bluuebunny/crossref_metadata_2025 huggingface dataset:
Rat/MgrA, a Regulator of Autolysis, Is a Regulator of Virulence Genes in
Staphylococcus aureus
Authors: Ingavale Susham , van Wamel Willem , Luong Thanh T. , Lee Chia Y. , Cheung Ambrose L.
Published: February 2005

We have previously identified
mgrA
(
rat
) as a regulator of autolysis in
Staphylococcus aureus
. Besides its effect on autolytic activity, we recently found alterations in the expression of regulator and target virulence genes in the
mgrA
mutant. Northern analysis and transcription fusion assays showed that inactivation of
mgrA
has led to the downregulation of RNAIII of
agr
and
hla
and upregulation of
sarS
and
spa
. Although both SarA and
agr
are activators of α-hemolysin and a repressors of protein A synthesis, we found that the transcription of
sarA
was not affected in the
mgrA
mutant and vice versa, indicating that MgrA likely regulates
hla
and
spa
in a SarA-independent manner. Previously we have shown that SarT, a SarA homolog, is a repressor of
hla
and an activator of
spa
, presumably by activating SarS, however, analysis of the double
sarT mgrA
mutant for
hla
and
spa
transcription indicated that the
mgrA-
mediated effect is not mediated via
sarT
. Our results further demonstrated that the
mgrA
gene product regulates
hla
and
spa
expression in a dual fashion, with the first being
agr
dependent and the second
agr
independent. In the
agr
-independent pathway, MgrA binds directly to
hla
and the
sarS
promoter to modulate α-hemolysin and protein A expression. Thus, our studies here have defined the nature of interaction of
mgrA
with other regulators such as
agr
,
sarS
, and
sarT
and its role in regulating
hla
and
spa
transcription within the virulence regulatory network of
S. aureus.
IpaD of
Shigella flexneri
Is Independently Required for Regulation of Ipa Protein Secretion and Efficient Insertion of IpaB and IpaC into Host Membranes
Authors: Picking Wendy L. , Nishioka Hiroaki , Hearn Patricia D. , Baxter M. Aaron , Harrington Amanda T. , Blocker Ariel , Picking William D.
Published: February 2005

Shigella flexneri
causes human dysentery after invading the cells of the colonic epithelium. The best-studied effectors of
Shigella
entry into colonocytes are the invasion plasmid antigens IpaC and IpaB. These proteins are exported via a type III secretion system (TTSS) to form a pore in the host membrane that may allow the translocation of other effectors into the host cytoplasm. TTSS-mediated secretion of IpaD is also required for translocation pore formation, bacterial invasion, and virulence, but the mechanistic role of this protein is unclear. IpaD is also known to be involved in controlling Ipa protein secretion, but here it is shown that this activity can be separated from its requirement for cellular invasion. Amino acids 40 to 120 of IpaD are not essential for IpaD-dependent invasion; however, deletions in this region still lead to constitutive IpaB/IpaC secretion. Meanwhile, a central deletion causes only a partial loss of control of Ipa secretion but completely eliminates IpaD's invasion function, indicating that IpaD's role in invasion is not a direct outcome of its ability to control Ipa secretion. As shigellae expressing
ipaD
N-terminal deletion mutations have reduced contact-mediated hemolysis activity and are less efficient at introducing IpaB and IpaC into erythrocyte membranes, it is possible that IpaD is responsible for insertion of IpaB/IpaC pores into target cell membranes. While efficient insertion of IpaB/IpaC pores is needed for optimal invasion efficiency, it may be especially important for Ipa-dependent membrane disruption and thus for efficient vacuolar escape and intercellular spread.
The Locus of Enterocyte Effacement-Encoded Effector Proteins All Promote Enterohemorrhagic
Escherichia coli
Pathogenicity in Infant Rabbits
Authors: Ritchie Jennifer M. , Waldor Matthew K.
Published: February 2005

The genes encoding the enterohemorrhagic
Escherichia coli
(EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of
tir
, which encodes one of these effector proteins, results in a profound reduction (∼10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in
escN
, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of
escN
reduced EHEC recovery from the rabbit intestine by ∼10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the
espH
and
espF
mutants was reduced throughout the intestine. In contrast, colonization by the
map
and
espG
mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC
espF
mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.
Translocation and Surface Expression of Lipidated Serogroup B Capsular Polysaccharide in
Neisseria meningitidis
Authors: Tzeng Yih-Ling , Datta Anup K. , Strole Cristy A. , Lobritz Michael A. , Carlson Russell W. , Stephens David S.
Published: February 2005

