bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutagenesis Analysis of the nsp4 Main Proteinase Reveals Determinants of Arterivirus Replicase Polyprotein Autoprocessing
Authors: van Aken Danny , Snijder Eric J. , Gorbalenya Alexander E. 
Published: March 2006

ABSTRACT

Nonstructural protein 4 (nsp4; 204 amino acids) is the chymotrypsin-like serine main proteinase of the arterivirus
Equine arteritis virus
(order
Nidovirales
), which controls the maturation of the replicase complex. nsp4 includes a unique C-terminal domain (CTD) connected to the catalytic two-β-barrel structure by the poorly conserved residues 155 and 156. This dipeptide might be part of a hinge region (HR) that facilitates interdomain movements and thereby regulates (in time and space) autoprocessing of replicase polyproteins pp1a and pp1ab at eight sites that are conserved in arteriviruses. To test this hypothesis, we characterized nsp4 proteinase mutants carrying either point mutations in the putative HR domain or a large deletion in the CTD. When tested in a reverse genetics system, three groups of mutants were recognized (wild-type-like, debilitated, and dead), which was in line with the expected impact of mutations on HR flexibility. When tested in a transient expression system, the effects of the mutations on the production and turnover of replicase proteins varied widely. They were cleavage product specific and revealed a pronounced modulating effect of moieties derived from the nsp1-3 region of pp1a. Mutations that were lethal affected the efficiency of polyprotein autoprocessing most strongly. These mutants may be impaired in the accumulation of nsp5-7 and/or suffer from delayed or otherwise perturbed processing at the nsp5/6 and nsp6/7 junctions. On average, the production of nsp7-8 seems to be the most resistant to debilitating nsp4 mutations. Our results further prove that the CTD is essential for a vital nsp4 property other than catalysis.
Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Modulates K1 Expression through Its
cis
-Acting Elements within the Terminal Repeats
Authors: Verma Subhash C. , Lan Ke , Choudhuri Tathagata , Robertson Erle S. 
Published: March 2006

ABSTRACT
K1 is the first open reading frame encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and lies positionally to the immediate right of the terminal repeats. K1 is a transmembrane glycoprotein having a functional immunoreceptor tyrosine-based activation motif (ITAM) capable of activating B-cell receptor signaling. K1 is expressed mostly during the lytic cycle of the virus and its promoter lies within the terminal repeat which contains the binding sites for latency-associated nuclear antigen (LANA). The K1 promoter (K1p) having LANA binding sites assayed by reporter assay demonstrated that LANA is capable of down-regulating K1 promoter transcriptional activity. However, the KSHV replication transcription activator RTA up-regulates K1p transcriptional activity. The promoter deleted of LANA binding sites showed loss in LANA-mediated down-regulation but was unaffected for RTA-mediated up-regulation. Increasing amounts of RTA rescued LANA-mediated repression of K1p transcriptional activity in cotransfection experiments. Reporter assay data suggest that LANA binding to its cognate sequence is critical for LANA-mediated repression of K1p as a LANA construct lacking the DNA binding domain was unable to repress K1p transcription. Additionally, KSHV primary infection experiments suggest that K1 is expressed during early infection but is repressed on the establishment of latency and so follows an expression profile similar to that of RTA during infection. Analysis of the promoter sequence revealed the presence of Oct-1 transcription factor binding sites within the −116 to +76 region. Mutational analysis of the Oct-1 sites abolished RTA-mediated transcriptional activation, suggesting that RTA up-regulates K1p transcription through binding to this transcription factor.
Interplay of Cellular and Humoral Immune Responses against BK Virus in Kidney Transplant Recipients with Polyomavirus Nephropathy
Authors: Chen Yiping , Trofe Jennifer , Gordon Jennifer , Du Pasquier Renaud A. , Roy-Chaudhury Prabir , Kuroda Marcelo J. , Woodle E. Steve , Khalili Kamel , Koralnik Igor J. 
Published: March 2006

