bluuebunny/crossref_metadata_2025 huggingface dataset:
Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family
Authors: Daugherty Matthew , Vonstein Veronika , Overbeek Ross , Osterman Andrei
Published: July 2002

Shikimate kinase (EC2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi‖1500322 inMethanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC2.7.1.39) of the GHMP-kinase superfamily. The latter functionality inM. jannaschiiis assigned to another gene (gi‖1591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed inEscherichia coliand purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases:Km,shikimate= 414 ± 33 μM,Km,ATP= 48 ± 4 μM, andkcat= 57 ± 2 s−1for the predicted shikimate kinase andKm,homoserine= 188 ± 37 μM,Km,ATP= 101 ± 7 μM, andkcat= 28 ± 1 s−1for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.
Regulation of
Staphylococcus aureus
Type 5 and Type 8 Capsular Polysaccharides by CO
2
Authors: Herbert Silvia , Newell Steven W. , Lee Chia , Wieland Karsten-Peter , Dassy Bruno , Fournier Jean-Michel , Wolz Christiane , Döring Gerd
Published: July 2002

Staphylococcus aureus
expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO
2
. Here we show that CO
2
reduces CP5 expression at the transcriptional level and that CO
2
regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO
2
caused a significant decrease in CP8 expression in four
S. aureus
strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine
S. aureus
strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of
cap5
and
cap8
. To test whether these sequence variations are responsible for the different responses to CO
2
, promoter regions from selected strains were fused to the reporter gene
xyl
E in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO
2
in all derivatives of strain Newman and was always positively regulated by CO
2
in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO
2
response.
trans
-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive
S. aureus
strains.
Lowering
S
-Adenosylmethionine Levels in
Escherichia coli
Modulates C-to-T Transition Mutations
Authors: Macintyre Georgina , Atwood C. Victoria , Cupples Claire G.
Published: July 2002

Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor,
S
-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM.
Escherichia coli
SAM levels were lowered by reducing SAM synthetase activity via the introduction of a
metK84
allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. The
metK84
strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible P
BAD
promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.
Identification of the Ω4514 Regulatory Region, a Developmental Promoter of
Myxococcus xanthus
That Is Transcribed In Vitro by the Major Vegetative RNA Polymerase
Authors: Hao Tong , Biran Dvora , Velicer Gregory J. , Kroos Lee
Published: July 2002

Ω4514 is the site of a Tn
5 lac
insertion in the
Myxococcus xanthus
genome that fuses
lacZ
expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and σ
A
RNA polymerase, the major form of RNA polymerase in growing
M. xanthus
, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Ω4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn
5 lac
Ω4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An
orf1
disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of
orf2
nor insertion Ω4514 in
orf3
caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an
orf1
mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Ω4514 is the first example of a developmentally regulated
M. xanthus
operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.
Genes Required for Plasmid R64 Thin-Pilus Biogenesis: Identification and Localization of Products of thepilK,pilM,pilO,pilP,pilR, andpilTGenes
Authors: Sakai Daisuke , Komano Teruya
Published: July 2002

We have previously shown that thepilL,pilN,pilQ,pilS,pilU, andpilVgenes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation. In this work, we characterized the products of the remaining essential genes,pilK,pilM,pilO,pilP,pilR, andpilT,with regard to their localization and processing. Overexpression systems containingpilM,pilO, andpilPgenes fused with N-terminal glutathioneS-transferase (GST) or a His tag were constructed. Overproduced proteins were purified and used to raise specific antibodies. Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively. ThepilK,pilR, andpilTproducts were produced with a C-terminal His tag and then detected by anti-His tag antibody. Subcellular fractionation experiments withEscherichia colicells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins. PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins. Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein. We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.
The IntI-Like Tyrosine Recombinase ofShewanella oneidensisIs Active as an Integron Integrase
Authors: Drouin François , Mélançon Josiane , Roy Paul H.
Published: July 2002

We have found an integron-like integrase gene and anattIsite inShewanella oneidensisas part of a small chromosomal integron. We have cloned this gene and tested the ability of the integrase to excise cassettes from various integrons. Most cassettes flanked by twoattCsites are readily excised, while cassettes in the “first” position, with anattI1orattI3site on one end, are not excised. An exception is a cassette withattI2on one end. TheattI2site, from Tn7, has greater similarity to theattIsite adjacent to the integrase ofS. oneidensisthan doattI1orattI3. We cloned theattIsite ofS. oneidensisand observed the integration of two different cassettes. We have, therefore, demonstrated the function of this integron-like integrase.
Bacillus subtilis
168 Contains Two Differentially Regulated Genes Encoding
l
-Asparaginase
Authors: Fisher Susan H. , Wray Lewis V.
Published: July 2002