The capsule of
N. meningitidis
serogroup B, (α2→8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal
cps
complex in
Escherichia coli
K-12 indicated that deletion of genes designated
lipA
and
lipB
caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of
lip
and
lipB
in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in
lipA
and
lipB
were generated in the serogroup B meningococcal strain NMB. The
lipA
and
lipB
genes are located on the 3′ end of the
ctr
operon and are most likely transcribed independently. Inactivation of
lipA
,
lipB
, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in
lipA
,
lipB
, and
lipA lipB
mutants. Capsular polymers purified from
lipA
,
lipB
, and
lipA lipB
mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus,
lipA
and
lipB
do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.
Inactivation of Membrane Tumor Necrosis Factor Alpha by Gingipains fromPorphyromonas gingivalis
Authors: Mężyk-Kopeć Renata , Bzowska Małgorzata , Potempa Jan , Bzowska Monika , Jura Natalia , Sroka Aneta , Black Roy A. , Bereta Joanna
Published: February 2005

Gingipains are cysteine proteinases produced byPorphyromonas gingivalis, a major causative bacterium of adult periodontitis. They consist of arginine-specific (HRgpA and RgpB) and lysine-specific (Kgp) proteinases. Gingipains strongly affect the host defense system by degrading some cytokines, components of the complement system, and several immune cell receptors. In an in vitro model, gingipains were shown to degrade soluble tumor necrosis factor alpha (TNF-α). However, since membrane TNF-α shows strong biological activity, especially in local inflammatory lesions, it was worth investigating whether gingipains might also destroy membrane TNF-α and limit its biological activities. To avoid a possible influence of gingipains on ADAM17, the secretase of TNF-α, the majority of experiments were performed using ADAM17−/−fibroblasts stably transfected with cDNA of human pro-TNF-α (ADAM17−/−TNF+). Arginine-specific gingipains (Rgp's) strongly diminished the level of TNF-α on the cell surface as measured by flow cytometry, and this process was not accompanied by an increased concentration of soluble TNF-α in the culture medium. Degradation of membrane TNF-α by Rgp's correlated with a strong decrease in TNF-α-mediated biological activities of ADAM17−/−TNF+cells. First, the activation state of transcription factor NF-κB was suppressed; second, the cells were no longer able to induce apoptosis in HL-60 cells. Kgp was also able to cleave membrane TNF-α, but its effect was much weaker than that of Rgp's. Gingipains also limited the binding of native TNF-α to the target cells. Thus, gingipains are able not only to cleave soluble TNF-α but also to destroy the membrane form of the cytokine, which may additionally dysregulate the cytokine network.
Rapid Method for the Construction of
Salmonella enterica
Serovar Typhimurium Vaccine Carrier Strains
Authors: Husseiny Mohamed I. , Hensel Michael
Published: February 2005

Salmonella enterica
serovar Typhimurium is a versatile organism for the generation of live recombinant vaccines for mucosal immunization. Various strategies have been devised for the stable and efficient expression of heterologous antigens by attenuated
S. enterica
strains, but these methods often require complex manipulations. Use of phage λ Red recombinase has recently been devised for gene replacements in
Escherichia coli
and
S. enterica
after introduction of PCR products. Based on this method, we have developed an approach that allows the integration of recombinant expression cassettes for heterologous antigens in a single step. The recombinant construct is integrated into the chromosome and is devoid of any selective marker such as antibiotic resistance. We observed the stable expression of model antigens without selective pressure. In addition, the method allows the simultaneous generation of attenuating mutations by gene deletions. The novel “knock-in” approach allows the rapid and efficient construction of recombinant
Salmonella
strains as vaccine carriers.
Gelatinase Is Important for Translocation of
Enterococcus faecalis
across Polarized Human Enterocyte-Like T84 Cells
Authors: Zeng Jing , Teng Fang , Murray Barbara E.
Published: February 2005

