bluuebunny/crossref_metadata_2025 huggingface dataset:
An Improved Time Domain Simulation for Dynamic Milling at Small Radial Immersions
Authors: Campomanes Marc L. , Altintas Yusuf
Published: July 2003

This paper presents an improved milling time domain model to simulate vibratory cutting conditions at very small radial widths of cut. The improved kinematics model allows simulation of very small radial immersions. The model can predict forces, surface finish, and chatter stability, accurately accounting for non-linear effects that are difficult to model analytically. The discretized cutter and workpiece kinematics and dynamic models are used to represent the exact trochoidal motion of the cutter, and to investigate the effects of forced vibrations and changing radial immersion due to deflection and vibrations on chatter stability. Three dimensional surface finish profiles are predicted and are compared to measured results. Stability lobes generated from the time domain simulation are also shown for various cases.
Formability Enhancement for Tailor-Welded Blanks Using Blank Holding Force Control
Authors: He Sijun , Wu Xin , Hu S. Jack
Published: July 2003

Tailor-welded blanks (TWB) are widely used for stamped auto body panels because of their great benefits in weight and cost reduction. However, the weld line in a tailor-welded blank causes serious concerns in formability because of material discontinuity and additional inhomogeneous stress/strain distribution. This paper proposes a blank holding force (BHF) control strategy to control the weld line movement, distribute the deformation more uniformly and thereby improve TWB formability. The control methodology is developed based on a simplified 2-D sectional analytical model that estimates the stress/strain distribution and the BHFs required for each side of the flange with dissimilar materials. The model can be further extended to 3-D analysis by superimposing the 2-D sectional analysis results around the entire binder ring and thus determining the required BHF for the whole panel. Finite element simulations are performed to study the effects of forming parameters on the weld line movement. Experiments have been conducted to verify the analytical model and partial finite element simulations. Both analysis and experiments demonstrated that a lower BHF should be applied on the thicker blank side to allow more metal to flow-in for obtaining more uniform strain distribution. The proposed BHF control is proven to be a good approach to enhancing TWB formability.
CBN-TiN Composite Coating Using a Novel Combinatorial Method—Structure and Performance in Metal Cutting
Authors: Russell William C. , Malshe Ajay P. , Yedave Sharad N. , Brown William D.
Published: July 2003

Cubic boron nitride coatings are under development for a variety of applications but stabilization of the pure CBN form and adhesion of films deposited by PVD and ion-based methods have been difficult. An alternate two-stage process comprising of ESC (electrostatic spray coating) and CVI (chemical vapor infiltration) has been developed for depositing CBN-TiN composite coatings for wear related applications. The process optimization yields fully dense films of CBN particles evenly dispersed in a continuous TiN matrix. Metal cutting tests (turning–4340 steel), using CBN-TiN composite coating thus developed, have shown an increase in tool life of three to seven times, depending on machining parameters, in comparison with CVD deposited TiN films.
A Volumetric Difference-based Adaptive Slicing and Deposition Method for Layered Manufacturing
Authors: Yang Y. , Fuh J. Y. H. , Loh H. T. , Wong Y. S.
Published: July 2003

Adaptive slicing capable of producing variable thickness is a useful means to improve the fabrication efficiency in layered manufacturing (LM) or Rapid Prototyping (RP) processes. Many approaches have been reported in this field; however, most of them are based on the cusp height criteria, which is not an effective representation of the staircase effect when the surface normal is near vertical. Furthermore, most of the existing methods slice the model without considering the local features in the plane of the sliced layer. This paper introduces a novel difference-based adaptive slicing and deposition method. The advantage of this slicing method is that the slicing error is independent of the surface normal. A new criterion for adaptive slicing is evaluated and compared with that based on cusp-height. An adaptive slicing algorithm, which uses the volumetric difference between two adjacent layers as the criterion for slicing, has been developed in this work. Different deposition strategies for the common area and the difference area are applied to layer fabrication while considering the local features of the sliced layer. The algorithm has been tested with a sample part, and the results indicate that a better surface finish can be achieved for both surfaces whose normals are nearly in the slicing plane and surfaces whose normals are nearly perpendicular to the slicing plane. It is found that the building time can be reduced by 40% compared with the traditional adaptive slicing. The proposed method has minimized the volumetric error between the built LM part and the original CAD model while achieving a higher efficiency. It is suitable for most commercialized LM systems due to its simplicity in implementation.
Design of a Slot-Coater-Based Layered-Composites Manufacturing System
Authors: Haberer M. , Zak G. , Park C. B. , Benhabib B.
Published: July 2003