Expression of the two
Bacillus subtilis
genes encoding
l
-asparaginase is controlled by independent regulatory factors. The
ansZ
gene (formerly
yccC
) was shown by mutational analysis to encode a functional
l
-asparaginase, the expression of which is activated during nitrogen-limited growth by the TnrA transcription factor. Gel mobility shift and DNase I footprinting experiments indicate that TnrA regulates
ansZ
expression by binding to a DNA site located upstream of the
ansZ
promoter. The expression of the
ansA
gene, which encodes the second
l
-asparaginase, was found to be induced by asparagine. The
ansA
repressor, AnsR, was shown to negatively regulate its own expression.
A Conserved C-Terminal Region in Gp71 of the Small Isometric-Head Phage LL-H and ORF474 of the Prolate-Head Phage JCL1032 Is Implicated in Specificity of Adsorption of Phage to Its Host,
Lactobacillus delbrueckii
Authors: Ravin Victor , Räisänen Liisa , Alatossava Tapani
Published: July 2002

Thirty-five phage-resistant mutants of
Lactobacillus delbrueckii
subsp.
lactis
ATCC 15808 were selected. Thirty-three of these mutants were assigned to the Bes group, while the remaining two were grouped under the Ads designation. Bes group mutants adsorbed phage LL-H but did not allow efficient phage development. Preliminary evidence suggests that these strains exhibit a mutation that changes the DNA specificity of a restriction-modification system. The Ads group mutants did not adsorb the small isometric-head phage LL-H. The results suggest that there are at least three different types of phage receptors in
L. delbrueckii
: two that are specific for small isometric-head phages and one that is specific for prolate-head phage JCL1032. Five LL-H host-range mutants which could overcome the adsorption block (a-type mutants) were selected and investigated by sequencing the genes
g71
and
g17
, which encode minor and major tail proteins, respectively. Each of the a-type mutants carried a nucleotide change at the 3′ end of gene
g71
. No mutations were observed in gene
g17
. Comparison of the gene product of
g71
of phage LL-H with its homolog in JCL1032 (ORF474) showed that these proteins had very similar C-terminal regions. No similarities were found at the N-terminal part of the proteins. We conclude that the C-terminal portion of the protein encoded by
g71
of phage LL-H and its homolog in phage JCL1032 determines the adsorption specificities of these phages on
L. delbrueckii
.
High Levels of Resistance to Metronidazole and Clarithromycin in
Helicobacter pylori
Strains in Children
Authors: Kalach Nicolas , Bergeret Michel , Benhamou Pierre Henri , Dupont Christophe , Raymond Josette
Published: July 2002

The aim of the study was to evaluate the prevalence of resistance to amoxicillin, metronidazole, and clarithromycin before treatment of
Helicobacter pylori
infection in children and to assess the evolution of resistance with time. The study was carried out between 1994 and 1999 with 150
H. pylori
-positive children through gastric culture (antimicrobial susceptibility) and histology. All cultured
H. pylori
strains were sensitive to amoxicillin, 64 (43%) were resistant to metronidazole, 32 (21%) were resistant to clarithromycin, and 14 (9%) were resistant to both metronidazole and clarithromycin. The overall prevalence of resistance to metronidazole and clarithromycin did not change significantly with time. The study highlights the generalized high-level and stable metronidazole and clarithromycin resistance of
H. pylori
strains from children.
Cutaneous Infection Caused by
Aspergillus ustus
, an Emerging Opportunistic Fungus in Immunosuppressed Patients
Authors: Gené Josepa , Azón-Masoliver Antoni , Guarro Josep , De Febrer Gabriel , Martı́nez Angels , Grau Cristina , Ortoneda Montserrat , Ballester Frederic
Published: July 2002

We report a case of primary cutaneous infection by the emerging fungus
Aspergillus ustus
in an immunosuppressed patient after a domestic accident. The patient failed to respond to itraconazole and died before receiving a new treatment with amphotericin B. There have been eight other cases reported since 1973, and only two patients survived the infection. In vitro susceptibility testing of seven antifungal drugs showed that terbinafine and the new azole derivative UR-9825 were the most active against this fungus.
Presence of
icaA
and
icaD
Genes and Slime Production in a Collection of Staphylococcal Strains from Catheter-Associated Infections
Authors: Arciola Carla Renata , Baldassarri Lucilla , Montanaro Lucio
Published: July 2002

