bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a DtxR-Regulated Operon That Is Essential for Siderophore-Dependent Iron Uptake inCorynebacterium diphtheriae
Authors: Qian Yilei , Lee John H. , Holmes Randall K.
Published: October 2002

The diphtheria toxin repressor (DtxR) uses Fe2+as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) inCorynebacterium diphtheriae. A new IRP, designated IRP6, was cloned fromC. diphtheriaeby a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in anEscherichia colisystem was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-typeC. diphtheriaeC7(β), but they were expressed constitutively in C7(β) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-typeirp6operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-typeirp1operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles ofirp6A,irp6C, andirp6Bwere responsible for the phenotypes of HC1, HC4, and HC5, respectively. Theirp6Aallele in HC1 and theirp6Ballele in HC5 encoded single amino acid substitutions in their predicted protein products, and theirp6Callele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation indtxRin addition to a missense mutation in itsirp6Ballele. These findings demonstrated that theirp6operon inC. diphtheriaeencodes a putative ABC transporter, that specific mutant alleles ofirp6A,irp6B, andirp6Care associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by theirp6operon.
A Novel Histidine-Rich CPx-ATPase from the Filamentous Cyanobacterium
Oscillatoria brevis
Related to Multiple-Heavy-Metal Cotolerance
Authors: Tong Liu , Nakashima Susumu , Shibasaka Mineo , Katsuhara Maki , Kasamo Kunihiro
Published: October 2002

A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium
Oscillatoria brevis
. Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases. The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix. However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site. Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu
+
and Ag
+
) and divalent (Zn
2+
and Cd
2+
) heavy-metal ions at similar levels. Experiments on heavy-metal tolerance in
Escherichia coli
with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals. This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.
Temperature- and H-NS-Dependent Regulation of a Plasmid-Encoded Virulence Operon Expressing
Escherichia coli
Hemolysin
Authors: Madrid Cristina , Nieto José M. , Paytubi Sònia , Falconi Maurizio , Gualerzi Claudio O. , Juárez Antonio
Published: October 2002

Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated
hly
operon of
Escherichia coli.
We have been able to identify two H-NS binding sites in the
hly
regulatory region. One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I). In contrast, Hha protein did not show any preference for specific sequences. In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of
hly
operon transcription. Deletion analysis of the
hly
regulatory region confirms the relevance of site I for thermoregulation of this operon. We present a model to explain the temperature-modulated repression of the
hly
operon, based on the experiments reported here and other, preexisting data.
A LuxR Homolog Controls Production of Symbiotically Active Extracellular Polysaccharide II by
Sinorhizobium meliloti
Authors: Pellock Brett J. , Teplitski Max , Boinay Ryan P. , Bauer W. Dietz , Walker Graham C.
Published: October 2002

Production of complex extracellular polysaccharides (EPSs) by the nitrogen-fixing soil bacterium
Sinorhizobium meliloti
is required for efficient invasion of root nodules on the host plant alfalfa. Any one of three
S. meliloti
polysaccharides, succinoglycan, EPS II, or K antigen, can mediate infection thread initiation and extension (root nodule invasion) on alfalfa. Of these three polysaccharides, the only symbiotically active polysaccharide produced by
S. meliloti
wild-type strain Rm1021 is succinoglycan. The
expR101
mutation is required to turn on production of symbiotically active forms of EPS II in strain Rm1021. In this study, we have determined the nature of the
expR101
mutation in
S. meliloti
. The
expR101
mutation, a spontaneous dominant mutation, results from precise, reading frame-restoring excision of an insertion sequence from the coding region of
expR
, a gene whose predicted protein product is highly homologous to the
Rhizobium leguminosarum
bv. viciae RhiR protein and a number of other homologs of
Vibrio fischeri
LuxR that function as receptors for
N
-acylhomoserine lactones (AHLs) in quorum-sensing regulation of gene expression.
S. meliloti
ExpR activates transcription of genes involved in EPS II production in a density-dependent fashion, and it does so at much lower cell densities than many quorum-sensing systems. High-pressure liquid chromatographic fractionation of
S. meliloti
culture filtrate extracts revealed at least three peaks with AHL activity, one of which activated ExpR-dependent expression of the
expE
operon.
Differential Expression and Localization of Mn and Fe Superoxide Dismutases in the Heterocystous Cyanobacterium
Anabaena
sp. Strain PCC 7120
Authors: Li Tao , Huang Xu , Zhou Ruanbao , Liu Yingfang , Li Bin , Nomura Chris , Zhao Jindong
Published: October 2002

Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms. The nitrogen-fixing heterocystous cyanobacterium
Anabaena
sp. strain PCC 7120 has two Sod-encoding genes: a
sodB
, encoding a soluble iron-containing Sod (FeSod), and a
sodA
, encoding a manganese-containing Sod (MnSod). The FeSod was purified and characterized. A recombinant FeSod was also obtained by overproduction in
Escherichia coli.
Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six- to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition. However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen. Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the
sodB
gene expression. The predicted preprotein of the
sodA
gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein. Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in
Anabaena
sp. strain PCC 7120 was located on thylakoids toward the lumenal side. Expression of the
sodA
gene in
E. coli
shows that the leader peptide was required for its activity and the membrane localization of the MnSod. Northern hybridization detected one 0.82-kb transcript of
sodA
. The
sodA
gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation. Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts. No MnSod was observed in isolated heterocysts. These results show that the two different Sod proteins have differentiated roles in
Anabaena
sp. strain PCC 7120.
Utility of Gram Staining for Evaluation of the Quality of Cystic Fibrosis Sputum Samples
Authors: Nair Bindu , Stapp Jenny , Stapp Lynn , Bugni Linda , Van Dalfsen Jill , Burns Jane L.
Published: October 2002

The microscopic examination of Gram-stained sputum specimens is very helpful in the evaluation of patients with community-acquired pneumonia and has also been recommended for use in cystic fibrosis (CF) patients. This study was undertaken to evaluate that recommendation. One hundred one sputum samples from CF patients were cultured for gram-negative bacilli and examined by Gram staining for both sputum adequacy (using the quality [Q] score) and bacterial morphology. Subjective evaluation of adequacy was also performed and categorized. Based on Q score evaluation, 41% of the samples would have been rejected despite a subjective appearance of purulence. Only three of these rejected samples were culture negative for gram-negative CF pathogens. Correlation between culture results and quantitative Gram stain examination was also poor. These data suggest that subjective evaluation combined with comprehensive bacteriology is superior to Gram staining in identifying pathogens in CF sputum.
In Vitro Comparison of Activities of Terbinafine and Itraconazole against
Paracoccidioides brasiliensis
Authors: Hahn R. C. , Fontes C. J. F. , Batista R. D. , Hamdan J. S.
Published: October 2002

In vitro, terbinafine is highly active against a broad spectrum of pathogenic fungi. We evaluated the activities of terbinafine and itraconazole against 31 isolates of
Paracoccidioides brasiliensis.
The tests were conducted by using a broth macrodilution procedure. MICs, in micrograms per milliliter, were as follows: terbinafine, 0.015 to 1.0 (geometric mean, 0.1188); itraconazole, 0.007 to 0.5 (geometric mean, 0.03165). The usual therapy for paracoccidioidomycosis is sulfonamides, amphotericin B, and azole derivatives (ketoconazole, itraconazole, and fluconazole). In comparison to amphotericin B, azole derivatives allow shorter treatment courses, can be administered orally, and are equally effective. Itraconazole has as high efficacy as ketoconazole, but with superior tolerance. It is the current drug of choice for treatment of paracoccidioidomycosis. The data obtained in this study indicate that terbinafine is active against
P. brasiliensis
in vitro and suggest that this allylamine can be considered a new option as drug therapy for paracoccidioidomycosis.
Inactivation of West Nile Virus during Serologic Testing and Transport
Authors: Mayo Donald R. , Beckwith William H.
Published: October 2002