This paper addresses the reinforcement of photopolymers, through the addition of short glass fibers, for Rapid Layered Composite parts Manufacturing (RLCM). Novel designs for an (external) fiber-resin-mixing subsystem and a (slot-coating-based) liquid-layer-formation subsystem are presented. These subsystems, when used as integral parts of a lithography-based RLCM system, successfully cope with typical difficulties encountered in the formation of thin layers from a highly viscous fiber-photopolymer composite liquid. Axiomatic Design theory was utilized for the analysis of both subsystem designs. Verification experiments run on an RLCM system confirmed (i) the ability of the fiber-resin-mixing subsystem to supply liquid composite with specified fiber content and to avoid fiber degradation through breakage, as well as (ii) the ability of the liquid-layer-formation subsystem to form solid layers with high fiber content and of uniform thickness.
Metal Embedded Fiber Bragg Grating Sensors in Layered Manufacturing
Authors: Li Xiaochun , Prinz Fritz
Published: July 2003

Layered Manufacturing can build functional “smart” parts with sensors, integrated circuits, complete functional assemblies, and actuators placed within the structure and fully embedded. These sensors can be used to gain data for validating or improving designs during the prototype stage or to obtain information on the performance and structural integrity of functional components in service. A sequence for embedding fiber optic sensors in metals via Layered Manufacturing is developed. Fiber Bragg Grating (FBG) sensors, embedded in nickel and stainless steel were characterized for temperature and strain measurements. Moreover, a decoupling technique that is capable of separating temperature and strain effects was also developed. This paper also presents the application of the embedded sensors to monitor residual strains during laser-assisted Layered Manufacturing.
A Model-Based Monitoring and Fault Diagnosis Methodology for Free-Form Surface Machining Process
Authors: Zhu Rixin , DeVor Richard E. , Kapoor Shiv G.
Published: July 2003

In this paper, a process model-based approach has been proposed for monitoring and fault diagnosis in the multi-axis ball end milling process. Besides its ability to deal with complex cutting geometry in free-form surface machining, the method has the capability of not only detecting the presence but also estimating the magnitudes of faults, which include flute chipping, breakage and spindle/cutter axes runout. A threshold-based fault detection method is developed based on the analysis of harmonic power distribution in the cutting force signal. A genetic algorithm approach is used to search and determine the fault pattern and magnitudes. The new approach is validated through both constant cross-section cut and free-form surface machining tests on 1018 steel.
Invertebrate Ecological Immunology
Authors: Rolff J. , Siva-Jothy M. T.
Published: July 2003

Ecological immunology is a rapidly expanding field that examines the causes and consequences of variation in immune function in the context of evolution and of ecology. Millions of invertebrate species rely solely on innate immunity, compared with only 45,000 vertebrate species that rely additionally on an acquired immune system. Despite this difference in diversity, most studies of ecological immunology focus on vertebrates. Here we review recent progress derived largely from the mechanistic analysis of invertebrate innate immunity. Using this empirical base, we pose general questions in areas that are of central importance for the development of ecological immunology.
The Ancients' Views of Science
Authors: Andrews Bridie
Published: July 2003

The Way and the Word
Science and Medicine in Early China and Greece. by Geoffrey Lloyd and Nathan Sivin. Yale University Press, New Haven, CT, 2002. 368 pp. $35, £25. ISBN 0-300-09297-0.

Comparing the cosmology, science, and medicine in Greek and Chinese civilizations in the six centuries from 400 B.C., Lloyd and Sivin discuss the social, political, and intellectual context of scientific concepts and thinkers and relate conceptual differences to these circumstances.
VDAC2 Inhibits BAK Activation and Mitochondrial Apoptosis
Authors: Cheng Emily H.-Y. , Sheiko Tatiana V. , Fisher Jill K. , Craigen William J. , Korsmeyer Stanley J.
Published: July 2003

The multidomain proapoptotic molecules BAK or BAX are required to initiate the mitochondrial pathway of apoptosis. How cells maintain the potentially lethal proapoptotic effector BAK in a monomeric inactive conformation at mitochondria is unknown. In viable cells, we found BAK complexed with mitochondrial outer-membrane protein VDAC2, a VDAC isoform present in low abundance that interacts specifically with the inactive conformer of BAK. Cells deficient in VDAC2, but not cells lacking the more abundant VDAC1, exhibited enhanced BAK oligomerization and were more susceptible to apoptotic death. Conversely, overexpression of VDAC2 selectively prevented BAK activation and inhibited the mitochondrial apoptotic pathway. Death signals activate “BH3-only” molecules such as tBID, BIM, or BAD, which displace VDAC2 from BAK, enabling homo-oligomerization of BAK and apoptosis. Thus, VDAC2, an isoform restricted to mammals, regulates the activity of BAK and provides a connection between mitochondrial physiology and the core apoptotic pathway.
Contributions of Anthropogenic and Natural Forcing to Recent Tropopause Height Changes
Authors: Santer B. D. , Wehner M. F. , Wigley T. M. L. , Sausen R. , Meehl G. A. , Taylor K. E. , Ammann C. , Arblaster J. , Washington W. M. , Boyle J. S. , Brüggemann W.
Published: July 2003

