bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutations within the P2 Domain of Norovirus Capsid Affect Binding to Human Histo-Blood Group Antigens: Evidence for a Binding Pocket
Authors: Tan Ming , Huang Pengwei , Meller Jaroslaw , Zhong Weiming , Farkas Tibor , Jiang Xi 
Published: November 2003

ABSTRACT

Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible “pocket” in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N
302
, T
337
, and Q
375
for VA387 and N
302
, N
338
, and E
378
for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T
337
(to A) in VA387 or a double amino acid mutation at RN
338
(to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N
302
or Q
375
in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E
378
from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.
Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Authors: Peyerl Fred W. , Barouch Dan H. , Yeh Wendy W. , Bazick Heidi S. , Kunstman Jennifer , Kunstman Kevin J. , Wolinsky Steven M. , Letvin Norman L. 
Published: November 2003

ABSTRACTVirus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A*01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infectedMamu-A*01+monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.
Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin
Authors: Jiao Junfang , Simmons Daniel T. 
Published: November 2003

ABSTRACT
Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.
Codelivery of CCR7 Ligands as Molecular Adjuvants Enhances the Protective Immune Response against Herpes Simplex Virus Type 1
Authors: Toka Felix N. , Gierynska Malgorzata , Rouse Barry T. 
Published: November 2003

ABSTRACTHumoral and cellular immunity, associated with long-term protective immunological memory, defines the efficacy of a given vaccine formulation. However, few vaccines achieve this target without the aid of a suitable adjuvant. Molecular adjuvants in vaccination against infectious agents offer a noninvasive means of enhancing the immune response against target antigens. To examine the potency of two β-chemokines as immunomodulators, plasmid DNA encoding β-chemokines CCL19 and CCL21 (CCR7L) was codelivered intranasally with plasmid DNA or recombinant vaccinia virus encoding herpes simplex virus (HSV) gB (HSV-gB) in a prime-and-boost vaccination strategy. This vaccination regimen increased serum and vaginal immunoglobulin G (IgG) and IgA, respectively, as well as the numbers of HSV-gB498-505peptide-specific gamma interferon-producing CD8+T cells. Distinctively, a high number of cytotoxic T lymphocytes was achieved when pCCR7L was applied at both prime and boost as opposed to omission of pCCR7L. A rapid-recall response was induced in the genital tract upon challenge with the HSV McKrae strain, affording a high level of protection and survival of vaccinated mice. Our results demonstrate that high innate immune kinetics and distribution of adaptive response induced in the nasal mucosa appears to be key factors in generating protective memory responses against HSV. Thus CCR7L expressed ectopically may serve as a molecular adjuvant to boost the immune response to a codelivered antigen in mucosal surfaces.
Covalently Linked Human Immunodeficiency Virus Type 1 gp120/gp41 Is Stably Anchored in Rhabdovirus Particles and Exposes Critical Neutralizing Epitopes
Authors: McKenna Philip M. , Pomerantz Roger J. , Dietzschold Bernhard , McGettigan James P. , Schnell Matthias J. 
Published: November 2003

ABSTRACTRabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Because the gp120 and gp41 portions are covalently attached in the gp140 SOS molecule, the protein is maintained on the surface of the RV virion throughout purification. Surface immunostaining and fluorescence-activated cell sorting analysis with anti-envelope antibodies show that the gp140 SOS protein is stably expressed on the surface of infected cells and maintains CD4 binding capabilities. Furthermore, Western blot and immunoprecipitation experiments with infected-cell lysates and purified virions show that a panel of neutralizing anti-envelope antibodies efficiently recognize the gp140 SOS protein. The antigenic properties of this recombinant RV particle containing covalently attached Env, as well as the ability to present Env in a membrane-bound form, suggest that this approach could be a useful component of a HIV-1 vaccine strategy.
Relationship between RNA Lariat Debranching and Ty1 Element Retrotransposition
Authors: Salem Laura A. , Boucher Christopher L. , Menees Thomas M. 
Published: November 2003

ABSTRACTTheSaccharomyces cerevisiae DBR1gene encodes a 2′-5′ phosphodiesterase that debranches intron RNA lariats following splicing. Yeastdbr1mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection ofdbr1mutant alleles to explore the relationship between the roles ofDBR1in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize toACT1intron RNA. Protection patterns confirm that sequences from the 5′ end of the intron to the lariat branch point accumulate indbr1mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generateddbr1mutant alleles are also deficient for debranching, further supporting a role for 2′-5′ phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently ofDBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.
The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity
Authors: Jeetendra E. , Ghosh Kakoli , Odell Derek , Li Jin , Ghosh Hara P. , Whitt Michael A. 
Published: November 2003