Both
Staphylococcus epidermidis
and
Staphylococcus aureus
are important causes of infections associated with catheters and other medical devices. It has recently been shown that not only
S. epidermidis
but also
S. aureus
can produce slime and carries the
ica
operon responsible for slime production. In the operon, coexpression of
icaA
and
icaD
is required for full slime synthesis. In this study, the presence of
icaA
and
icaD
was determined in a collection of 91 staphylococcal (68
S. epidermidis
and 23
S. aureus
) strains from intravenous catheter-associated infections, in 10 strains from the skin and mucosa of healthy volunteers, and in two reference strains by a PCR method. Slime-forming ability was tested on Congo red agar plates; 49% of
S. epidermidis
strains from catheters and, surprisingly, 61% of
S. aureus
strains were
icaA
and
icaD
positive and slime forming. All the saprophytic strains turned out to be negative for both
icaA
and
icaD
and also non-slime forming. Two
S. aureus
and one
S. epidermidis
strain from catheters, detected as
icaA
and
icaD
positive by PCR analysis and as slime forming (black colonies) at 24 h on Congo red agar, at 48 h exhibited tiny red spikes at the center of black colonies. The onset of these variants could not be ascribed to a mutagenic potential of Congo red, which, in the Ames test, was devoid of mutagenicity. PCR analysis showed that these red variants were negative for both
icaA
and
icaD
and even lacking the entire
icaADBC
operon. The data reported indicate an important role of
ica
genes as a virulence marker in staphylococcal infections from intravenous catheters.
Two Different Extended-Spectrum β-Lactamases (ESBLs) in One of the First ESBL-Producing Salmonella Isolates in Poland
Authors: Baraniak Anna , Sadowy Ewa , Hryniewicz Waleria , Gniadkowski Marek
Published: July 2002

Two extended-spectrum β-lactamase (ESBL)-producing salmonella isolates,
Salmonella
enterica
serovar Enteritidis and
Salmonella
enterica
serovar Typhimurium, were analyzed. Both isolates produced the CTX-M-3 ESBL; however, their
bla
CTX-M-3
genes were located on different plasmids. The serovar Typhimurium isolate also expressed another ESBL, SHV-2a, and probably the two ESBL genes had been acquired independently by the strain.
Detection by Reverse Transcription-PCR and Genetic Characterization of Field Isolates of Swine Hepatitis E Virus from Pigs in Different Geographic Regions of the United States
Authors: Huang F. F. , Haqshenas G. , Guenette D. K. , Halbur P. G. , Schommer S. K. , Pierson F. W. , Toth T.E. , Meng X. J.
Published: July 2002

Hepatitis E virus (HEV) is an important public health concern in many developing countries. HEV is also endemic in some industrialized counties, including the United States. With our recent discovery of swine HEV in pigs that is genetically closely related to human HEV, hepatitis E is now considered a zoonotic disease. Human strains of HEV are genetically heterogenic. So far in the United States, only one strain of swine HEV has been identified and characterized from a pig. To determine the extent of genetic variations and the nature of swine HEV infections in U.S. pigs, we developed a universal reverse transcription-PCR (RT-PCR) assay that is capable of detecting genetically divergent strains of HEV. By using this universal RT-PCR assay, we tested fecal and serum samples of pigs of 2 to 4 months of age from 37 different U.S. swine farms for the presence of swine HEV RNA. Thirty-four of the 96 pigs (35%) and 20 of the 37 swine herds (54%) tested were positive for swine HEV RNA. The sequences of a 348-bp region within the ORF2 gene of 27 swine HEV isolates from different geographic regions were determined. Sequence analyses revealed that the 27 U.S. swine HEV isolates shared 88 to 100% nucleotide sequence identities with each other and 89 to 98% identities with the prototype U.S. strain of swine HEV. These U.S. swine HEV isolates are only distantly related to the Taiwanese strains of swine HEV, with about 74 to 78% nucleotide sequence identities; to most known human strains of HEV worldwide, with <79% sequence identities; and to avian HEV, with 54 to 56% sequence identities. Phylogenetic analysis showed that all the U.S. swine HEV isolates identified in this study clustered in the same genotype with the prototype U.S. swine HEV and the two U.S. strains of human HEV. The data from this study indicated that swine HEV is widespread and enzoonotic in U.S. swine herds and that, as is with human HEV, swine HEV isolates from different geographic regions of the world are also genetically heterogenic. These data further raise potential concerns for zoonosis, xenozoonosis, and food safety.
Serratia ficaria
Endophthalmitis
Authors: Badenoch P. R. , Thom A. L. , Coster D. J.
Published: July 2002