Inactivation of West Nile virus (WNV) in enzyme-linked immunosorbent assay (ELISA) wash buffer at 37°C was studied, as well as inactivation of WNV in cell culture medium over several days at an ambient temperature (28°C). Aliquots of WNV were removed from the 37°C ELISA wash buffer at 5, 15, 30, and 60 min for the former experiment, while daily aliquots of medium were sampled for the latter experiment. No virus was detected in the wash buffer at 30 and 60 min, while virus was readily detected from cell culture medium over this time. In addition, titers of WNV consistently dropped over a 7-day period at 28°C compared to control suspensions of virus held at 4°C. These observations indicate that WNV is readily inactivated in the presence of detergent-containing buffers. Furthermore, the viability loss at ambient temperature suggests that WNV is easily inactivated during routine transportation and testing of human body fluids such as serum and cerebrospinal fluid.
Corneal Ulcer Caused by the New Fungal SpeciesSarcopodium oculorum
Authors: Guarro Josep , Höfling-Lima Ana Luisa , Gené Josepa , De Freitas Denise , Godoy Patricio , Zorat-Yu Maria Luisa , Zaror Luis , Fischman Olga
Published: October 2002

We describe a case of keratitis caused by a new species of the hyphomycetous genusSarcopodium,S. oculorum. The corneal ulcer developed after 5 months of treatment with corticosteroids in a Brazilian boy diagnosed with allergic conjunctivitis. Fungal hyphae and conidia were detected in corneal scrapings, and repeated cultures were positive for this fungus. The infection was resolved with natamycin and ketoconazole. Eleven antifungals were tested against this fungus, and all except flucytosine and fluconazole showed in vitro activity.
Chronic Diarrhea, Hemorrhagic Colitis, and Hemolytic-Uremic Syndrome Associated with HEp-2 Adherent
Escherichia coli
in Adults Infected with Human Immunodeficiency Virus in Bangui, Central African Republic
Authors: Mossoro Christian , Glaziou Philippe , Yassibanda Simon , Lan Nguyen Thi Phuong , Bekondi Claudine , Minssart Pierre , Bernier Christine , Le Bouguénec Chantal , Germani Yves
Published: October 2002

In human immunodeficiency virus (HIV)-infected adults from the Central African Republic, the occurrence of chronic diarrhea due to HEp-2 adherent
Escherichia coli
(EAEC) harboring virulence markers (
eaeA
, BFP, EAF,
astA
determinant of EAST/1, positive FAS test, enteropathogenic
E. coli
O serogroup) was shown to be associated with AIDS. We also show that EAEC that produce verotoxin (Stx2) but do not harbor the genetic markers for classical enterohemorrhagic
E. coli
are involved in hemorrhagic colitis and hemolytic-uremic syndrome in patients with HIV.
Unique Case of
Helicobacter
sp. Osteomyelitis in an Immunocompetent Child Diagnosed by Broad-Range 16S PCR
Authors: Harris Kathryn A. , Fidler Katy J. , Hartley John C. , Vogt Julie , Klein Nigel J. , Monsell Fergal , Novelli Vas M.
Published: October 2002

We report the first case of
Helicobacter
sp. osteomyelitis in an immunocompetent child. The infection was diagnosed by broad-range 16S PCR followed by sequencing of the resulting amplicon. All other microbiological investigations proved negative. This case highlights the importance of molecular methods in the diagnosis of unsuspected etiological agents and the potential role of
Helicobacter
sp. in human infection.
Identification of VP1/2A and 2C as Virulence Genes of Hepatitis A Virus and Demonstration of Genetic Instability of 2C
Authors: Emerson Suzanne U. , Huang Ying K. , Nguyen Hanh , Brockington Alicia , Govindarajan Sugantha , St. Claire Marisa , Shapiro Max , Purcell Robert H.
Published: October 2002

Fourteen different chimeric virus genomes were constructed from two infectious cDNA clones encoding a virulent and an attenuated isolate, respectively, of the HM175 strain of hepatitis A virus. The ability of each recombinant virus to infect tamarins and to cause acute hepatitis was determined. Comparisons of the genotype and phenotype of each virus suggested that VP1/2A and 2C genes were responsible for virulence. The 2C gene derived from the attenuated parent virus was unstable, and one or more mutations arose in this gene during the first passage in tamarins.
Mechanisms of Inhibition of Nuclear Hormone Receptor-Dependent Hepatitis B Virus Replication by Hepatocyte Nuclear Factor 3β
Authors: Tang Hong , McLachlan Alan
Published: October 2002