ABSTRACTReactivation of the polyomavirus BK (BKV) causes polyomavirus nephropathy (PVN) in kidney transplant (KTx) recipients and may lead to loss of the renal allograft. We have identified two HLA-A*0201-restricted nine-amino-acid cytotoxic T lymphocyte (CTL) epitopes of the BKV major capsid protein VP1, VP1p44, and VP1p108.Using tetramer staining assays, we showed that these epitopes were recognized by CTLs in 8 of 10 (VP1p44) and 5 of 10 (VP1p108) HLA-A*0201+healthy individuals, while both epitopes elicited a CTL response in 10 of 10 KTx recipients with biopsy-proven PVN, although at variable levels. After in vitro stimulation with the respective peptides, CTLs directed against VP1p44were more abundant than against VP1p108in most healthy individuals, while the converse was true in KTx recipients with PVN, suggesting a shift in epitope immunodominance in the setting of active BKV infection. A strong CTL response in KTx recipients with PVN appeared to be associated with decreased BK viral load in blood and urine and low anti-BKV antibody titers, while a low or undetectable CTL response correlated with viral persistence and high anti-BKV antibody titers. These results suggest that this cellular immune response is present in most BKV-seropositive healthy individuals and plays an important role in the containment of BKV in KTx recipients with PVN. Interestingly, the BKV CTL epitopes bear striking homology with the recently described CTL epitopes of the other human polyomavirus JC (JCV), JCV VP1p36and VP1p100. A high degree of epitope cross-recognition was present between BKV and corresponding JCV-specific CTLs, which indicates that the same population of cells is functionally effective against these two closely related viruses.
Partial Delipidation Improves the T-Cell Antigenicity of Hepatitis B Virus Surface Antigen
Authors: Desombere Isabelle , Willems Annick , Gijbels Yvonne , Leroux-Roels Geert 
Published: March 2006

ABSTRACTHepatitis B virus surface antigen (HBsAg) is a complex macromolecular particle composed of glycoproteins and lipids. The latter, representing 25% of the particle mass, are of host origin and determine the solubility, stability, and, indirectly, B-cell immunogenicity of HBsAg. HBsAg is a T-cell-dependent immunogen that does not elicit a detectable humoral immune response in 5% of HBsAg vaccine recipients and in most subjects suffering from chronic hepatitis B. We investigated the influence of the lipid content on the antigenicity of the particle. Lipids were partially removed from HBsAg by treatment with β-d-octyl glucoside and density centrifugation. Sham treatment consisted of density centrifugation of HBsAg only. We compared the in vitro proliferative responses of established T-cell lines and nonfractionated peripheral blood mononuclear cells (PBMC) from HBsAg vaccinees and chronic HBV patients when stimulated with partially delipidated HBsAg, untreated HBsAg, or sham-treated HBsAg. In all experiments, delipidated HBsAg turned out to be 10 to 100 times more antigenic than its untreated or sham-treated counterpart. Remarkably, PBMC from vaccine nonresponders or chronic HBV patients displayed a proliferative response towards delipidated HBsAg, whereas native HBsAg never induced a response. A series of control experiments demonstrated that this enhancement of T-cell antigenicity was HBsAg specific and directly linked to lipid extraction. Nonspecific adjuvant effects of any kind could be ruled out. In vivo evaluation in mice demonstrated that delipidated particles lose most of their B-cell antigenicity. However, when native and delipidated particles were mixed, these mixtures induced equal or slightly superior anti-HBs responses to those induced by the same quantity of native HBsAg alone. In conclusion, our data show that partial delipidation of HBsAg strikingly increases the T-cell antigenicity of this unique viral antigen.
Replication Properties of Human Adenovirus In Vivo and in Cultures of Primary Cells from Different Animal Species
Authors: Jogler Christian , Hoffmann Dennis , Theegarten Dirk , Grunwald Thomas , Überla Klaus , Wildner Oliver 
Published: March 2006

ABSTRACTOncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.
Adenovirus-Mediated RNA Interference against Foot-and-Mouth Disease Virus Infection both In Vitro and In Vivo
Authors: Chen Weizao , Liu Mingqiu , Jiao Ye , Yan Weiyao , Wei Xuefeng , Chen Jiulian , Fei Liang , Liu Yang , Zuo Xiaoping , Yang Fugui , Lu Yonggan , Zheng Zhaoxin 
Published: March 2006

ABSTRACT

Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10
6
PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID
50
) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 × 10
9
PFU each and challenged 24 h later with 100 ID
50
of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing
Escherichia coli
galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.
Herpes Simplex Virus Type 1 Accumulation, Envelopment, and Exit in Growth Cones and Varicosities in Mid-Distal Regions of Axons
Authors: Saksena Monica Miranda , Wakisaka Hiroyuki , Tijono Bibing , Boadle Ross A. , Rixon Frazer , Takahashi Hirotaka , Cunningham Anthony L. 
Published: March 2006