Observations indicate that the height of the tropopause—the boundary between the stratosphere and troposphere—has increased by several hundred meters since 1979. Comparable increases are evident in climate model experiments. The latter show that human-induced changes in ozone and well-mixed greenhouse gases account for ∼80% of the simulated rise in tropopause height over 1979–1999. Their primary contributions are through cooling of the stratosphere (caused by ozone) and warming of the troposphere (caused by well-mixed greenhouse gases). A model-predicted fingerprint of tropopause height changes is statistically detectable in two different observational (“reanalysis”) data sets. This positive detection result allows us to attribute overall tropopause height changes to a combination of anthropogenic and natural external forcings, with the anthropogenic component predominating.
Role of Subplate Neurons in Functional Maturation of Visual Cortical Columns
Authors: Kanold Patrick O. , Kara Prakash , Reid R. Clay , Shatz Carla J.
Published: July 2003

The subplate forms a transient circuit required for development of connections between the thalamus and the cerebral cortex. When subplate neurons are ablated, ocular dominance columns do not form in the visual cortex despite the robust presence of thalamic axons in layer 4. We show that subplate ablation also prevents formation of orientation columns. Visual responses are weak and poorly tuned to orientation. Furthermore, thalamocortical synaptic transmission fails to strengthen, whereas intracortical synapses are unaffected. Thus, subplate circuits are essential not only for the anatomical segregation of thalamic inputs but also for key steps in synaptic remodeling and maturation needed to establish the functional architecture of visual cortex.
Whales Before Whaling in the North Atlantic
Authors: Roman Joe , Palumbi Stephen R.
Published: July 2003

It is well known that hunting dramatically reduced all baleen whale populations, yet reliable estimates of former whale abundances are elusive. Based on coalescent models for mitochondrial DNA sequence variation, the genetic diversity of North Atlantic whales suggests population sizes of approximately 240,000 humpback, 360,000 fin, and 265,000 minke whales. Estimates for fin and humpback whales are far greater than those previously calculated for prewhaling populations and 6 to 20 times higher than present-day population estimates. Such discrepancies suggest the need for a quantitative reevaluation of historical whale populations and a fundamental revision in our conception of the natural state of the oceans.
Enhanced Optical Emission During Crab Giant Radio Pulses
Authors: Shearer A. , Stappers B. , O'Connor P. , Golden A. , Strom R. , Redfern M. , Ryan O.
Published: July 2003

We detected a correlation between optical and giant radio pulse emission from the Crab pulsar. Optical pulses coincident with the giant radio pulses were on average 3% brighter than those coincident with normal radio pulses. Combined with the lack of any other pulse profile changes, this result indicates that both the giant radio pulses and the increased optical emission are linked to an increase in the electron-positron plasma density.
30,000 Years of Hydrothermal Activity at the Lost City Vent Field
Authors: Früh-Green Gretchen L. , Kelley Deborah S. , Bernasconi Stefano M. , Karson Jeffrey A. , Ludwig Kristin A. , Butterfield David A. , Boschi Chiara , Proskurowski Giora
Published: July 2003

Strontium, carbon, and oxygen isotope data and radiocarbon ages document at least 30,000 years of hydrothermal activity driven by serpentinization reactions at Lost City. Serpentinization beneath this off-axis field is estimated to occur at a minimum rate of 1.2 × 10
–
4
cubic kilometers per year. The access of seawater to relatively cool, fresh peridotite, coupled with faulting, volumetric expansion, and mass wasting processes, are crucial to sustain such systems. The amount of heat produced by serpentinization of peridotite massifs, typical of slow and ultraslow spreading environments, has the potential to drive Lost City–type systems for hundreds of thousands, possibly millions, of years.
Dense Packing and Symmetry in Small Clusters of Microspheres
Authors: Manoharan Vinothan N. , Elsesser Mark T. , Pine David J.
Published: July 2003