The nuclear hormone receptors hepatocyte nuclear factor 4 (HNF4) and the retinoid X α (RXRα) plus the peroxisome proliferator-activated receptor α (PPARα) heterodimer support hepatitis B virus (HBV) replication in nonhepatoma cells. Hepatocyte nuclear factor 3 (HNF3) inhibits nuclear hormone receptor-mediated viral replication. Inhibition of HBV replication by HNF3β is associated with the preferential reduction in the level of the pregenomic RNA compared with that of precore RNA. Hepatitis B e antigen (HBeAg), encoded by the precore RNA, mediates part of the inhibition of viral replication by HNF3β. The amino-terminal transcriptional activation domain of HNF3β is essential for the inhibition of HBV replication. The activation of transcription by HNF3 from HBV promoters downstream from the nucleocapsid promoter appears to contribute indirectly to the reduction in the steady-state level of 3.5-kb HBV RNA, possibly by interfering with the elongation rate of these transcripts. Therefore, transcriptional interference mediated by HNF3 may also regulate HBV RNA synthesis and viral replication.
Modulation of Adenovirus Vector Tropism via Incorporation of Polypeptide Ligands into the Fiber Protein
Authors: Belousova Natalya , Krendelchtchikova Valentina , Curiel David T. , Krasnykh Victor
Published: October 2002

The efficacy of adenovirus (Ad)-based gene therapy might be significantly improved if viral vectors capable of tissue-specific gene delivery could be developed. Previous attempts to genetically modify the tropism of Ad vectors have been only partially successful, largely due to the limited repertoire of ligands that can be incorporated into the Ad capsid. Early studies identified stringent size limitations imposed by the structure of the Ad fiber protein on ligands incorporated into its carboxy terminus and thus limited the range of potential ligand candidates to short peptides. We have previously identified the HI loop of the fiber knob domain as a preferred site for the incorporation of targeting ligands and hypothesized that the structural properties of this loop would allow for the insertion of a wide variety of ligands, including large polypeptide molecules. In the present study we have tested this hypothesis by deriving a family of Ad vectors whose fibers contain polypeptide inserts of incrementally increasing lengths. By assessing the levels of productivity and infectivity and the receptor specificities of the resultant viruses, we show that polypeptide sequences exceeding by 50% the size of the knob domain can be incorporated into the fiber with only marginal negative consequences on these key properties of the vectors. Our study has also revealed a negative correlation between the size of the ligand used for vector modification and the infectivity and yield of the resultant virus, thereby predicting the limits beyond which further enlargement of the fiber knob would not be compatible with the virion's integrity.
Nelfinavir-Resistant, Amprenavir-Hypersusceptible Strains of Human Immunodeficiency Virus Type 1 Carrying an N88S Mutation in Protease Have Reduced Infectivity, Reduced Replication Capacity, and Reduced Fitness and Process the Gag Polyprotein Precursor Aberrantly
Authors: Resch Wolfgang , Ziermann Rainer , Parkin Neil , Gamarnik Andrea , Swanstrom Ronald
Published: October 2002