Previously, in our laboratory, we established a two-chamber system to study translocation of
Enterococcus faecalis
across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of
gelE
of strain OG1RF, which also has a polar effect on the cotranscribed
sprE
, as well as disruption of its regulatory system (
fsrA
,
fsrB
, and
fsrC
) resulted in a loss of detectable translocation by
E. faecalis
OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A
gelE
deletion mutant of OG1RF (GelE
−
SprE
+
) also showed that significantly reduced translocation and complementation with the
gelE
gene (pTEX5438) in
trans
restored gelatinase and translocation, demonstrating that gelatinase is important for
E. faecalis
translocation. Complementation of
fsrA
,
fsrB
, and
fsrC
mutants with all three
fsr
genes also resulted in production of gelatinase and translocation. Furthermore, introduction of
fsr
genes into two non-gelatinase-producing
E. faecalis
isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have
gelE
but not
fsrA
or
fsrB
, are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 (
gelE
) showed little or no translocation and no detectable gelatinase, confirming the importance of both
fsr
and gelatinase for
E. faecalis
translocation. The importance of gelatinase production was also corroborated among 20
E. faecalis
human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of
E. faecalis
across human enterocyte-like T84 cells.
Transcriptional Control of the
rhuIR-bhuRSTUV
Heme Acquisition Locus in
Bordetella avium
Authors: King Natalie D. , Kirby Amy E. , Connell Terry D.
Published: February 2005

Iron (Fe) is an essential nutrient for most bacterial pathogens. In these organisms, a variety of regulatory systems that respond to specific Fe complexes found within their vertebrate hosts have evolved. In
Bordetella avium
, the heme utilization locus encoded by
rhuIR-bhuRSTUV
mediates efficient acquisition of Fe from heme and hemoproteins. Control of
bhuRSTUV
expression is promulgated at two levels. When Fe is abundant, expression is repressed in a Fur-dependent manner which is partially relieved when Fe is limiting. In the presence of heme or hemoproteins, expression of the
bhuRSTUV
operon is induced via a three-component signal transduction cascade composed of RhuI, RhuR, and BhuR. Herein, we report the identification of two promoters (P

rhuI

and P

bhuR

) that control expression of the
rhuIR-bhuRSTUV
cluster. Primer extension analysis identified the transcriptional start site of P

rhuI

within a putative Fur box. Transcriptional initiation of P

bhuR

mapped within the
rhuR-bhuR
intergenic region. Maximal transcription from P

bhuR

required Fe-limiting conditions, the presence of heme (or hemoglobin), and
rhuI
; however, analysis of transcripts produced from the
rhuIR-bhuRSTUV
locus revealed a pattern of low-level
bhuR
transcription in the absence of heme which originated from both P

bhuR

and P

rhuI

. Transcription from P

rhuI

was repressed by Fe in the presence of
fur
and somewhat enhanced by the addition of hemin to Fe-limited media. The nature of this hemin-associated P

rhuI

stimulation was
rhuI
independent and therefore not induced by heme via the BhuR-RhuR-RhuI signal cascade. Fe also repressed transcription from P

bhuR

in a
fur
-dependent manner; however, activation from this promoter, in the presence or absence of heme, did not occur without
rhuI
.
Interactions of OspA Monoclonal Antibody C3.78 withBorrelia burgdorferiwithin Ticks
Authors: Gipson Clay L. , de Silva Aravinda M.
Published: February 2005

TheBorrelia burgdorferiouter surface protein A (OspA) vaccine induces antibodies that prevent transmission from the tick to the host. Here we describe studies with an OspA monoclonal antibody (C3.78) to understand the mechanism by which antibodies entering the tick blockBorreliatransmission. Host complement in the tick's blood meal did not contribute to protection because the antibody was equally effective whether infected ticks fed on normal or complement-deficient mice. Antibody-mediated cross-linking of bacteria or cross-linking of OspA molecules was not required for protection because C3.78 Fab′ fragments were as effective as whole antibody molecules. At low C3.78 concentrations, transmission was blocked despite the presence of many live spirochetes within the tick, confirming that clearance ofBorreliaorganisms was not required to block transmission. We propose that OspA antibody binding to the surface of spirochetes blocks transmission by a mechanism that does not require bacterial killing.
Role of the Type III Secreted Exoenzymes S, T, and Y in Systemic Spread of
Pseudomonas aeruginosa
PAO1 In Vivo
Authors: Vance Russell E. , Rietsch Arne , Mekalanos John J.
Published: February 2005

