation designated conserved regions (CR) 1, 2, and 3. As expected, these regions are critical for a variety of E1A functions. Since that time, the sequences of several other human and simian adenovirus E1A proteins have been determined. Using these, and two additional sequences that we determined, we report here a detailed comparison of the sequences of 15 E1A proteins representing each of the six hAd subgroups and several simian adenoviruses. These analyses refine the positioning of CR1, 2, and 3; define a fourth CR located near the carboxyl terminus of E1A; and suggest several new functions for E1A.
Survey of Transcript Expression in Rainbow Trout Leukocytes Reveals a Major Contribution of Interferon-Responsive Genes in the Early Response to a Rhabdovirus Infection
Authors: O'Farrell Caroline , Vaghefi Nikta , Cantonnet Monique , Buteau Bénédicte , Boudinot Pierre , Benmansour Abdenour
Published: July 2002

Virus infections induce changes in the expression of host cell genes. A global knowledge of these modifications should help to better understand the virus/host cell interactions. To obtain a more comprehensive view of the rainbow trout response to a viral infection, we used the subtractive suppressive hybridization methodology in the viral hemorrhagic septicemia model of infection. We infected rainbow trout leukocytes with viral hemorrhagic septicemia virus (VHSV), and total RNA from infected and mock-infected cells was compared at 40 h postinfection. Twenty-four virus-induced genes were ultimately retrieved from the subtracted cDNA library, and their differential expression was further confirmed by semiquantitative reverse transcription-PCR and Northern blot analysis. Among these sequences, three were already described as VHSV-induced genes. Eight sequences with known homologs were extended to full-length cDNA using 5′ and 3′ rapid amplification of cDNA ends, and they were subsequently divided into three functional subsets. Four genes were homologous to mammalian interferon responsive genes, three were similar to chemo-attractant molecules (CXC chemokine, galectin), and two had nucleic acid binding domains. All of the virus-induced genes were also induced by rainbow trout interferon, indicating that the interferon pathway is the predominant component of the anti-VHSV response. They were also expressed in vivo in experimentally infected fish, indicating their biological relevance in natural infection.