The evolution of human immunodeficiency virus type 1 (HIV-1) strains with reduced susceptibility to protease inhibitors (PIs) is a major cause of PI treatment failure. A subset of subjects failing a therapy regimen containing the PI nelfinavir developed mutations at position 88 in the protease region. The N88S mutation occurring in some of these subjects induces amprenavir hypersusceptibility and a reduction of fitness and replication capacity. Here we demonstrate that substitutions L63P and V77I in protease, in combination, partially compensate for the loss of fitness, loss of replication capacity, loss of specific infectivity, and aberrant Gag processing induced by the N88S mutation. In addition, these mutations partially ablate amprenavir hypersusceptibility. Addition of mutation M46L to a strain harboring mutations L63P, V77I, and N88S resulted in a reduction of fitness and infectivity without changing Gag-processing efficiency, while amprenavir hypersusceptibility was further diminished. The ratio of reverse transcriptase activity to p24 protein was reduced in this strain compared to that in the other variants, suggesting that the M46L effect on fitness occurred through a mechanism different from a Gag-processing defect. We utilized these mutant strains to undertake a systematic comparison of indirect, single, cycle-based measures of fitness with direct, replication-based fitness assays and demonstrated that both yield consistent results. However, we observed that the magnitude of the fitness loss for one of the mutants varied depending on the assay used.
Consistent Patterns in the Development and Immunodominance of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific CD8+T-Cell Responses following Acute HIV-1 Infection
Authors: Yu Xu G. , Addo Marylyn M. , Rosenberg Eric S. , Rodriguez William R. , Lee Paul K. , Fitzpatrick Cecily A. , Johnston Mary N. , Strick Daryld , Goulder Philip J. R. , Walker Bruce D. , Altfeld Marcus
Published: October 2002

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+T-cell responses generated during acute infection play a critical role in the initial control of viremia. However, little is known about the viral T-cell epitopes targeted during acute infection or about their hierarchy in appearance and relative immunodominance over time. In this study, HIV-1-specific CD8+T-cell responses in 18 acutely infected individuals expressing HLA-A3 and/or -B7 were characterized. Detailed analysis of CD8 responses in one such person who underwent treatment of acute infection followed by reexposure to HIV-1 through supervised treatment interruptions (STI) revealed recognition of only two cytotoxic T-lymphocyte (CTL) epitopes during symptomatic acute infection. HIV-1-specific CD8+T-cell responses broadened significantly during subsequent exposure to the virus, ultimately targeting 27 distinct CTL epitopes, including 15 different CTL epitopes restricted by a single HLA class I allele (HLA-A3). The same few peptides were consistently targeted in an additional 17 persons expressing HLA-A3 and/or -B7 during acute infection. These studies demonstrate a consistent pattern in the development of epitope-specific responses restricted by a single HLA allele during acute HIV-1 infection, as well as persistence of the initial pattern of immunodominance during subsequent STI. In addition, they demonstrate that HIV-1-specific CD8+T-cell responses can ultimately target a previously unexpected and unprecedented number of epitopes in a single infected individual, even though these are not detectable during the initial exposure to virus. These studies have important implications for vaccine design and evaluation.
Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein
Authors: Zhang Xinsheng , Glendening Candace , Linke Hawley , Parks Christopher L. , Brooks Charles , Udem Stephen A. , Oglesbee Michael
Published: October 2002

The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.
Viral Interferon Regulatory Factor 1 of Kaposi's Sarcoma-Associated Herpesvirus Interacts with a Cell Death Regulator, GRIM19, and Inhibits Interferon/Retinoic Acid-Induced Cell Death
Authors: Seo Taegun , Lee Daeyoup , Shim Young Sam , Angell Jon E. , Chidambaram Natesa V. , Kalvakolanu Dhananjaya V. , Choe Joonho
Published: October 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) plays a significant role in the development of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The KSHV open reading frame K9 encodes the viral interferon (IFN) factor 1 (vIRF1), which downregulates IFN- and IRF-mediated transcriptional activation, and leads to cellular transformation in rodent fibroblasts and induction of tumors in nude mice. Using the yeast two-hybrid assay, we identified genes associated with retinoid-IFN-induced mortality-19 (GRIM19), which interacts directly with vIRF1, both in vivo and in vitro. The N-terminal region of vIRF1 is required for binding GRIM19. Colocalization of vIRF1 and GRIM19 was observed in 293T cells. The vIRF1 protein deregulates GRIM19-induced apoptosis in the presence of IFN/all-trans-retinoic acid (RA) and inhibits IFN/RA-induced cell death. Another DNA tumor viral protein, human papillomavirus type 16 E6, also binds GRIM19, suggesting that this is a general target of viral proteins. Our results collectively indicate that vIRF1 modulates IFN/RA-cell death signals via interactions with GRIM19.
Human Immunodeficiency Virus Type 1 Bound to B Cells: Relationship to Virus Replicating in CD4+T Cells and Circulating in Plasma
Authors: Malaspina Angela , Moir Susan , Nickle David C. , Donoghue Eileen T. , Ogwaro Kisani M. , Ehler Linda A. , Liu Shuying , Mican Jo Ann M. , Dybul Mark , Chun Tae-Wook , Mullins James I. , Fauci Anthony S.
Published: October 2002