We report a case of
Serratia ficaria
endophthalmitis in a 73-year-old man. The patient's ocular history included a chemical burn, glaucoma, and corneal transplantation.
S. ficaria
is part of the fig tree ecosystem and is rarely isolated from clinical specimens. When it has been previously implicated as an agent of disease, the patients have been treated successfully and there have been no complications. In our patient, however, the infection resulted in the loss of the infected eye. This case illustrates that
S. ficaria
infection in a compromised patient can have serious consequences.
Vancomycin Susceptibility of Oxacillin-Resistant
Staphylococcus aureus
Isolates Causing Nosocomial Bloodstream Infections
Authors: Tallent Sandra M. , Bischoff Tammy , Climo Michael , Ostrowsky Belinda , Wenzel Richard P. , Edmond Michael B.
Published: July 2002

Nosocomial oxacillin-resistant
Staphylococcus aureus
(ORSA) bloodstream isolates were tested to determine the prevalence of vancomycin heteroresistance. We screened 619 ORSA nosocomial bloodstream isolates from 36 hospitals between 1997 and 2000. Only one isolate exhibiting heterotypic resistance was detected. Thus, vancomycin heteroresistance in clinical bloodstream isolates remains rare in the United States.
The Role of CD8+T Cells and Major Histocompatibility Complex Class I Expression in the Central Nervous System of Mice Infected with Neurovirulent Sindbis Virus
Authors: Kimura Takashi , Griffin Diane E.
Published: July 2002

Little is known about the role of CD8+T cells infiltrating the neural parenchyma during encephalitis induced by neurovirulent Sindbis virus (NSV). NSV preferentially infects neurons in the mouse brain and spinal cord; however, it is generally accepted that neurons can express few if any major histocompatibility complex (MHC) class I molecules. We evaluated the possible roles and interactions of CD8+T cells during NSV encephalitis and demonstrated that MHC class I antigen (H2K/D) was expressed on endothelial cells, inflammatory cells, and ependymal cells after intracerebral inoculation of NSV. No immunoreactivity was observed in neurons. On the other hand, in situ hybridization with probes for MHC class I heavy chain, β2 microglobulin, and TAP1 and TAP2 mRNAs revealed increased expression in a majority of neurons, as well as in inflammatory cells, endothelial cells, and ependymal cells in the central nervous system of infected mice. NSV-infected neurons may fail to express MHC class I molecules due to a posttranscriptional block or may express only nonclassical MHC class I genes. To better understand the role CD8+T cells play during fatal encephalitis induced by NSV, mice lacking functional CD8+T cells were studied. The presence or absence of CD8 did not alter outcome, but absence of β2 microglobulin improved survival. Interestingly, the intracellular levels of viral RNA decreased more rapidly in immunocompetent mice than in mice without functional CD8+T cells. These observations suggest that CD8+T cells may act indirectly, possibly via cytokines, to contribute to the clearance of viral RNA in neurons.
Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?
Authors: Hoffmann Erich , Stech Juergen , Leneva Irina , Krauss Scott , Scholtissek Christoph , Chin Po San , Peiris Malik , Shortridge Kennedy F. , Webster Robert G.
Published: July 2002

In 1997, an H5N1 influenza virus outbreak occurred in chickens in Hong Kong, and the virus was transmitted directly to humans. Because there is limited information about the avian influenza virus reservoir in that region, we genetically characterized virus strains isolated in Hong Kong during the 1997 outbreak. We sequenced the gene segments of a heterogeneous group of viruses of seven different serotypes (H3N8, H4N8, H6N1, H6N9, H11N1, H11N9, and H11N8) isolated from various bird species. The phylogenetic relationships divided these viruses into several subgroups. An H6N1 virus isolated from teal (A/teal/Hong Kong/W312/97 [H6N1]) showed very high (>98%) nucleotide homology to the human influenza virus A/Hong Kong/156/97 (H5N1) in the six internal genes. The N1 neuraminidase sequence showed 97% nucleotide homology to that of the human H5N1 virus, and the N1 protein of both viruses had the same 19-amino-acid deletion in the stalk region. The deduced hemagglutinin amino acid sequence of the H6N1 virus was most similar to that of A/shearwater/Australia/1/72 (H6N5). The H6N1 virus is the first known isolate with seven H5N1-like segments and may have been the donor of the neuraminidase and the internal genes of the H5N1 viruses. The high homology between the internal genes of H9N2, H6N1, and the H5N1 isolates indicates that these subtypes are able to exchange their internal genes and are therefore a potential source of new pathogenic influenza virus strains. Our analysis suggests that surveillance for influenza A viruses should be conducted for wild aquatic birds as well as for poultry, pigs, and humans and that H6 isolates should be further characterized.
Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA
Authors: Burke Emily , Mahoney Nicole M. , Almo Steven C. , Barik Sailen
Published: July 2002