Pseudomonas aeruginosa
uses a dedicated type III secretion system to deliver toxins directly into the cytoplasm of host cells. While progress has been made in elucidating the function of type III-secreted toxins in vitro, the in vivo functions of the type III-secreted exoenzymes are less well understood, particularly for the sequenced strain PAO1. Therefore, we have systematically deleted the genes for the three known type III effector molecules (
exoS
,
exoT
, and
exoY
) in
P. aeruginosa
PAO1 and assayed the effect of the deletions, both singly and in combination, on cytotoxicity in vitro and in vivo. We found that the type III secretion system acts differently on different cell types, causing an
exoST
-dependent rounding of a lung epithelial-like cell line in contrast to causing an
exoSTY
-independent but translocase (
popB
)-dependent lysis of a macrophage cell line. We utilized an in vivo competitive infection model to test each of our mutants, examining replication in the lung and spread to secondary sites such as the blood and spleen. Type III mutants inoculated intranasally exhibited only a minor defect in replication and survival in the lung, but
popB
and
exoSTY
triple mutants were profoundly defective in their ability to spread systemically. Intravenous injection of the mutants indicated that the type III secretion machinery is required for survival in the blood. Furthermore, our findings suggest that the effector-independent
popB
-dependent cytotoxicity that we and others have observed in vitro in macrophage cell lines may not be of great importance in vivo.
Plasmodium falciparumMalaria: Reduction of Endothelial Cell Apoptosis In Vitro
Authors: Hemmer Christoph Josef , Lehr Hans Anton , Westphal Kathi , Unverricht Marcus , Kratzius Manja , Reisinger Emil Christian
Published: February 2005

Organ failure inPlasmodium falciparummalaria is associated with neutrophil activation and endothelial damage. This study investigates whether neutrophil-induced endothelial damage involves apoptosis and whether it can be prevented by neutralization of neutrophil secretory products. Endothelial cells from human umbilical veins were coincubated with neutrophils from healthy donors and with sera from eight patients withP. falciparummalaria, three patients withP. vivaxmalaria, and three healthy controls. Endothelial apoptosis was demonstrated by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and annexin V staining. The rate of apoptosis of cells was markedly increased after incubation with patient serum compared to that with control serum. Apoptosis was most pronounced after incubation with sera from two patients with fatal cases ofP. falciparummalaria, followed by sera of survivors with severeP. falciparummalaria and, finally, by sera of patients with mildP. falciparumandP. vivaxmalaria. Ascorbic acid, tocopherol, and ulinastatin reduced the apoptosis rate, but gabexate mesilate and pentoxifylline did not. Furthermore, in fatalP. falciparummalaria, apoptotic endothelial cells were identified in renal and pulmonary tissue by TUNEL staining. These findings show that apoptosis caused by neutrophil secretory products plays a major role in endothelial cell damage in malaria. The antioxidants ascorbic acid and tocopherol and the protease inhibitor ulinastatin can reduce malaria-associated endothelial apoptosis in vitro.
Phenotypic Switching ofCryptococcus neoformansCan Produce Variants That Elicit Increased Intracranial Pressure in a Rat Model of Cryptococcal Meningoencephalitis
Authors: Fries B. C. , Lee S. C. , Kennan R. , Zhao W. , Casadevall A. , Goldman D. L.
Published: February 2005

Increased intracranial pressure (ICP) plays an important role in the morbidity and mortality of cryptococcal meningoencephalitis. The microbial and host factors that contribute to the development of increased ICP are poorly understood. We found that phenotypic switch variants ofCryptococcus neoformans(smooth and mucoid) differed in their abilities to promote increased ICP in a rat model of cryptococcal meningitis. Rats infected with the mucoid variant developed increased ICP, whereas rats infected with the smooth parent did not. This trend correlated with a shorter survival time and a higher cerebrospinal fluid (CSF) fungal burden for mucoid variant-infected rats, although brain fungal burdens were comparable between mucoid variant- and smooth parent-infected rats. Magnetic resonance imaging revealed enhanced T2 signal intensity over the surfaces of the brains of mucoid variant-infected rats. In addition, more polysaccharide accumulated in the CSF and brains of mucoid variant-infected rats. The accumulation of glucorunoxylomannan was associated with elevated levels of MCP-1 (CCL2) and, accordingly, a more pronounced but ineffective monocytic inflammatory response in the meninges of mucoid variant-infected rats. In summary, these findings suggest that strain-specific characteristics can influence the development of increased ICP and indicate a manner in which phenotypic switching could influence the outcome of a central nervous system infection.
The Gamma Interferon Receptor Is Required for the Protective Pulmonary Inflammatory Response toCryptococcus neoformans
Authors: Chen Gwo-Hsiao , McDonald Roderick A. , Wells Jason C. , Huffnagle Gary B. , Lukacs Nicholas W. , Toews Galen B.
Published: February 2005

