bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α1→2 Mannose Residues on the Outer Face of gp120
Authors: Scanlan Christopher N. , Pantophlet Ralph , Wormald Mark R. , Ollmann Saphire Erica , Stanfield Robyn , Wilson Ian A. , Katinger Hermann , Dwek Raymond A. , Rudd Pauline M. , Burton Dennis R. 
Published: July 2002

ABSTRACT

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N→A substitution produced significant decreases in 2G12 binding affinity to gp120
JR-CSF
. Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120
JR-CSF
. Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with
Aspergillus saitoi
mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Manα1→2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or
N
-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue
N
-butyl-deoxynojirimycin similarly implicated Manα1→2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Manα1→2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.
Genetic Evidence that Interhelical Packing Interactions in the gp41 Core Are Critical for Transition of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein to the Fusion-Active State
Authors: Follis Kathryn E. , Larson Scott J. , Lu Min , Nunberg Jack H. 
Published: July 2002

ABSTRACT
The envelope glycoprotein complex (gp120-gp41) of human immunodeficiency virus type 1 (HIV-1) promotes the fusion of viral and cellular membranes through formation of the fusion-active six-helix bundle in the gp41 ectodomain. This gp41 core structure consists of three C-terminal helices packed in an antiparallel manner into hydrophobic grooves on the surface of the N-terminal trimeric coiled coil. Alanine mutations that destabilize the N- and C-terminal interhelical packing interactions also reduce viral infectivity. Here we show that viruses bearing these mutations exhibit a marked potentiation of inhibition by peptides that make up the gp41 core. By contrast, these viruses are unchanged in their sensitivities to soluble CD4, the CXCR4 coreceptor ligand SDF-1α, and human anti-HIV immunoglobulin, reagents that impact the initial, receptor-induced conformational changes in the envelope glycoprotein. Our results support the notion that these alanine mutations specifically affect the conformational transition to the fusion-active gp41 structure. The mutations also increase viral sensitivity to the gp41-directed monoclonal antibody 2F5, suggesting that this broadly neutralizing antibody may also interfere with this transition. The conformational activation of the HIV-1 envelope glycoprotein likely represents a viable target for vaccine and antiviral drug development.
Analysis of Deletion Mutants Indicates that the 2A Polypeptide of Hepatitis A Virus Participates in Virion Morphogenesis
Authors: Cohen Lisette , Bénichou Danièle , Martin Annette 
Published: July 2002

ABSTRACT

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C
pro
. The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C
pro
to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C
pro
-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C
pro
and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C
pro
-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.
Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex
Authors: Ochsenbauer-Jambor Christina , Delos Sue E. , Accavitti Mary Ann , White Judith M. , Hunter Eric 
Published: July 2002

ABSTRACT
We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.
Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCR
Authors: Hüser Daniela , Weger Stefan , Heilbronn Regine 
Published: July 2002

ABSTRACT
Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.
Comparison of Predicted Scaffold-Compatible Sequence Variation in the Triple-Hairpin Structure of Human Immunodeficiency Virus Type 1 gp41 with Patient Data
Authors: Boutonnet Nathalie , Janssens Wouter , Boutton Carlo , Verschelde Jean-Luc , Heyndrickx Leo , Beirnaert Els , van der Groen Guido , Lasters Ignace 
Published: July 2002

ABSTRACT
It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.
Cell Clones Selected from the Huh7 Human Hepatoma Cell Line Support Efficient Replication of a Subgenomic GB Virus B Replicon
Authors: De Tomassi Amedeo , Pizzuti Maura , Graziani Rita , Sbardellati Andrea , Altamura Sergio , Paonessa Giacomo , Traboni Cinzia 
Published: July 2002

ABSTRACT

Tamarins (
Saguinus
species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.
Oligomeric Structure of the Human Immunodeficiency Virus Type 1 Envelope Protein on the Virion Surface
Authors: Center Rob J. , Leapman Richard D. , Lebowitz Jacob , Arthur Larry O. , Earl Patricia L. , Moss Bernard 
Published: July 2002

ABSTRACT
The envelope protein (Env) of human immunodeficiency virus type 1 forms homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained after cleavage in a Golgi compartment and transport to the surfaces of infected cells, where incorporation into budding virions takes place. Here, we use biophysical techniques to assess the oligomeric valency of virion-associated Env prior to fusion activation. Virion-associated Env oligomers were stabilized by chemical cross-linking prior to detergent extraction and were purified by immunoaffinity chromatography. Gel filtration revealed a single predominant oligomeric species, and sedimentation equilibrium analysis-derived mass values indicated a trimeric structure. Determination of the masses of individual Env molecules by scanning transmission electron microscopy demonstrated that virion-associated Env was trimeric, and a triangular morphology was observed in 20 to 30% of the molecules. These results, which firmly establish the oligomeric structure of human immunodeficiency virus virion-associated Env, parallel those of our previous analysis of the simian immunodeficiency virus Env.
Murine Cytomegalovirus (CMV) M33 and Human CMV US28 Receptors Exhibit Similar Constitutive Signaling Activities
Authors: Waldhoer Maria , Kledal Thomas N. , Farrell Helen , Schwartz Thue W. 
Published: July 2002