ABSTRACT
The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.
In Vitro and In Vivo Interactions between the Hepatitis B Virus Protein P22 and the Cellular Protein gC1qR
Authors: Lainé S. , Thouard A. , Derancourt J. , Kress M. , Sitterlin D. , Rossignol J.-M. 
Published: November 2003

ABSTRACTgC1qR, a mitochondrial matrix protein, was identified as the main cellular partner of the hepatitis B virus P22 protein. We demonstrated by immunofluorescence studies that some P22 molecules were colocalized with the endogenous gC1qR in both the cytoplasm and the nucleus but never in the mitochondria. We also showed that the last 34 amino acids of P22 were involved in the association with gC1qR.
A Pseudorabies Virus Recombinant Simultaneously Lacking the Major Tegument Proteins Encoded by the UL46, UL47, UL48, and UL49 Genes Is Viable in Cultured Cells
Authors: Fuchs Walter , Granzow Harald , Mettenleiter Thomas C. 
Published: November 2003

ABSTRACT
The UL46, UL47, UL48, and UL49 genes, which encode major tegument proteins, are conserved in most alphaherpesvirus genomes. However, the relative importance of each of these proteins for replication of individual alphaherpesviruses appears to be different. Recently, we demonstrated that single deletions of UL47 or UL48 impair maturation and egress of pseudorabies virus (PrV) particles to different extents, whereas deletions of UL46 or UL49 have no significant effects on virus replication in cell culture (W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp, and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002; M. Kopp, B. G. Klupp, H. Granzow, W. Fuchs, and T. C. Mettenleiter, J. Virol. 76:8820-8833, 2002). To test for possible functional redundancy between the four tegument proteins, a quadruple gene deletion mutant (PrV-ΔUL46-49) was generated and characterized in vitro. Although plaque formation by this mutant was almost abolished and maximum titers were reduced more than 100-fold compared to those of parental wild-type virus, PrV-ΔUL46-49 could be propagated and serially passaged in noncomplementing porcine and rabbit kidney cells. Electron-microscopic studies revealed that nucleocapsid formation and egress of PrV-ΔUL46-49 from the host cell nucleus were not affected, but secondary envelopment of nucleocapsids in the cytoplasm was only rarely observed. The replication defect of PrV-ΔUL46-49 could be fully corrected by reinsertion of the UL46-to-UL49 gene cluster. Plaque sizes and virus titers were only slightly increased after restoration of only UL47 expression, whereas repair of only UL48 resulted in a significant increase in replication capacity to the level of a UL47 deletion mutant. In conclusion, we show that none of the UL46 to UL49 tegument proteins is absolutely required for productive replication of PrV. Moreover, our data indicate that the UL47 and UL48 proteins function independently during cell-to-cell spread and virus egress.
Potential High-Throughput Assay for Screening Inhibitors of West Nile Virus Replication
Authors: Lo Michael K. , Tilgner Mark , Shi Pei-Yong 
Published: November 2003

ABSTRACTPrevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encodingRenillaluciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.
COSMOLOGICAL ISSUES FOR REVISED CANONICAL QUANTUM GRAVITY
Authors: MONTANI GIOVANNI 
Published: November 2003

In a recent work1 we presented a reformulation of the canonical quantum gravity, based on adding the so-called kinematical term to the gravity-matter action. This revised approach leads to a self-consistent canonical quantization of the 3-geometries, which referred to the external time as provided via the added term. Here, we show how the kinematical term can be interpreted in terms of a non-relativistic dust fluid which plies the role of a "real clock" for the quantum gravity theory, and, in the WKB limit of a cosmological problem, makes account for a dark matter component which, at present time, could play a dynamical role.
Phase 1 study of low-dose prolonged exposure schedules of the hypomethylating agent 5-aza-2′-deoxycytidine (decitabine) in hematopoietic malignancies
Authors: Issa Jean-Pierre J. , Garcia-Manero Guillermo , Giles Francis J. , Mannari Rajan , Thomas Deborah , Faderl Stefan , Bayar Emel , Lyons John , Rosenfeld Craig S. , Cortes Jorge , Kantarjian Hagop M. 
Published: November 2003