Mice with a null deletion mutation in the gamma interferon (IFN-γ) receptor gene were used to study the role of IFN-γ responsiveness during experimental pulmonary cryptococcosis.Cryptococcus neoformanswas inoculated intratracheally into mice lacking the IFN-γ receptor gene (IFN-γR−/−) and into control mice (IFN-γR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-γR−/−mice are markedly more susceptible toC. neoformansinfection than IFN-γR+/+mice. (ii) In the absence of IFN-γ signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-γ receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-γR−/−mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-γ signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- γ responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-γ is not affected, IFN-γR−/−mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-γ signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.
Use of Genome-Wide Expression Profiling and Mutagenesis To Study the Intestinal Lifestyle of
Campylobacter jejuni
Authors: Stintzi Alain , Marlow Denver , Palyada Kiran , Naikare Hemant , Panciera Roger , Whitworth Lisa , Clarke Cyril
Published: February 2005

Campylobacter jejuni
is the most common bacterial cause of diarrhea worldwide. To colonize the gut and cause infection,
C. jejuni
must successfully compete with endogenous microbes for nutrients, resist host defenses, persist in the intestine, and ultimately infect the host. These challenges require the expression of a battery of colonization and virulence determinants. In this study, the intestinal lifestyle of
C. jejuni
was studied using whole-genome microarray, mutagenesis, and a rabbit ileal loop model. Genes associated with a wide range of metabolic, morphological, and pathological processes were expressed in vivo. The in vivo transcriptome of
C. jejuni
reflected its oxygen-limited, nutrient-poor, and hyperosmotic environment. Strikingly, the expression of several
C. jejuni
genes was found to be highly variable between individual rabbits. In particular, differential gene expression suggested that
C. jejuni
extensively remodels its envelope in vivo by differentially expressing its membrane proteins and by modifying its peptidoglycan and glycosylation composition. Furthermore, mutational analysis of seven genes,
hspR
,
hrcA
,
spoT
, Cj0571, Cj0178, Cj0341, and
fliD
, revealed an important role for the stringent and heat shock response in gut colonization. Overall, this study provides new insights on the mechanisms of gut colonization, as well as possible strategies employed by
Campylobacter
to resist or evade the host immune responses.
Biofilm Formation,
icaADBC
Transcription, and Polysaccharide Intercellular Adhesin Synthesis by Staphylococci in a Device-Related Infection Model
Authors: Fluckiger Ursula , Ulrich Martina , Steinhuber Andrea , Döring Gerd , Mack Dietrich , Landmann Regine , Goerke Christiane , Wolz Christiane
Published: February 2005

Biofilm formation of
Staphylococcus epidermidis
and
S. aureus
is mediated by the polysaccharide intercellular adhesin (PIA) encoded by the
ica
operon. We used a device-related animal model to investigate biofilm formation, PIA expression (immunofluorescence), and
ica
transcription (quantitative transcript analysis) throughout the course of infection by using two prototypic
S. aureus
strains and one
S. epidermidis
strain as well as corresponding
ica
mutants. During infection, the
ica
mutants were growth attenuated when inoculated in competition with the corresponding wild-type strains but not when grown singly. A typical biofilm was observed at the late course of infection. Only in
S. aureus
RN6390, not in
S. aureus
Newman, were PIA and
ica-
specific transcripts detectable after anaerobic growth in vitro. However, both
S. aureus
strains were PIA positive in vivo by day 8 of infection.
ica
transcription preceded PIA expression and biofilm formation in vivo. In
S. epidermidis
, both PIA and
ica
expression levels were elevated compared to those in the
S. aureus
strains in vitro as well as in vivo and were detectable throughout the course of infection. In conclusion, in
S. aureus
, PIA expression is dependent on the genetic background of the strain as well as on strong inducing conditions, such as those dominating in vivo. In
S. epidermidis
, PIA expression is elevated and less vulnerable to environmental conditions.
Both CD1d Antigen Presentation and Interleukin-12 Are Required To Activate Natural Killer T Cells duringTrypanosoma cruziInfection
Authors: Duthie Malcolm S. , Kahn Maria , White Maria , Kapur Raj P. , Kahn Stuart J.
Published: February 2005

Mechanisms of natural killer T (NKT)-cell activation remain unclear. Here, we report that duringTrypanosoma cruziinfection, interleukin-12 (IL-12) deficiency or anti-CD1d antibody treatment prevents normal activation. The required IL-12 arises independently of MyD88. The data support a model of normal NKT-cell activation that requires IL-12 and TCR stimulation.
Signature for Long-Term Vaccine-Mediated Control of a Simian and Human Immunodeficiency Virus 89.6P Challenge: Stable Low-Breadth and Low-Frequency T-Cell Response Capable of Coproducing Gamma Interferon and Interleukin-2
Authors: Sadagopal Shanmugalakshmi , Amara Rama Rao , Montefiori David C. , Wyatt Linda S. , Staprans Silvija I. , Kozyr Natalia L. , McClure Harold M. , Moss Bernard , Robinson Harriet L.
Published: February 2005