When small numbers of colloidal microspheres are attached to the surfaces of liquid emulsion droplets, removing fluid from the droplets leads to packings of spheres that minimize the second moment of the mass distribution. The structures of the packings range from sphere doublets, triangles, and tetrahedra to exotic polyhedra not found in infinite lattice packings, molecules, or minimum–potential energy clusters. The emulsion system presents a route to produce newcolloidal structures and a means to study howdifferent physical constraints affect symmetry in small parcels of matter.
First-Principles Calculations of PuO
2
±

x
Authors: Petit L. , Svane A. , Szotek Z. , Temmerman W. M.
Published: July 2003

The electronic structure of PuO
2
±

x

was studied using first-principles quantum mechanics, realized with the self-interaction corrected local spin density method. In the stoichiometric PuO
2
compound, Pu occurs in the Pu(IV) oxidation state, corresponding to a localized f
4
shell. If oxygen is introduced onto the octahedral interstitial site, the nearby Pu atoms turn into Pu(V) (f
3
) by transferring electrons to the oxygen. Oxygen vacancies cause Pu(III) (f
5
) to form by taking up electrons released by oxygen. At
T
= 0, the PuO
2
compound is stable with respect to free oxygen, but the delicate energy balance suggests the possible deterioration of the material during long-term storage.
The Archaeology of Ushki Lake, Kamchatka, and the Pleistocene Peopling of the Americas
Authors: Goebel Ted , Waters Michael R. , Dikova Margarita
Published: July 2003

The Ushki Paleolithic sites of Kamchatka, Russia, have long been thought to contain information critical to the peopling of the Americas, especially the origins of Clovis. New radiocarbon dates indicate that human occupation of Ushki began only 13,000 calendar years ago—nearly 4000 years later than previously thought. Although biface industries were widespread across Beringia contemporaneous to the time of Clovis in western North America, these data suggest that late-glacial Siberians did not spreadinto Beringia until the endof the Pleistocene, perhaps too recently to have been ancestral to proposedpre-Clovis populations in the Americas.
The Distribution of Active Force Generators Controls Mitotic Spindle Position
Authors: Grill Stephan W. , Howard Jonathon , Schäffer Erik , Stelzer Ernst H. K. , Hyman Anthony A.
Published: July 2003

During unequal cell divisions a mitotic spindle is eccentrically positioned before cell cleavage. To determine the basis of the net force imbalance that causes spindle displacement in one-cell
Caenorhabditis elegans
embryos, we fragmented centrosomes with an ultraviolet laser. Analysis of the mean and variance of fragment speeds suggests that the force imbalance is due to a larger number of force generators pulling on astral microtubules of the posterior aster relative to the anterior aster. Moreover, activation of heterotrimeric guanine nucleotide– binding protein (Gprotein) α subunits is required to generate these astral forces.
Can One Forget to Remember?
Authors: Ceci Stephen J.
Published: July 2003

Remembering Trauma
. by Richard J. McNally. Belknap (Harvard University Press), Cambridge, MA, 2003. 430 pp. $35.00, £23.50. ISBN 0-674-01082-5.

McNally provides a dispassionate evaluation of findings from cognitive, behavioral, and neurological studies of how people remember and forget traumatic experiences.
Potent, Persistent Induction and Modulation of Cellular Immune Responses in Rhesus Macaques Primed with Ad5hr-Simian Immunodeficiency Virus (SIV)
env/rev
,
gag
, and/or
nef
Vaccines and Boosted with SIV gp120
Authors: Patterson L. Jean , Malkevitch Nina , Pinczewski Joel , Venzon David , Lou Yuanmei , Peng Bo , Munch Cindy , Leonard Melissa , Richardson Ersell , Aldrich Kristine , Kalyanaraman V. S. , Pavlakis George N. , Robert-Guroff Marjorie
Published: July 2003