Human immunodeficiency virus type 1 (HIV-1) virions bind to B cells in the peripheral blood and lymph nodes through interactions between CD21 on B cells and complement-complexed virions. B-cell-bound virions have been shown to be highly infectious, suggesting a unique mode of HIV-1 dissemination by B cells circulating between peripheral blood and lymphoid tissues. In order to investigate the relationship between B-cell-bound HIV-1 and viruses found in CD4+T cells and in plasma, we examined the genetic relationships of HIV-1 found in the blood and lymph nodes of chronically infected patients with heteroduplex mobility and tracking assays and DNA sequence analysis. In samples from 13 of 15 patients examined, HIV-1 variants in peripheral blood-derived B cells were closely related to virus in CD4+T cells and more divergent from virus in plasma. In samples from five chronically viremic patients for whom analyses were extended to include lymph node-derived HIV-1 isolates, B-cell-associated HIV-1 and CD4+-T-cell-associated HIV-1 in the lymph nodes were equivalent in their divergence from virus in peripheral blood-derived B cells and generally more distantly related to virus in peripheral blood-derived CD4+T cells. These results indicates virologic cross talk between B cells and CD4+T cells within the microenvironment of lymphoid tissues and, to a lesser extent, between cells in lymph nodes and the peripheral blood. These findings also indicate that most of the virus in plasma originates from cells other than CD4+T cells in the peripheral blood and lymph nodes.
Stabilization of the Soluble, Cleaved, Trimeric Form of the Envelope Glycoprotein Complex of Human Immunodeficiency Virus Type 1
Authors: Sanders Rogier W. , Vesanen Mika , Schuelke Norbert , Master Aditi , Schiffner Linnea , Kalyanaraman Roopa , Paluch Maciej , Berkhout Ben , Maddon Paul J. , Olson William C. , Lu Min , Moore John P.
Published: October 2002

The envelope glycoprotein (Env) complex of human immunodeficiency virus type 1 has evolved a structure that is minimally immunogenic while retaining its natural function of receptor-mediated virus-cell fusion. The Env complex is trimeric; its six individual subunits (three gp120 and three gp41 subunits) are associated by relatively weak, noncovalent interactions. The induction of neutralizing antibodies after vaccination with individual Env subunits has proven very difficult, probably because they are inadequate mimics of the native complex. Our hypothesis is that a stable form of the Env complex, perhaps with additional modifications to rationally alter its antigenic structure, may be a better immunogen than the individual subunits. A soluble form of Env, SOS gp140, can be made that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41. However, the gp41-gp41 interactions in SOS gp140 are too weak to maintain the protein in a trimeric configuration. Consequently, purified SOS gp140 is a monomer (N. Schülke, M. S. Vesanen, R. W. Sanders, P. Zhu, D. J. Anselma, A. R. Villa, P. W. H. I. Parren, J. M. Binley, K. H. Roux, P. J. Maddon, J. P. Moore, and W. C. Olson, J. Virol. 76:7760-7776, 2002). Here we describe modifications of SOS gp140 that increase its trimer stability. A variant SOS gp140, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41. This protein is fully cleaved, has favorable antigenic properties, and is predominantly trimeric. SOSIP gp140 trimers are noncovalently associated and can be partially purified by gel filtration chromatography. These gp140 trimers are dissociated into monomers by anionic detergents or heat but are relatively resistant to nonionic detergents, high salt concentrations, or exposure to a mildly acidic pH. SOSIP gp140 should be a useful reagent for structural and immunogenicity studies.
The Crown and Stem of the V3 Loop Play Distinct Roles in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Interactions with the CCR5 Coreceptor
Authors: Cormier Emmanuel G. , Dragic Tatjana
Published: October 2002