In 2001, we reported 20 weeks of control of challenge with the virulent 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) by a Gag-Pol-Env vaccine consisting of DNA priming and modified vaccinia virus Ankara boosting. Here we report that 22 out of 23 of these animals successfully controlled their viremia until their time of euthanasia at 200 weeks postchallenge. At euthanasia, all animals had low to undetectable viral loads and normal CD4 counts. During the long period of viral control, gamma interferon (IFN-γ)-producing antiviral T cells were present at unexpectedly low breadths and frequencies. Most animals recognized two CD8 and one CD4 epitope and had frequencies of IFN-γ-responding T cells from 0.01 to 0.3% of total CD8 or CD4 T cells. T-cell responses were remarkably stable over time and, unlike responses in most immunodeficiency virus infections, maintained good functional characteristics, as evidenced by coproduction of IFN-γ and interleukin-2. Overall, high titers of binding and neutralizing antibody persisted throughout the postchallenge period. Encouragingly, long-term control was effective in macaques of diverse histocompatibility types.
Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes
Authors: Brunner Jo Ellen , Nguyen Joseph H. C. , Roehl Holger H. , Ho Tri V. , Swiderek Kristine M. , Semler Bert L.
Published: February 2005

We had previously demonstrated that a cellular protein specifically interacts with the 3′ end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione
S
-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D
pol
, as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1ΔC) diminished these protein-protein interactions. When GST/hnRNP C1ΔC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.
Single-Amino-Acid Substitutions in Open Reading Frame (ORF) 1b-nsp14 and ORF 2a Proteins of the Coronavirus Mouse Hepatitis Virus Are Attenuating in Mice
Authors: Sperry Steven M. , Kazi Lubna , Graham Rachel L. , Baric Ralph S. , Weiss Susan R. , Denison Mark R.
Published: February 2005

A reverse genetic system was recently established for the coronavirus mouse hepatitis virus strain A59 (MHV-A59), in which cDNA fragments of the RNA genome are assembled in vitro into a full-length genome cDNA, followed by electroporation of in vitro-transcribed genome RNA into cells with recovery of viable virus. The “in vitro-assembled” wild-type MHV-A59 virus (icMHV-A59) demonstrated replication identical to laboratory strains of MHV-A59 in tissue culture; however, icMHV-A59 was avirulent following intracranial inoculation of C57BL/6 mice. Sequencing of the cloned genome cDNA fragments identified two single-nucleotide mutations in cloned genome fragment F, encoding a Tyr6398His substitution in open reading frame (ORF) 1b p59-nsp14 and a Leu94Pro substitution in the ORF 2a 30-kDa protein. The mutations were repaired individually and together in recombinant viruses, all of which demonstrated wild-type replication in tissue culture. Following intracranial inoculation of mice, the viruses encoding Tyr6398His/Leu94Pro substitutions and the Tyr6398His substitution alone demonstrated log
10
50% lethal dose (LD
50
) values too great to be measured. The Leu94Pro mutant virus had reduced but measurable log
10
LD
50
, and the “corrected” Tyr6398/Leu94 virus had a log
10
LD
50
identical to wild-type MHV-A59. The experiments have defined residues in ORF 1b and ORF 2a that attenuate virus replication and virulence in mice but do not affect in vitro replication. The results suggest that these proteins serve roles in pathogenesis or virus survival in vivo distinct from functions in virus replication. The study also demonstrates the usefulness of the reverse genetic system to confirm the role of residues or proteins in coronavirus replication and pathogenesis.
Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication
Authors: Daikoku Tohru , Kudoh Ayumi , Fujita Masatoshi , Sugaya Yutaka , Isomura Hiroki , Shirata Noriko , Tsurumi Tatsuya
Published: February 2005

Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1
oriLyt
binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.
Systematic Pathogenesis and Replication of Avian Hepatitis E Virus in Specific-Pathogen-Free Adult Chickens
Authors: Billam P. , Huang F. F. , Sun Z. F. , Pierson F. W. , Duncan R. B. , Elvinger F. , Guenette D. K. , Toth T. E. , Meng X. J.
Published: February 2005