Abstract
Decitabine (5-aza-2′-deoxycytidine) inhibits DNA methylation and has dual effects on neoplastic cells, including the reactivation of silenced genes and differentiation at low doses and cytotoxicity at high doses. We evaluated, in a phase 1 study, low-dose prolonged exposure schedules of decitabine in relapsed/refractory leukemias. Patient cohorts received decitabine at 5, 10, 15, or 20 mg/m2 intravenously over one hour daily, 5 days a week for 2 consecutive weeks, doses 5- to approximately 30-fold lower than the maximum tolerated dose (MTD). There were 2 groups that also received 15 mg/m2 daily for 15 or 20 days. A total of 50 patients were treated (44 with acute myelogenous leukemia [AML]/myelodysplasia [MDS], 5 with chronic myelogenous leukemia [CML], and 1 with acute lymphocytic leukemia [ALL]), and the drug was well tolerated at all dose levels, with myelosuppression being the major side effect. Responses were seen at all dose levels. However, the dose of 15 mg/m2 for 10 days appeared to induce the most responses (11 of 17 or 65%), with fewer responses seen when the dose was escalated or prolonged (2 of 19 or 11%). There was no correlation between P15 methylation at baseline or after therapy and response to decitabine. We conclude that decitabine is effective in myeloid malignancies, and low doses are as or more effective than higher doses.
Mechanisms of homing in the fiddler crabUca rapax1. Spatial and temporal characteristics of a system of small-scale navigation
Authors: Layne John E. , Barnes W. Jon P. , Duncan Lindsey M. J. 
Published: November 2003

SUMMARYFiddler crabs Uca rapax are central-place foragers, making feeding excursions of up to 2 m from their burrows. We describe the natural feeding excursions of path-integrating fiddler crabs and analyze their paths for signs of significant systematic or random navigation errors. No signs of any systematic errors are evident. Random errors are small, probably due to a combination of the short length and low sinuosity of the foraging paths, as well as the fiddler crabs' unique method of locomotion that allows them to remain oriented to their burrows throughout the foraging path and to minimize large body turns. We further examined the extent to which their body orientation during foraging (transverse body axis pointing more or less towards home) accurately represented their stored home vector. By examining sequences of fast escape, we have shown that crabs can correct for deviations of their transverse body axis from home during their escape path. Thus their stored home vector is independent of their moment-to-moment body orientation.Crabs were subjected to passive translational displacements and barrier obstructions. Responses to translational displacements were identical to those observed by previous authors, namely that crabs returned in the correct egocentric direction and distance as though no displacement had occurred. Covering the burrow entrance resulted in crabs returning to the correct position of the burrow, and then beginning to search. When a barrier was placed between foraging crabs and their burrow, crabs oriented their bodies toward the burrow as accurately as with no barrier.
Mechanics of wing-assisted incline running (WAIR)
Authors: Bundle Matthew W. , Dial Kenneth P. 
Published: November 2003

SUMMARYA recently discovered locomotor behavior, wing-assisted incline running(WAIR), allows fully volant animals to `run' up vertical obstacles. Such a task would appear to be especially formidable for bipeds, yet WAIR is used preferentially by ground-dwelling birds, specifically chukar partridge Alectoris chukar, to reach refugia. The basic locomotor mechanics that enable this behavior are not fully understood. For instance, are there functional differences at the level of the wing during WAIR and free flight,and do the hindlimbs actively participate in propulsion during WAIR? To investigate wing function during these activities we used accelerometry to compare the instantaneous whole-body acceleration during WAIR and ascending free flights at a similar climb angle. Throughout a substantial portion of the wingbeat cycle, chukars engaged in WAIR experienced an acceleration oriented towards the substrate, whereas during ascending free flights the acceleration of the center of mass was parallel to the direction of travel. We investigated whether the animals were using their hindlimbs for propulsion, rather than for some other function (e.g. to maintain balance), by measuring ground reaction forces (GRF) during bouts of WAIR. Estimates of the contribution of the hindlimbs towards the vertical external work done by the bird were 98±8% of the total at an incline of 60° (the steepest angle that birds were able to negotiate without the use of their forelimbs). During vertical (90°) bouts of WAIR the hindlimb contribution was 37±5% of the total external work. Yet, the magnitude of the peak GRF at 90° was 175% of the value generated during level walking, revealing that birds engaged in WAIR do generate sizeable hindlimb forces even during vertical ascents. These data support the hypothesis that forelimbs are enabling hindlimb function, and we argue that this represents a locomotor strategy which may have been used by proto-birds during the evolution of flight.