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.
The M184V Mutation in Reverse Transcriptase Can Delay Reversion of Attenuated Variants of Simian Immunodeficiency Virus
Authors: Whitney James B. , Oliveira Maureen , Detorio Mervi , Guan Yongjun , Wainberg Mark A.
Published: October 2002

We previously constructed a series of simian immunodeficiency virus (SIV) mutants containing deletions within a 97-nucleotide region of the SIVmac239 untranslated region or leader sequence. However, as is common with live attenuated viruses, several of the mutants exhibited a moderate propensity for reversion. Since the M184V mutation in human immunodeficiency virus type 1 reverse transcriptase is associated with diminished fitness as well as lamivudine resistance, we introduced this substitution into several of our deletion mutants to determine its effects on viral replication and compensatory reversion. Our results indicate that M184V impaired viral fitness in pair-wise comparisons of mutants that contained or lacked this substitution. We also observed that M184V significantly impaired the potential for both compensatory mutagenesis and reversion in these mutants both in cell lines and in peripheral blood mononuclear cells.
Inhibition of Beta Interferon Transcription by Noncytopathogenic Bovine Viral Diarrhea Virus Is through an Interferon Regulatory Factor 3-Dependent Mechanism
Authors: Baigent Susan J. , Zhang Gang , Fray Martin D. , Flick-Smith Helen , Goodbourn Stephen , McCauley John W.
Published: October 2002

The induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-α/β or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-β induction pathway have been analyzed. The transcription factor NF-κB was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-κB by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.
The Immunogenicity and Protective Efficacy of Bovine Herpesvirus 1 Glycoprotein D plus Emulsigen Are Increased by Formulation with CpG Oligodeoxynucleotides
Authors: Ioannou X. P. , Griebel P. , Hecker R. , Babiuk L. A. , van Drunen Littel-van den Hurk S.
Published: October 2002

The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.
A Balanced Type 1/Type 2 Response Is Associated with Long-Term Nonprogressive Human Immunodeficiency Virus Type 1 Infection
Authors: Imami Nesrina , Pires Antonio , Hardy Gareth , Wilson Jamie , Gazzard Brian , Gotch Frances
Published: October 2002

Previous reports have emphasized the requirements for strong type 1 cell-mediated responses in the control of human immunodeficiency virus type 1 (HIV-1). HIV-1 Gag p24-specific CD4 helper T-lymphocyte (HTL) responses have been shown to inversely correlate with viral burden in HIV-1-infected individuals. In this study, peripheral blood mononuclear cells from 70 individuals with chronic progressive HIV-1 infection (clinical progressors), 10 clinical nonprogressors, and 3 immunologically discordant progressors were assessed for HTL proliferation and type 1/type 2 cytokine production. Clinical progressors lacked functional HIV-1-specific HTLs with proliferative and cytokine-producing capacity. Clinical nonprogressors were found to respond to a wide range of HIV-1 antigens from different clades, producing both type 1 and type 2 cytokines. Immunologically discordant progressors responded strongly to clade B Gag p24 with a type 1 cytokine profile but not to other antigens. Thus, in contrast to clinical nonprogressors, neither progressors nor immunologically discordant progressors secreted interleukin-4 (IL-4) in response to HIV-1 antigens. Both clinical nonprogressors and immunologically discordant progressors responded broadly to B clade Gag p24-overlapping peptides. However, IL-4 production in the nonprogressors was restricted to a limited number of p24 peptides. No HIV-1-specific T-cell responses were seen in 20 seronegative controls. Additionally, we observed a rapid type 1 to type 2 shift in the response of one immunologically discordant progressor upon onset of clinical symptoms. These results suggest that a balanced type 1/type 2 profile correlates with successful long-term control of HIV-1.
Human Monoclonal Antibodies Specific for Conformation-Sensitive Epitopes of V3 Neutralize Human Immunodeficiency Virus Type 1 Primary Isolates from Various Clades
Authors: Gorny Miroslaw K. , Williams Constance , Volsky Barbara , Revesz Kathy , Cohen Sandra , Polonis Victoria R. , Honnen William J. , Kayman Samuel C. , Krachmarov Chavdar , Pinter Abraham , Zolla-Pazner Susan
Published: October 2002