Hepatitis E virus (HEV) is an important human pathogen. Due to the lack of a cell culture system and a practical animal model for HEV, little is known about its pathogenesis and replication. The discovery of a strain of HEV in chickens, designated avian HEV, prompted us to evaluate chickens as a model for the study of HEV. Eighty-five 60-week-old specific-pathogen-free chickens were randomly divided into three groups. Group 1 chickens (
n
= 28) were each inoculated with 5 × 10
4.5
50% chicken infectious doses of avian HEV by the oronasal route, group 2 chickens (
n
= 29) were each inoculated with the same dose by the intravenous (i.v.) route, and group 3 chickens (
n
= 28) were not inoculated and were used as controls. Two chickens from each group were necropsied at 1, 3, 5, 7, 10, 13, 16, 20, 24, 28, 35, and 42 days postinoculation (dpi), and the remaining chickens were necropsied at 56 dpi. Serum, fecal, and various tissue samples, including liver and spleen samples, were collected at each necropsy for pathological and virological testing. By 21 dpi, all oronasally and i.v. inoculated chickens had seroconverted. Fecal virus shedding was detected variably from 1 to 20 dpi for the i.v. group and from 10 to 56 dpi for the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples from both i.v. and oronasally inoculated chickens. Gross liver lesions, characterized by subcapsular hemorrhages or enlargement of the right intermediate lobe, were observed in 7 of 28 oronasally and 7 of 29 i.v. inoculated chickens. Microscopic liver lesions were mainly lymphocytic periphlebitis and phlebitis. The lesion scores were higher for oronasal (
P
= 0.0008) and i.v. (
P
= 0.0029) group birds than for control birds. Slight elevations of the plasma liver enzyme lactate dehydrogenase were observed in infected chickens. The results indicated that chickens are a useful model for studying HEV replication and pathogenesis. This is the first report of HEV transmission via its natural route in a homologous animal model.
A Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 ORF50 Deletion Mutant Is Defective for Reactivation of Latent Virus and DNA Replication
Authors: Xu Yiyang , AuCoin David P. , Huete Alicia Rodriguez , Cei Sylvia A. , Hanson Lisa J. , Pari Gregory S.
Published: February 2005

Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36Δ50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta intransin BAC36Δ50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36Δ50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36Δ50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36Δ50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.
Biological Differences between Vesicular Stomatitis Virus Indiana and New Jersey Serotype Glycoproteins: Identification of Amino Acid Residues Modulating pH-Dependent Infectivity
Authors: Martinez Isidoro , Wertz Gail W.
Published: February 2005

We previously generated recombinant vesicular stomatitis viruses (VSV) based on the Indiana serotype genome which contained either the homologous glycoprotein gene from the Indiana serotype (VSIV-GI) or the heterologous glycoprotein gene from the New Jersey serotype (VSIV-GNJ). The virus expressing the GNJgene was more pathogenic than the parental VSIV-GIvirus in swine, a natural host (26). For the present study, we investigated the biological differences between the GIand GNJproteins that may be related to the differences in pathogenesis between VSIV-GIand VSIV-GNJ. We show that the capacities of viruses with either the GNJor GIglycoprotein to infect cultured cells differ depending on the pH. VSIV-GNJcould infect cells at acidic pHs, while the infectivity of VSIV-GIwas severely reduced. VSIV-GNJinfection was also more sensitive to inhibition by ammonium chloride, indicating that the GNJprotein had a lower pH threshold for membrane fusion. We applied selective pressure to VSIV-GIby growing it at successively lower pH values and isolated variant viruses in which we identified amino acid changes that conferred low-pH-resistant infectivity. Repeated passage in cell culture at pH 6.8 resulted in the selection of a VSIV-GIvariant (VSIV-6.8) that was similar to VSIV-GNJregarding its pH- and ammonium chloride-dependent infectivity. Sequence analysis of VSIV-6.8 revealed that it had a single amino acid substitution in the amino-terminal region of the glycoprotein (F18L). This alteration was shown to be responsible for the observed phenotype by site-directed mutagenesis of a VSIV-GIfull-length cDNA and analysis of the recovered engineered virus. A further adaptation of VSIV-6.8 to pHs 6.6 and 6.4 resulted in additional amino acid substitutions in areas of the glycoprotein that were not previously implicated in attachment or fusion.
The Influenza A Virus M2Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging
Authors: McCown Matthew F. , Pekosz Andrew
Published: February 2005