ABSTRACT
The mechanism of anterograde transport of alphaherpesviruses in axons remains controversial. This study examined the transport, assembly, and egress of herpes simplex virus type 1 (HSV-1) in mid- and distal axons of infected explanted human fetal dorsal root ganglia using confocal microscopy and transmission electron microscopy (TEM) at 19, 24, and 48 h postinfection (p.i.). Confocal-microscopy studies showed that although capsid (VP5) and tegument (UL37) proteins were not uniformly present in axons until 24 h p.i., they colocalized with envelope (gG) proteins in axonal varicosities and in growth cones at 24 and 48 h p.i. TEM of longitudinal sections of axons in situ showed enveloped and unenveloped capsids in the axonal varicosities and growth cones, whereas in the midregion of the axons, predominantly unenveloped capsids were observed. Partially enveloped capsids, apparently budding into vesicles, were observed in axonal varicosities and growth cones, but not during viral attachment and entry into axons. Tegument proteins (VP22) were found associated with vesicles in growth cones, either alone or together with envelope (gD) proteins, by transmission immunoelectron microscopy. Extracellular virions were observed adjacent to axonal varicosities and growth cones, with some virions observed in crescent-shaped invaginations of the axonal plasma membrane, suggesting exit at these sites. These findings suggest that varicosities and growth cones are probable sites of HSV-1 envelopment of at least a proportion of virions in the mid- to distal axon. Envelopment probably occurs by budding of capsids into vesicles with associated tegument and envelope proteins. Virions appear to exit from these sites by exocytosis.
Mechanism of Substrate Recognition by Drug-Resistant Human Immunodeficiency Virus Type 1 Protease Variants Revealed by a Novel Structural Intermediate
Authors: Prabu-Jeyabalan Moses , Nalivaika Ellen A. , Romano Keith , Schiffer Celia A. 
Published: March 2006

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual “intermediate” conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.
Bioluminescence Imaging of Live Infected Salmonids Reveals that the Fin Bases Are the Major Portal of Entry for
Novirhabdovirus
Authors: Harmache Abdallah , LeBerre Monique , Droineau Stéphanie , Giovannini Marco , Brémont Michel 
Published: March 2006

ABSTRACT

Although
Novirhabdovirus
viruses, like the
Infectious hematopietic necrosis virus
(IHNV), have been extensively studied, limited knowledge exists on the route of IHNV entry during natural infection. A recombinant IHNV (rIHNV) expressing the
Renilla
luciferase gene was generated and used to infect trout. A noninvasive bioluminescence assay was developed so that virus replication in live fish could be followed hours after infection. We provide here evidence that the fin bases are the portal of entry into the fish. Confirmation was brought by the use of a nonpathogenic rIHNV, which was shown to persist in fins for 3 weeks postinfection.
Functional Central Polypurine Tract Provides Downstream Protection of the Human Immunodeficiency Virus Type 1 Genome from Editing by APOBEC3G and APOBEC3B
Authors: Wurtzer Sebastien , Goubard Armelle , Mammano Fabrizio , Saragosti Sentob , Lecossier Denise , Hance Allan J. , Clavel François 
Published: March 2006

ABSTRACT
Lentiviruses utilize two polypurine tracts for initiation of plus-strand viral DNA synthesis. We have examined to what extent human immunodeficiency virus type 1 plus-strand initiation at the central polypurine tract (cPPT) could protect the viral genome from DNA editing by APOBEC3G and APOBEC3B. The presence of a functional cPPT, but not of a mutated cPPT, extensively reduced editing by both APOBEC3G and APOBEC3B of sequences downstream, but not upstream, of the cPPT, with significant protection observed as far as 400 bp downstream. Thus, in addition to other potential functions, the cPPT could help protect lentiviruses from editing by cytidine deaminases of the APOBEC family.
Diazepam-induced disruption of classically-conditioned fear-potentiation of late-latency auditory evoked potentials is prevented by flumazenil given before, but not after, CS/US pairing
Authors: Scaife J. C. , Hou R. H. , Samuels E. R. , Baqui F. , Langley R. W. , Bradshaw C. M. , Szabadi E. 
Published: March 2006