Immunity elicited by multicomponent vaccines delivered by replication-competent Ad5hr-simian immunodeficiency virus (SIV) recombinants was systematically investigated. Rhesus macaques were immunized mucosally at weeks 0 and 12 with Ad5hr-SIV
smH4
env/rev
, with or without Ad5hr-SIV
mac239
gag
or Ad5hr-SIV
mac239
nef
, or with all three recombinants. The total Ad5hr dosage was comparably adjusted among all animals with empty Ad5hr-ΔE3 vector. The macaques were boosted with SIV gp120 in monophosphoryl A-stable emulsion adjuvant at 24 and 36 weeks. Controls received Ad5hr-ΔE3 vector or adjuvant only. By ELISPOT analysis, all four SIV gene products elicited potent cellular immune responses that persisted 42 weeks post-initial immunization. Unexpectedly, modulation of this cellular immune response was observed among macaques receiving one, two, or three Ad5hr-SIV recombinants. Env responses were significantly enhanced throughout the immunization period in macaques immunized with Ad5hr-SIV
env/rev
plus Ad5hr-SIV
gag
and tended to be higher in macaques that also received Ad5hr-SIV
nef
. Macaques primed with all three recombinants displayed significant down-modulation in numbers of gamma interferon (IFN-γ)-secreting cells specific for SIV Nef, and the Env- and Gag-specific responses were also diminished. Modulation of antibody responses was not observed. Down-modulation was seen only during the period of Ad5hr-recombinant priming, not during subunit boosting, although SIV-specific IFN-γ-secreting cells persisted. The effect was not attributable to Ad5hr replication differences among immunization groups. Vaccine delivery via replication-competent live vectors, which can persistently infect new cells and continuously present low-level antigen, may be advantageous in overcoming competition among complex immunogens for immune recognition. Effects of current multicomponent vaccines on individual immune responses should be evaluated with regard to future vaccine design.
Diminished RNA Primer Usage Associated with the L74V and M184V Mutations in the Reverse Transcriptase of Human Immunodeficiency Virus Type 1 Provides a Possible Mechanism for Diminished Viral Replication Capacity
Authors: Diallo Karidia , Marchand Bruno , Wei Xin , Cellai Luciano , Götte Matthias , Wainberg Mark A.
Published: July 2003

The emergence of drug resistance-conferring mutations can severely compromise the success of chemotherapy directed against human immunodeficiency virus type 1 (HIV-1). The M184V and/or L74V mutation in the reverse transcriptase (RT) gene are frequently found in viral isolates from patients treated with the nucleoside RT inhibitors lamivudine (3TC), abacavir (ABC), and didanosine (ddI). However, the effectiveness of combination therapy with regimens containing these compounds is often not abolished in the presence of these mutations; it has been conjectured that diminished fitness of HIV-1 variants containing L74V and M184V may contribute to sustained antiviral effects in such cases. We have determined that viruses containing both L74V and M184V are more impaired in replication capacity than viruses containing either mutation alone. To understand the biochemical mechanisms responsible for this diminished fitness, we generated a series of recombinant mutated enzymes containing either or both of the L74V and M184V substitutions. These enzymes were tested for their abilities to bypass important rate-limiting steps during the complex process of reverse transcription. We studied both the initiation of minus-strand DNA synthesis with the cognate replication primer human tRNA
3
Lys
and the initiation of plus-strand DNA synthesis, using a short RNA primer derived from the viral polypurine tract. We observed that the efficiencies of both reactions were diminished with enzymes containing either L74V or M184V and that these effects were significantly amplified with the double mutant. We also show that release from intrinsic pausing sites during reverse transcription appears to be a major obstacle that cannot be efficiently bypassed. Our data suggest that the efficiency of RNA-primed DNA synthesis represents an important consideration that can affect viral replication kinetics.
Identification of Foot-and-Mouth Disease Virus-Specific Linear B-Cell Epitopes To Differentiate between Infected and Vaccinated Cattle
Authors: Höhlich Bettina-Judith , Wiesmüller Karl-Heinz , Schlapp Tobias , Haas Bernd , Pfaff Eberhard , Saalmüller Armin
Published: July 2003

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. For several years, vaccination of animals, which had proven to be successful for the eradication of the disease, has been forbidden in the United States and the European Community because of the difficulty of differentiating between vaccinated and infected animals. In this study, detailed investigations of the bovine humoral immune response against FMD virus (FMDV) were performed with the aim of identifying viral epitopes recognized specifically by sera derived from FMDV-infected animals. The use of overlapping 15-mer synthetic peptides, covering the whole open reading frame of FMDV strain O
1
K in a peptide enzyme-linked immunosorbent assay, allowed the identification of 12 FMDV strain O
1
K-specific linear B-cell epitopes. Six of these linear B-cell epitopes, located in the nonstructural proteins, were used in further assays to compare the reactivities of sera from vaccinated and infected cattle. Antibodies recognizing these peptides could be detected only in sera derived from infected cattle. In further experiments, the reactivity of the six peptides with sera from animals infected with different strains of FMDV was tested, and strain-independent infection-specific epitopes were identified. Thus, these results clearly demonstrate the ability of a simple peptide-based assay to discriminate between infected and conventionally FMD-vaccinated animals.
Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Proteolytic Processing by Subtilase SKI-1
Authors: Vincent Martin J. , Sanchez Angela J. , Erickson Bobbie R. , Basak Ajoy , Chretien Michel , Seidah Nabil G. , Nichol Stuart T.
Published: July 2003