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-
l
-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-
l
-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-
l
-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome.
Varicella-Zoster Virus Gene Expression in Latently Infected and Explanted Human Ganglia
Authors: Kennedy Peter G. E. , Grinfeld Esther , Bell Jeanne E.
Published: July 2002

A consistent feature of varicella-zoster virus (VZV) latency is the restricted pattern of viral gene expression in human ganglionic tissues. To understand further the significance of this gene restriction, we used in situ hybridization (ISH) to detect the frequency of RNA expression for nine VZV genes in trigeminal ganglia (TG) from 35 human subjects, including 18 who were human immunodeficiency virus (HIV) positive. RNA for VZV gene 21 was detected in 7 of 11 normal and 6 of 10 HIV-positive subjects, RNA for gene 29 was detected in 5 of 14 normal and 11 of 11 HIV-positive subjects, RNA for gene 62 was detected in 4 of 10 normal and 6 of 9 HIV-positive subjects, and RNA for gene 63 was detected in 8 of 17 normal and 12 of 15 HIV-positive subjects. RNA for VZV gene 4 was detected in 2 of 13 normal and 4 of 9 HIV-positive subjects, and RNA for gene 18 was detected in 4 of 15 normal and 5 of 15 HIV-positive subjects. By contrast, RNAs for VZV genes 28, 40, and 61 were rarely or never detected. In addition, immunocytochemical analysis detected the presence of VZV gene 63-encoded protein in five normal and four HIV-positive subjects. VZV RNA was also analyzed in explanted fresh human TG and dorsal root ganglia from five normal human subjects over a period of up to 11 days in culture. We found a very different pattern of gene expression in these explants, with transcripts for VZV genes 18, 28, 29, 40, and 63 all frequently detected, presumably as a result of viral reactivation. Taken together, these data provide further support for the notion of significant and restricted viral gene expression in VZV latency.
The Human Immunodeficiency Virus Type 1 Tat Protein Up-Regulates the Promoter Activity of the Beta-Chemokine Monocyte Chemoattractant Protein 1 in the Human Astrocytoma Cell Line U-87 MG: Role of SP-1, AP-1, and NF-κB Consensus Sites
Authors: Lim Siew Pheng , Garzino-Demo Alfredo
Published: July 2002

It has been shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can specifically enhance expression and release of monocyte chemoattractant protein 1 (MCP-1) from human astrocytes. In this study, we show evidence that Tat-induced MCP-1 expression is mediated at the transcriptional level. Transient transfection of an expression construct encoding the full-length Tat into the human glioblastoma-astrocytoma cell line U-87 MG enhances reporter gene activity from cotransfected deletion constructs of the MCP-1 promoter. HIV-1 Tat exerts its effect through a minimal construct containing 213 nucleotides upstream of the translational start site. Site-directed mutagenesis studies indicate that an SP1 site (located between nucleotides −123 and −115) is critical for both constitutive and Tat-enhanced expression of the human MCP-1 promoter, as mutation of this SP1 site significantly diminished reporter gene expression in both instances. Gel retardation experiments further demonstrate that Tat strongly enhances the binding of SP1 protein to its DNA element on the MCP-1 promoter. Moreover, we also observe an increase in the binding activities of transcriptional factors AP1 and NF-κB to the MCP-1 promoter following Tat treatment. Mutagenesis studies show that an upstream AP1 site and an adjacent NF-κB site (located at −128 to −122 and −150 to −137, respectively) play a role in Tat-mediated transactivation. In contrast, a further upstream AP1 site (−156 to −150) does not appear to be crucial for promoter activity. We postulate that a Tat-mediated increase in SP1 binding activities augments the binding of AP1 and NF-κB, leading to synergistic activation of the MCP-1 promoter.
Relative Sensitivity of Hepatitis B Virus and Other Hepatotropic Viruses to the Antiviral Effects of Cytokines
Authors: McClary Heike , Koch Rick , Chisari Francis V. , Guidotti Luca G.
Published: July 2002