Classical fear conditioning involves pairing a neutral conditional stimulus (CS) with an aversive unconditional stimulus (US). Subsequent presentation of the CS alone induces fear responses. Acquisition of conditioned fear is thought to involve learning of the CS/US association, followed by memory consolidation. Recently we reported that the N1/P2 auditory evoked potential was enhanced by fear conditioning in humans. Diazepam 10mg, given before CS/US pairing, prevented subsequent expression of fear potentiation when the response was elicited, 1 week later, in the presence of the CS. In this experiment, we examined whether this effect of diazepam was caused by disruption of the formation of CS/US associations or by disruption of consolidation. The benzodiazepine antagonist flumazenil was used to block the effect of diazepam either during the association period or during subsequent consolidation. Forty-two male volunteers (18-35 years) participated in two sessions separated by 7 days. In Session One, they ingested diazepam 10 mg or placebo: 60 minutes later they received flumazenil 1 mg or saline intravenously (i.v.). Then they received 20 presentations of a light (CS), 50% of which terminated with electric shock (US). This was followed by a second infusion of flumazenil or saline. Subjects received placebo/saline/saline (Group 1), diazepam/saline/saline (Group 2), diazepam/flumazenil/saline (Group 3) and diazepam/saline/flumazenil (Group 4). In Session Two, the CS was presented without the US; 50% of CS presentations terminated with a sound pulse; an equal number of sound pulses were presented without the CS. Auditory evoked potentials were recorded at Cz. In Session Two, CS presentation enhanced the auditory N1/P2 potential in placebo-treated subjects (Group 1). This enhancement was prevented by diazepam (Group 2). Flumazenil reversed diazepam’s effect on fear potentiation if it was administered before conditioning (Group 3), but not if it was administered afterwards (Group 4). The results confirm that diazepam prevents the acquisition of fear conditioning in humans, and suggest that it disrupts the formation of CS/US associations, rather than the consolidation of fear memory.
A double-blind, placebo-controlled, multi-dose evaluation of the acute behavioural effects of guaraná in humans
Authors: Haskell C. F. , Kennedy D. O. , Wesnes K. A. , Milne A. L. , Scholey A. B. 
Published: March 2006

The present study aimed to systematically assess acute, dose-related behavioural effects of an extract of guaraná plant for the first time in humans.This double-blind, counterbalanced, placebo-controlled study ( n = 26) assessed the acute mood and cognitive effects throughout the day of four different doses (37.5mg, 75mg, 150mg and 300mg) of a standardised guaraná extract (PC-102). Assessment included the Cognitive Drug Research computerized test battery and Bond-Lader mood scales.Guaraná improved secondary memory performance and increased alert and content mood ratings. The two lower doses produced more positive cognitive effects than the higher doses.This research supports previous findings of cognitive improvements following 75mg guaraná and provides the first exploration of different dose effects of guaraná in humans. The findings suggest that the effects cannot be attributed to caffeine alone.
A validation of the 7.5% CO2 model of GAD using paroxetine and lorazepam in healthy volunteers
Authors: Bailey Jayne E. , Kendrick Adrian , Diaper Alison , Potokar John P. , Nutt David J. 
Published: March 2006

The inhalation of 7.5% carbon dioxide (CO2) in healthy subjects produces an increase in blood pressure and heart rate, and increased feelings of anxiety, fear and tension (Bailey et al. 2005). As this state is similar to that of general anxiety rather than panic, we further validated this by examining the effects of anxiolytic medication. Two separate studies in healthy volunteers are described; study one is a double-blind, placebo-controlled study of a single dose of 2mg lorazepam and study two describes the effects of 21 days of treatment with paroxetine. Gas challenges were air and 7.5% CO2 inhaled for 20 minutes, delivered on day 0 (before treatment) and day 21 (after treatment) in the paroxetine study. Subjective effects were measured using visual analogue scales and questionnaires. When compared with placebo, lorazepam 2mg significantly reduced peak CO2-induced subjective fear, feelings of wanting to leave, tension and worry. In the paroxetine study, when compared with day 0, day 21 showed a significantly attenuated peak CO2-induced nervousness and a trend for reduced ratings of anxiety, fear, feel like leaving, tense and worried. In these studies we have shown that this CO2 model of anxiety is sensitive to lorazepam and to a lesser extent paroxetine. This gives support to its utility as an experimental model of general anxiety disorder in healthy volunteers.
How to access structure and dynamics of solutions: The capabilities of computational methods (Special Topic Article)
Authors: Rode Bernd M. , Hofer Thomas S. 
Published: March 2006

Abstract
The progress of computational chemistry in the treatment of liquid systems is outlined, and the combination of the statistical methods (Monte Carlo, MC, and molecular dynamics, MD) with quantum mechanics as the main foundation of this progress is emphasized. The difficulties of experimental studies of liquid systems without having obtained sophisticated theoretical models describing the structural entities and the dynamical behavior of these liquids demonstrate that chemistry research is in a transition phase, where theory and high-performance computing have not only become a valuable supplement, but an essential and almost indispensable component to secure a correct interpretation of measured data.