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus
Nairovirus
, family
Bunyaviridae
. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol.
76:
7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family
Arenaviridae
) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-
cis
Golgi compartments and was dependent on an R at the −4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.
Reverse Genetic System for the Analysis of Parvovirus Telomeres Reveals Interactions between Transcription Factor Binding Sites in the Hairpin Stem
Authors: Burnett Erik , Tattersall Peter
Published: July 2003

The left-hand or 3′-terminal hairpin of minute virus of mice (MVM) contains sequence elements essential for both viral DNA replication at the left-hand origin (oriL) and for the modulation of the P4 promoter, from which the viral nonstructural gene cassette is transcribed. This hairpin sequence has proven difficult to manipulate in the context of the viral genome. Here we describe a system for generating mutant viruses using synthetic hairpin oligonucleotides and a truncated form of the infectious clone. This allows manipulation of the sequence of the left-hand hairpin and examination of the effects in the context of the viral life cycle. We have confirmed the requirement for a functional parvovirus initiation factor (PIF) binding site and determined that an optimized PIF binding site, with 6 bases between the half-sites, was actually detrimental to viral growth. The distal PIF half-site overlaps a cyclic AMP-responsive element (CRE), which was shown to play an important role in initiating infection, particularly in 324K simian virus 40-transformed human fibroblasts. Interestingly, reducing the spacing of the PIF half-sites, and thus the affinity of the binding site for PIF, increased viral fitness relative to wild type in 324K cells, but not in murine A9 cells. These results indicate that the relative importance of factor binding to the CRE and PIF sites during the establishment of an infection differs markedly between these two host cells and suggest that the suboptimal spacing of PIF half-sites found in wild-type virus represents a necessary reduction in the affinity of the PIF interaction in favor of CRE function.
Nonstructural Proteins NS1 and NS2 of Bovine Respiratory Syncytial Virus Block Activation of Interferon Regulatory Factor 3
Authors: Bossert Birgit , Marozin Sabrina , Conzelmann Karl-Klaus
Published: July 2003

We have previously shown that the nonstructural (NS) proteins NS1 and NS2 of bovine respiratory syncytial virus (BRSV) mediate resistance to the alpha/beta interferon (IFN)-mediated antiviral response. Here, we show that they, in addition, are able to prevent the induction of beta IFN (IFN-β) after virus infection or double-stranded RNA stimulation. In BRSV-infected MDBK cells upregulation of IFN-stimulated genes (ISGs) such as MxA did not occur, although IFN signaling via JAK/STAT was found intact. In contrast, infection with recombinant BRSVs lacking either or both NS genes resulted in efficient upregulation of ISGs. Biological IFN activity and IFN-β were detected only in supernatants of cells infected with the NS deletion mutants but not with wild-type (wt) BRSV. Subsequent analyses of IFN-β promoter activity showed that infection of cells with the double deletion mutant BRSV ΔNS1/2, but not with BRSV wt, resulted in a significant increase in
IFN-β
gene promoter activity. Induction of the IFN-β promoter depends on the activation of three distinct transcription factors, NF-κB, ATF-2/c-Jun, and IFN regulatory factor 3 (IRF-3). Whereas NF-κB and ATF-2/c-Jun activities were readily detectable and comparable in both wt BRSV- and BRSV ΔNS1/2-infected cells, phosphorylation and transcriptional activity of IRF-3, however, were observed only after BRSV ΔNS1/2 infection. NS protein-mediated inhibition of IRF-3 activation and IFN induction should have considerable impact on the pathogenesis and immunogenicity of BRSV.
Identification of a Region of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replication Proteins Which Binds Specifically to the Viral 3′-Terminal tRNA-Like Structure
Authors: Osman T. A. M. , Buck K. W.
Published: July 2003

UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L [TMV-L]) RNA-dependent RNA polymerase complex and the TMV-L RNA 3′-terminal region (3′-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3′-TR. Using bothEscherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3′-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3′-TR. Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3′-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA. The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3′-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3′-terminal CCCA initiation site.
Newcastle Disease Virus V Protein Is Associated with Viral Pathogenesis and Functions as an Alpha Interferon Antagonist
Authors: Huang Zhuhui , Krishnamurthy Sateesh , Panda Aruna , Samal Siba K.
Published: July 2003