We have previously shown that hepatitis B virus (HBV) replication is inhibited noncytopathically in the livers of transgenic mice following injection of HBV-specific cytotoxic T lymphocytes (CTLs) or infection with unrelated hepatotropic viruses, including lymphocytic choriomeningitis virus (LCMV) and adenovirus. These effects are mediated by gamma interferon (IFNγ), tumor necrosis factor alpha (TNFα), and IFNα/β. In the present study, we crossed HBV transgenic mice with mice genetically deficient for IFNγ (IFNγKO), the TNFα receptor (TNFαRKO), or the IFNα/β receptor (IFNα/βRKO) in order to determine the relative contribution of each cytokine to the antiviral effects observed in each of these systems. Interestingly, we showed that HBV replicates in unmanipulated IFNγKO and IFNα/βRKO mice at levels higher than those observed in control mice, implying that baseline levels of these cytokines control HBV replication in the absence of inflammation. We also showed that IFNγ mediates most of the antiviral effect of the CTLs while IFNα/β is primarily responsible for the early inhibitory effect of LCMV and adenovirus on HBV replication. In addition, we showed that the hepatic induction of IFNα/β observed after injection of poly(I · C) is sufficient to inhibit HBV replication and that a similar antiviral effect is achieved by systemic administration of very high doses of IFNα. We also compared the relative sensitivity of LCMV and adenovirus to control by IFNγ, TNFα, or IFNα/β in these animals. Importantly, IFNα/βRKO mice, and to a lesser extent IFNγKO mice, showed higher hepatic levels of LCMV RNA and adenovirus DNA and RNA than control mice, underscoring the importance of both interferons in controlling these other viral infections as well.
Recombinant Adeno-Associated Virus Expressing Human Papillomavirus Type 16 E7 Peptide DNA Fused with Heat Shock Protein DNA as a Potential Vaccine for Cervical Cancer
Authors: Liu Dai-Wei , Tsao Yeou-Ping , Kung John T. , Ding Yu-An , Sytwu Huey-Kang , Xiao Xiao , Chen Show-Li
Published: July 2002

In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242–2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine.
Feline Immunodeficiency Virus Cell Entry
Authors: Frey Susan C. S. , Hoover Edward A. , Mullins James I.
Published: July 2002

The process of feline immunodeficiency virus (FIV) cell entry was examined using assays for virus replication intermediates. FIV subtype B was found to utilize the chemokine receptor CXCR4, but not CCR5, as a cellular receptor. Zidovudine blocked formation of late viral replication products most effectively, including circular DNA genome intermediates. Our findings extend the role of CXCR4 as a primary receptor for CD4-independent cell entry by FIV.
Potential Sources of the 1995 Venezuelan Equine Encephalitis Subtype IC Epidemic
Authors: Brault Aaron C. , Powers Ann M. , Medina Gladys , Wang Eryu , Kang Wenli , Salas Rosa Alba , De Siger Julieta , Weaver Scott C.
Published: July 2002

Venezuelan equine encephalitis viruses (VEEV) belonging to subtype IC have caused three (1962–1964, 1992–1993 and 1995) major equine epizootics and epidemics. Previous sequence analyses of a portion of the envelope glycoprotein gene demonstrated a high degree of conservation among isolates from the 1962–1964 and the 1995 outbreaks, as well as a 1983 interepizootic mosquito isolate from Panaquire, Venezuela. However, unlike subtype IAB VEEV that were used to prepare inactivated vaccines that probably initiated several outbreaks, subtype IC viruses have not been used for vaccine production and their conservation cannot be explained in this way. To characterize further subtype IC VEEV conservation and to evaluate potential sources of the 1995 outbreak, we sequenced the complete genomes of three isolates from the 1962–1964 outbreak, the 1983 Panaquire interepizootic isolate, and two isolates from 1995. The sequence of the Panaquire isolate, and that of virus isolated from a mouse brain antigen prepared from subtype IC strain P676 and used in the same laboratory, suggested that the Panaquire isolate represents a laboratory contaminant. Some authentic epizootic IC strains isolated 32 years apart showed a greater degree of sequence identity than did isolates from the same (1962–1964 or 1995) outbreak. If these viruses were circulating and replicating between 1964 and 1995, their rate of sequence evolution was at least 10-fold lower than that estimated during outbreaks or that of closely related enzootic VEEV strains that circulate continuously. Current understanding of alphavirus evolution is inconsistent with this conservation. This subtype IC VEEV conservation, combined with phylogenetic relationships, suggests the possibility that the 1995 outbreak was initiated by a laboratory strain.
The Hemagglutinin of Canine Distemper Virus Determines Tropism and Cytopathogenicity
Authors: von Messling Veronika , Zimmer Gert , Herrler Georg , Haas Ludwig , Cattaneo Roberto
Published: July 2002

Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV
5804
), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDV
OS
and CDV
OL
, respectively), and the MV vaccine strain Edmonston B (MV
Edm
). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDV
OS
. The coding regions of the H proteins of all CDV strains and MV
Edm
were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H
5804
, rCDV-H
OL
, rCDV-H
Edm
, rMV-H
5804
, rMV-H
OL
, and rMV-H
OS
were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the family
Paramyxoviridae
, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used.
Activity of the Hepatitis A Virus IRES Requires Association between the Cap-Binding Translation Initiation Factor (eIF4E) and eIF4G
Authors: Ali Iraj K. , McKendrick Linda , Morley Simon J. , Jackson Richard J.
Published: July 2002