The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3JR-CSFsequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P< 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.
Characterization of the
cis-
Acting Contributions to Avian Hepadnavirus RNA Encapsidation
Authors: Ostrow Kristin M. , Loeb Daniel D.
Published: October 2002

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two
cis
-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), ε and region II. ε, an RNA stem-loop near the 5′ end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, ε and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between ε and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between ε and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.
In Vivo Association of Adenovirus Large E1A Protein with the Human Mediator Complex in Adenovirus-Infected and -Transformed Cells
Authors: Wang Gang , Berk Arnold J.
Published: October 2002

The adenovirus large E1A protein activates transcription from early viral promoters by a mechanism that requires a forty amino acid zinc finger activation domain in E1A conserved region 3 (CR3). Recent results indicate that activation by a Gal4 DNA-binding domain-E1A-CR3 fusion requires an interaction between the E1A-CR3 zinc finger and the Sur2 subunit of the mammalian Mediator (of transcription) complex. Although several host proteins have been shown to bind stably to E1A proteins in adenovirus-infected and -transformed cells, an in vivo interaction with Mediator complex subunits has not been described previously. Using immunoprecipitation and gel filtration analyses of nuclear extracts prepared from HeLa cells infected with adenovirus 5 or mutants that express either large or small E1A specifically and from adenovirus 5-transformed cells, we report here that large E1A, but not small E1A, binds to Mediator complex in vivo. Only ∼1 to 10% of large E1A is bound to Mediator complex at 18 h postinfection and in transformed cells, probably explaining why Mediator complex subunits were not identified among cellular E1A-binding proteins described earlier. Surprisingly, even though extracted Mediator can quantitatively bind to an E1A-CR3 affinity column, only on the order of 1% of cellular Mediator complex is bound by E1A in vivo. Much of the large E1A bound to Mediator in 293 cells is in a stable complex that includes RNA polymerase II, leading us to suggest that the interaction of E1A-CR3 with Mediator stabilizes the interaction of Mediator with the polymerase. This stabilization of the interaction between Mediator and RNA polymerase II may contribute to the mechanism of activation by E1A-CR3.
Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference
Authors: Coburn Glen A. , Cullen Bryan R.
Published: October 2002

Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle.
Upregulation of Surface Feline CXCR4 Expression following Ectopic Expression of CCR5: Implications for Studies of the Cell Tropism of Feline Immunodeficiency Virus
Authors: Willett Brian J. , Cannon Celia A. , Hosie Margaret J.
Published: October 2002

Feline CXCR4 and CCR5 were expressed in feline cells as fusion proteins with enhanced green fluorescent protein (EGFP). Expression of the EGFP fusion proteins was localized to the cell membrane, and surface expression of CXCR4 was confirmed by using a cross-species-reactive anti-CXCR4 monoclonal antibody. Ectopic expression of feline CCR5 enhanced expression of either endogenous feline CXCR4 or exogenous feline or human CXCR4 expressed from a retrovirus vector, indicating that experiments investigating the effect of CCR5 expression on feline immunodeficiency virus (FIV) infection must be interpreted with caution. Susceptibility to infection with cell culture-adapted strains of FIV or to syncytium formation following transfection with a eukaryotic vector expressing an
env
gene from a cell culture-adapted strain of virus correlated with expression of either human or feline CXCR4, whereas feline CCR5 had no effect. In contrast, neither CXCR4 nor CCR5 rendered cells permissive to either productive infection with primary strains of FIV or syncytium formation following transfection with primary
env
gene expression vectors. Screening a panel of Ghost cell lines expressing diverse human chemokine receptors confirmed that CXCR4 alone supported fusion mediated by the FIV Env from cell culture-adapted viruses. CXCR4 expression was upregulated in Ghost cells coexpressing CXCR4 and CCR5 or CXCR4, CCR5, and CCR3, and susceptibility to FIV infection could be correlated with the level of CXCR4 expression. The data suggest that β-chemokine receptors may influence FIV infection by modulating the expression of CXCR4.