ABSTRACT
Cellular infection by cytomegalovirus (CMV) is associated with very early G-protein-mediated signal transduction and reprogramming of gene expression. Here we investigated the involvement of human CMV (HCMV)-encoded US27, US28, and UL33 receptors as well as murine CMV-encoded M33 transmembrane (7TM) receptors in host cell signaling mechanisms. HCMV-encoded US27 did not show any constitutive activity in any of the studied signaling pathways; in contrast, US28 and M33 displayed ligand-independent, constitutive signaling through the G protein q (Gq)/phospholipase C pathway. In addition, M33 and US28 also activated the transcription factor NF-κB as well as the cyclic AMP response element binding protein (CREB) in a ligand-independent, constitutive manner. The use of specific inhibitors indicated that the p38 mitogen-activated protein (MAP) kinase but not the extracellular signal-regulated kinase 1/2-MAP kinase pathway is involved in M33- and US28-mediated CREB activation but not NF-κB activation. Interestingly, UL33—the HCMV-encoded structural homologue of M33—was only marginally constitutively active in the Gq/phospholipase C turnover and CREB activation assays and did not show any constitutive activity in the NF-κB pathway, where M33 and US28 were highly active. Hence, CMVs appear to have conserved mechanisms for regulating host gene transcription, i.e., constitutive activation of certain kinases and transcription factors through the constitutive activities of 7TM proteins. These data, together with the previous identification of the incorporation of such proteins in the viral envelope, suggest that these proteins could be involved in the very early reprogramming of the host cell during viral infection.
The Long Noncoding Region of the Human Parainfluenza Virus Type 1 F Gene Contributes to the Read-Through Transcription at the M-F Gene Junction
Authors: Bousse Tatiana , Matrosovich Tatyana , Portner Allen , Kato Atsushi , Nagai Yoshiyuki , Takimoto Toru 
Published: July 2002

ABSTRACT

Sendai virus (SV) and human parainfluenza virus type 1 (hPIV1) have genomes consisting of nonsegmented negative-sense RNA in which the six genes are separated by well-conserved intergenic (IG) sequences and transcriptional start (S) and end signals. In hPIV1-infected cells, transcriptional termination at the M-F gene junction is ineffective; a large number of M-F read-through transcripts are produced (T. Bousse, T. Takimoto, K. G. Murti, and A. Portner, Virology
232
:44-52, 1997). In contrast, few M-F read-through transcripts are detected in SV-infected cells. Sequence analysis indicated that the hPIV1 IG and S sequences in the M-F junction differ from those of SV. Furthermore, the hPIV1 F gene contains an unusually long noncoding sequence. To identify the
cis
-acting elements that prevent transcriptional termination at the M-F junction, we rescued recombinant SV (rSVhMFjCG) in which its M-F gene junction was replaced by that of hPIV1. Cells infected with rSVhMFjCG produced an abundance of M-F read-through transcripts; this result indicated that the hPIV1 M-F junction is responsible for inefficient termination. When one or both of the IG and S sites in rSVhMFjCG were replaced by those of SV, the efficiency of transcriptional termination increased but not to the level observed in wild-type SV-infected cells. Deletion of most of the long noncoding region of the hPIV1 F gene in rSVhMFjCG in addition to the mutations in IG and S signals resulted in efficient termination that was equivalent to the level observed in wild-type virus-infected cells. Therefore, the long noncoding sequence of the hPIV1 F gene contains
cis
-acting element(s) that affects transcriptional termination. Our evaluation of the effect of inefficient transcriptional termination on viral replication in culture revealed that cells infected with rSVhMFjCG produced less F protein than cells infected with wild-type SV and that assembly of the recombinant SV in culture was less efficient. These phenotypes seem to be responsible for the extended survival of mice infected with rSVhMFjCG.
Mutations of the RNase H C Helix of the Moloney Murine Leukemia Virus Reverse Transcriptase Reveal Defects in Polypurine Tract Recognition
Authors: Lim David , Orlova Marianna , Goff Stephen P. 
Published: July 2002

ABSTRACT

Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and
Escherichia coli
RNase H possess a positively charged α-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (ΔC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5′ and 3′ viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5′ end.
Dengue Virus Selectively Induces Human Mast Cell Chemokine Production
Authors: King Christine A. , Anderson Robert , Marshall Jean S. 
Published: July 2002

ABSTRACTSevere dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1α, and MIP-1β, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P< 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1α, or MIP-1β response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.
Neurological complications of coeliac disease
Authors: Pengiran Tengah D S N A , Wills A J , Holmes G K T 
Published: July 2002

Abstract
A variety of neurological disorders have been reported in association with coeliac disease including epilepsy, ataxia, neuropathy, and myelopathy. The nature of this association is unclear and whether a specific neurological complication occurs in coeliac disease remains unproved. Malabsorption may lead to vitamin and trace element deficiencies. Therefore, patients who develop neurological dysfunction should be carefully screened for these. However, malabsorption does not satisfactorily explain the pathophysiology and clinical course of many of the associated neurological disorders. Other mechanisms proposed include altered autoimmunity, heredity, and gluten toxicity. This review attempts to summarise the literature and suggests directions for future research.
Cullen’s sign in amoebic liver abscess
Authors: Misra A , Agrahari D , Gupta R 
Published: July 2002

Abstract
A 45 year old woman presented with pain in her right upper abdomen and fever. Ultrasound of her abdomen showed a large liver abscess with subhepatic collection. She had Cullen’s sign. The liver abscess was managed by percutaneous catheter drainage.