Newcastle disease virus (NDV) edits its P gene by inserting one or two G residues at the conserved editing site (UUUUUCCC, genome sense) and transcribes the P mRNA (unedited), the V mRNA (with a +1 frameshift), and the W mRNA (with a +2 frameshift). All three proteins are amino coterminal but vary at their carboxyl terminus in length and amino acid composition. Little is known about the role of the V and W proteins in NDV replication and pathogenesis. We have constructed and recovered two recombinant viruses in which the expression of the V or both the V and W proteins has been abolished. Compared to the parental virus, the mutant viruses showed impaired growth in cell cultures, except in Vero cells. However, transient expression of the carboxyl-terminal portion of the V protein enhanced the growth of the mutant viruses. In embryonated chicken eggs, the parental virus grew to high titers in embryos of different gestational ages, whereas the mutant viruses showed an age-dependent phenomenon, growing to lower titer in more-developed embryos. An interferon (IFN) sensitivity assay showed that the parental virus was more resistant to the antiviral effect of IFN than the mutant viruses. Moreover, infection with the parental virus resulted in STAT1 protein degradation, but not with the mutant viruses. These findings indicate that the V protein of NDV possesses the ability to inhibit alpha IFN and that the IFN inhibitory function lies in the carboxyl-terminal domain. Pathogenicity studies showed that the V protein of NDV significantly contributes to the virus virulence.
PML Residue Lysine 160 Is Required for the Degradation of PML Induced by Herpes Simplex Virus Type 1 Regulatory Protein ICP0
Authors: Boutell Chris , Orr Anne , Everett Roger D.
Published: July 2003

During the early stages of herpes simplex virus type 1 (HSV-1) infection, viral immediate-early regulatory protein ICP0 localizes to and disrupts cellular nuclear structures known as PML nuclear bodies or ND10. These activities correlate with the functions of ICP0 in stimulating lytic infection and reactivating quiescent HSV-1. The disruption of ND10 occurs because ICP0 induces the loss of the SUMO-1-modified forms of PML and the subsequent proteasome-mediated degradation of the PML protein. The functions of ICP0 are largely dependent on the integrity of its zinc-binding RING finger domain. Many RING finger proteins have been found to act as ubiquitin E3 ligase enzymes, stimulating the production of conjugated polyubiquitin chains in the presence of ubiquitin, the ubiquitin-activating enzyme E1, and the appropriate E2 ubiquitin-conjugating enzyme. Substrate proteins that become polyubiquitinated are then subject to degradation by proteasomes. We have previously shown that purified full-length ICP0 acts as an efficient E3 ligase in vitro, producing high-molecular-weight polyubiquitin chains in a RING finger-dependent but substrate-independent manner. In this paper we report on investigations into the factors governing the degradation of PML induced by ICP0 in a variety of in vivo and in vitro assays. We found that ICP0 expression increases the levels of ubiquitinated PML in transfected cells. However, ICP0 does not interact with or directly ubiquitinate either unmodified PML or SUMO-1-modified PML in vitro, suggesting either that additional factors are required for the ICP0-mediated ubiquitination of PML in vivo or that PML degradation is an indirect consequence of some other activity of ICP0 at ND10. Using a transfection-based approach and a family of deletion and point mutations of PML, we found that efficient ICP0-induced PML degradation requires sequences within the C-terminal part of PML and lysine residue 160, one of the principal targets for SUMO-1 modification of the protein.
Selection of Retroviral Reverse Transcription Primer Is Coordinated with tRNA Biogenesis
Authors: Kelly Nathan J. , Palmer Matthew T. , Morrow Casey D.
Published: July 2003

Initiation of retrovirus reverse transcription requires the selection of a tRNA primer from the intracellular milieu. To investigate the features of primer selection, a human immunodeficiency virus type 1 (HIV-1) and a murine leukemia virus (MuLV) were created that require yeast tRNA
Phe
to be supplied in
trans
for infectivity. Wild-type yeast tRNA
Phe
expressed in mammalian cells was transported to the cytoplasm and aminoacylated. In contrast, tRNA
Phe
without the D loop (tRNA
Phe
D
−
) was retained within the nucleus and did not complement infectivity of either HIV-1 or MuLV; however, infectivity was restored when tRNA
Phe
D
−
was directly transfected into the cytoplasm of cells. A tRNA
Phe
mutant (tRNA
Phe
UUA) that did not have the capacity to be aminoacylated was transported to the cytoplasm and did complement infectivity of both HIV-1 and MuLV, albeit at a level less than that with wild-type tRNA
Phe
. Collectively, our results demonstrate that the tRNA primer captured by HIV-1 and MuLV occurs after nuclear export of tRNA and supports a model in which primer selection for retroviruses is coordinated with tRNA biogenesis at the intracellular site of protein synthesis.
Oral Immunogenicity of Human Papillomavirus-Like Particles Expressed in Potato
Authors: Warzecha Heribert , Mason Hugh S. , Lane Christopher , Tryggvesson Anders , Rybicki Edward , Williamson Anna-Lise , Clements John D. , Rose Robert C.
Published: July 2003