The question of whether translation initiation factor eIF4E and the complete eIF4G polypeptide are required for initiation dependent on the IRES (internal ribosome entry site) of hepatitis A virus (HAV) has been examined using in vitro translation in standard and eIF4G-depleted rabbit reticulocyte lysates. In agreement with previous publications, the HAV IRES is unique among all picornavirus IRESs in that it was inhibited if translation initiation factor eIF4G was cleaved by foot-and-mouth disease L-proteases. In addition, the HAV IRES was inhibited by addition of eIF4E-binding protein 1, which binds tightly to eIF4E and sequesters it, thus preventing its association with eIF4G. The HAV IRES was also inhibited by addition of m
7
GpppG cap analogue, irrespective of whether the RNA tested was capped or not. Thus, initiation on the HAV IRES requires that eIF4E be associated with eIF4G and that the cap-binding pocket of eIF4E be empty and unoccupied. This suggests two alternative models: (i) initiation requires a direct interaction between an internal site in the IRES and eIF4E/4G, an interaction which involves the cap-binding pocket of eIF4E in addition to any direct eIF4G-RNA interactions; or (ii) it requires eIF4G in a particular conformation which can be attained only if eIF4E is bound to it, with the cap-binding pocket of the eIF4E unoccupied.
High Resistance of Human Parainfluenza Type 2 Virus Protein-Expressing Cells to the Antiviral and Anti-Cell Proliferative Activities of Alpha/Beta Interferons: Cysteine-Rich V-Specific Domain Is Required for High Resistance to the Interferons
Authors: Nishio Machiko , Tsurudome Masato , Ito Morihiro , Kawano Mitsuo , Komada Hiroshi , Ito Yasuhiko
Published: July 2002

Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-α/β) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-γ. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-α/β. HeLa cells expressing the N-terminally truncated V protein show resistance to IFN-α/β, showing that the IFN resistance determinant maps to the cysteine-rich V-specific domain. A complete defect of Stat2 is found in HeLa-CA and HeLa-V cells, whereas the levels of Stat1 expression are not significantly different among HeLa, HeLa-CA, HeLa-P, and HeLa-V cells, indicating that IFN-α/β resistance of HeLa-CA and HeLa-V cells is due to a defect of Stat2. HeLa-SV41V cells show high resistance to all IFNs, and no expression of Stat1 can be detected. Stat2 mRNA is fully detected in HeLa-V cells. Stat2 was scarcely pulse-labeled in the HeLa-V cells, indicating that synthesis of Stat2 is suppressed or Stat2 is very rapidly degraded in HeLa-V cells. The V protein suppresses the in vitro translation of Stat2 mRNA more extensively than that of Stat1 mRNA. An extremely small amount of Stat2 can be detected in HeLa-V cells treated with proteasome inhibitors. The half-life of Stat2 is approximately 3.5 and 2 h in uninfected and hPIV-2-infected HeLa cells, respectively. This study shows that synthesis of Stat2 may be suppressed and Stat2 degradation is also enhanced in hPIV-2-infected HeLa and HeLa-V cells.
Genome Sequence of Bovine Herpesvirus 4, a Bovine
Rhadinovirus,
and Identification of an Origin of DNA Replication
Authors: Zimmermann Wolfgang , Broll Hermann , Ehlers Bernhard , Buhk Hans-Jörg , Rosenthal André , Goltz Michael
Published: July 2002

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus of cattle. The complete long unique coding region (LUR) of BoHV-4 strain 66-p-347 was determined by a shotgun approach. Together with the previously published noncoding terminal repeats, the entire genome sequence of BoHV-4 is now available. The LUR consists of 108,873 bp with an overall G+C content of 41.4%. At least 79 open reading frames (ORFs) are present in this coding region, 17 of them unique to BoHV-4. In contrast to herpesvirus saimiri and human herpesvirus 8, BoHV-4 has a reduced set of ORFs homologous to cellular genes. Gene arrangement as well as phylogenetic analysis confirmed that BoHV-4 is a member of the genus
Rhadinovirus
. In addition, an origin of replication (
ori
) in the genome of BoHV-4 was identified by
Dpn
I assays. A minimum of 1.69 kbp located between ORFs 69 and 71 was sufficient to act as a
cis
signal for replication.
Yellow Fever Virus Encephalitis: Properties of the Brain-Associated T-Cell Response during Virus Clearance in Normal and Gamma Interferon-Deficient Mice and Requirement for CD4+Lymphocytes
Authors: Liu Ting , Chambers Thomas J.
Published: July 2002