The M2integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2protein intransrestored the replication of the M2truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.
Analysis of Nucleotide Sequences and Multimeric Forms of a Novel Satellite RNA Associated with Beet Black Scorch Virus
Authors: Guo Li-Hua , Cao Yun-He , Li Da-Wei , Niu Sheng-Niao , Cai Zhu-Nan , Han Cheng-Gui , Zhai Ya-Feng , Yu Jia-Lin
Published: February 2005

The full-length sequence of a satellite RNA (sat-RNA) of
Beet black scorch virus
isolate X (BBSV-X) was determined. This agent is 615 nucleotides long and lacks extensive sequence homology with its helper virus or with other reported viruses. Purified virus particles contained abundant single-stranded plus-sense monomers and smaller amounts of dimers. Single-stranded RNAs from total plant RNA extracts also included primarily monomers and smaller amounts of dimers that could be revealed by hybridization, and preparations of purified double-stranded RNAs also contained monomers and dimers. Coinoculation of in vitro transcripts of sat-RNA to
Chenopodium amaranticolor
with BBSV RNAs was used to assess the replication and accumulation of various forms of sat-RNA, including monomers, dimers, and tetramers. Dimeric sat-RNAs with 5- or 10-base deletions or 15-base insertions within the junction regions accumulated preferentially. In contrast, the replication of monomeric sat-RNA was severely inhibited by five-nucleotide deletions in either the 5′ or the 3′ termini. Therefore, sequences at both the 5′ and the 3′ ends of the monomers or the presence of intact juxtaposed multimers is essential for the replication of sat-RNA and for the predomination of monomeric progeny. Comparisons of the time courses of replication initiated by in vitro-synthesized monomeric or multimeric sat-RNAs raised the possibility that the dimeric form has an intermediate role in replication. We propose that replication primarily involves multimers, possibly as dimeric forms. These forms may revert to monomers by a termination of replication at 5′ end sequences and/or by internal initiation at the 3′ ends of multimeric junctions.
Amsacta moorei
Entomopoxvirus Inhibitor of Apoptosis Suppresses Cell Death by Binding Grim and Hid
Authors: Li Qianjun , Liston Peter , Schokman Natasha , Ho Jenny Mei , Moyer Richard W.
Published: February 2005

Inhibitor of apoptosis (
iap
) genes have been identified in the genomes of two independent families of insect viruses, the
Baculoviridae
and the
Entomopoxvirinae
. In this report, we examined the functional attributes of the
Amsacta moorei
entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic
Drosophila
proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.
Characterization of Highly Pathogenic H5N1 Avian Influenza A Viruses Isolated from South Korea
Authors: Lee Chang-Won , Suarez David L. , Tumpey Terrence M. , Sung Haan-Woo , Kwon Yong-Kuk , Lee Youn-Jeong , Choi Jun-Gu , Joh Seong-Joon , Kim Min-Chul , Lee Eun-Kyoung , Park Jong-Myung , Lu Xiuhua , Katz Jacqueline M. , Spackman Erica , Swayne David E. , Kim Jae-Hong
Published: February 2005

An unprecedented outbreak of H5N1 highly pathogenic avian influenza (HPAI) has been reported for poultry in eight different Asian countries, including South Korea, since December 2003. A phylogenetic analysis of the eight viral genes showed that the H5N1 poultry isolates from South Korea were of avian origin and contained the hemagglutinin and neuraminidase genes of the A/goose/Guangdong/1/96 (Gs/Gd) lineage. The current H5N1 strains in Asia, including the Korean isolates, share a gene constellation similar to that of the Penfold Park, Hong Kong, isolates from late 2002 and contain some molecular markers that seem to have been fixed in the Gs/Gd lineage virus since 2001. However, despite genetic similarities among recent H5N1 isolates, the topology of the phylogenetic tree clearly differentiates the Korean isolates from the Vietnamese and Thai isolates which have been reported to infect humans. A representative Korean isolate was inoculated into mice, with no mortality and no virus being isolated from the brain, although high titers of virus were observed in the lungs. The same isolate, however, caused systemic infections in chickens and quail and killed all of the birds within 2 and 4 days of intranasal inoculation, respectively. This isolate also replicated in multiple organs and tissues of ducks and caused some mortality. However, lower virus titers were observed in all corresponding tissues of ducks than in chicken and quail tissues, and the histological lesions were restricted to the respiratory tract. This study characterizes the molecular and biological properties of the H5N1 HPAI viruses from South Korea and emphasizes the need for comparative analyses of the H5N1 isolates from different countries to help elucidate the risk of a human pandemic from the strains of H5N1 HPAI currently circulating in Asia.