Viral encephalitis caused by neuroadapted yellow fever 17D virus (PYF) was studied in parental and gamma interferon (IFN-γ)-deficient (IFN-γ knockout [GKO]) C57BL/6 mice. The T-cell responses which enter the brain during acute fatal encephalitis of nonimmunized mice, as well as nonfatal encephalitis of immunized mice, were characterized for relative proportions of CD4+and CD8+cells, their proliferative responses, and antigen-specific expression of cytokines during stimulation in vitro. Unimmunized mice accumulated only low levels of T cells within the brain during fatal disease, whereas the brains of immunized mice contained higher levels of both T-cell subsets in response to challenge, with CD8+cells increased relative to the CD4+subset. The presence of T cells correlated with the time at which virus was cleared from the central nervous system in both parental and GKO mice. Lymphocytes isolated from the brains of challenged immunized mice failed to proliferate in vitro in response to T-cell mitogens or viral antigens; however, IFN-γ, interleukin 4 (IL-4), and, to a lesser extent, IL-2 were detectable after stimulation. The levels of IFN-γ, but not IL-2 or IL-4, were augmented in response to viral antigen, and this specificity was detectable in the CD4+compartment. When tested for the ability to survive both immunization and challenge with PYF virus, GKO and CD8 knockout mice did not differ from parental mice (80 to 85% survival), although GKO mice exhibited a defect in virus clearance. In contrast, CD4 knockout and Igh-6 mice were unable to resist challenge. The data implicate antibody in conjunction with CD4+lymphocytes bearing a Th1 phenotype as the critical factors involved in virus clearance in this model.
Human Immunodeficiency Virus Type 1 Induces Expression of Complement Factors in Human Astrocytes
Authors: Speth Cornelia , Stöckl Gabriele , Mohsenipour Iradji , Würzner Reinhard , Stoiber Heribert , Lass-Flörl Cornelia , Dierich Manfred P.
Published: July 2002

Since the brain is separated from the blood immune system by a tight barrier, the brain-resident complement system may represent a central player in the immune defense of this compartment against human immunodeficiency virus (HIV). Chronic complement activation, however, may participate in HIV-associated neurodegeneration. Since the level of complement factors in the cerebrospinal fluid is known to be elevated in AIDS-associated neurological disorders, we evaluated the effect of HIV type 1 (HIV-1) on the complement synthesis of brain astrocytes. Incubation of different astrocytic cell lines and primary astrocytes with HIV-1 induced a marked upregulation of the expression of the complement factors C2 and C3. The synthesis of other secreted or membrane-bound complement proteins was not found to be altered. The enhancement of C3 production was measured both on the mRNA level and as secreted protein in the culture supernatants. HIV-1 laboratory strains as well as primary isolates were capable of inducing C3 production with varied effectiveness. The usage of viral coreceptors by HIV-1 was proved to be a prerequisite for the upregulation of C3 synthesis, which was modulated by the simultaneous addition of cytokines. The C3 protein which is secreted after incubation of the cells with HIV was shown to be biologically active as it can participate in the complement cascade.
GRP78, a Coreceptor for Coxsackievirus A9, Interacts with Major Histocompatibility Complex Class I Molecules Which Mediate Virus Internalization
Authors: Triantafilou Kathy , Fradelizi Didier , Wilson Keith , Triantafilou Martha
Published: July 2002

It is becoming apparent that over the years cell infection by virus seems to have evolved into a multistep process in which many viruses employ distinct cell surface molecules for their attachment and cell entry. In this study the attachment and entry pathway of coxsackievirus A9 (CAV-9), a member of thePicornaviridaefamily, was investigated. It has been known that, although integrin αvβ3 is utilized as a receptor, its presence alone is insufficient for CAV-9 infection and that CAV-9 also requires a 70-kDa major histocompatibility complex class I (MHC-I)-associated protein (MAP-70) as a coreceptor molecule. We document by protein isolation and peptide sequencing that the 70-kDa protein is GRP78, a member of the heat shock protein 70 family of stress proteins. Furthermore we show by using fluorescence resonance energy transfer (FRET) that GRP78 is also expressed on the cell surface and associates with MHC-I molecules. In addition CAV-9 infection of permissive cells requires GRP78 and also MHC-I molecules, which are essential for virus internalization. The identification of GRP78 as a coreceptor for CAV-9 and the revelation of GRP78 and MHC-I associations have provided new insights into the life cycle of CAV-9, which utilizes integrin αvβ3 and GRP78 as receptor molecules whereas MHC-I molecules serve as the internalization pathway of this virus to mammalian cells.