Human papillomavirus-like particles (HPV VLPs) have shown considerable promise as a parenteral vaccine for the prevention of cervical cancer and its precursor lesions. Parenteral vaccines are expensive to produce and deliver, however, and therefore are not optimal for use in resource-poor settings, where most cervical HPV disease occurs. Transgenic plants expressing recombinant vaccine immunogens offer an attractive and potentially inexpensive alternative to vaccination by injection. For example, edible plants can be grown locally and can be distributed easily without special training or equipment. To assess the feasibility of an HPV VLP-based edible vaccine, in this study we synthesized a plant codon-optimized version of the HPV type 11 (HPV11) L1 major capsid protein coding sequence and introduced it into tobacco and potato. We show that full-length L1 protein is expressed and localized in plant cell nuclei and that expression of L1 in plants is enhanced by removal of the carboxy-terminal nuclear localization signal sequence. We also show that plant-expressed L1 self-assembles into VLPs with immunological properties comparable to those of native HPV virions. Importantly, ingestion of transgenic L1 potato was associated with activation of an anti-VLP immune response in mice that was qualitatively similar to that induced by VLP parenteral administration, and this response was enhanced significantly by subsequent oral boosting with purified insect cell-derived VLPs. Thus, papillomavirus L1 protein can be expressed in transgenic plants to form immunologically functional VLPs, and ingestion of such material can activate potentially protective humoral immune responses.
Replication-Competent Bacterial Artificial Chromosomes of Marek's Disease Virus: Novel Tools for Generation of Molecularly Defined Herpesvirus Vaccines
Authors: Petherbridge Lawrence , Howes Ken , Baigent Susan J. , Sacco Melanie A. , Evans Simon , Osterrieder Nikolaus , Nair Venugopal
Published: July 2003

Marek's disease (MD), a highly infectious disease caused by an oncogenic herpesvirus, is one of the few herpesvirus diseases against which live attenuated vaccines are used as the main strategy for control. We have constructed bacterial artificial chromosomes (BACs) of the CVI988 (Rispens) strain of the virus, the most widely used and effective vaccine against MD. Viruses derived from the BAC clones were stable after in vitro and in vivo passages and showed characteristics and growth kinetics similar to those of the parental virus. Molecular analysis of the individual BAC clones showed differences in the structure of the
meq
gene, indicating that the commercial vaccine contains virus populations with distinct genomic structures. We also demonstrate that, contrary to the published data, the sequence of the L-
meq
of the BAC clone did not show any frameshift. Virus stocks derived from one of the BAC clones (clone 10) induced 100 percent protection against infection by the virulent strain RB1B, indicating that BAC-derived viruses could be used with efficacies similar to those of the parental CVI988 vaccines. As a DNA vaccine, this BAC clone was also able to induce protection in 6 of 20 birds. Isolation of CVI988 virus from all of these six birds suggested that immunity against challenge was probably dependent on the reconstitution of the virus in vivo and that such viruses are also as immunogenic as the in vitro-grown BAC-derived or parental vaccine viruses. Although the reasons for the induction of protection only in a proportion of birds (33.3%) that received the DNA vaccine are not clear, this is most likely to be related to the suboptimal method of DNA delivery. The construction of the CVI988 BAC is a major step towards understanding the superior immunogenic features of CVI988 and provides the opportunity to exploit the power of BAC technology for generation of novel molecularly defined vaccines.
Induction of Protective Immune Responses against R5 Human Immunodeficiency Virus Type 1 (HIV-1) Infection in hu-PBL-SCID Mice by Intrasplenic Immunization with HIV-1-Pulsed Dendritic Cells: Possible Involvement of a Novel Factor of Human CD4+T-Cell Origin
Authors: Yoshida Atsushi , Tanaka Reiko , Murakami Tsutomu , Takahashi Yoshiaki , Koyanagi Yoshio , Nakamura Masataka , Ito Mamoru , Yamamoto Naoki , Tanaka Yuetsu
Published: July 2003

The potential of a dendritic cell (DC)-based vaccine against human immunodeficiency virus type 1 (HIV-1) infection in humans was explored with SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC). HIV-1-negative normal human PBMC were transplanted directly into the spleens of SCID mice (hu-PBL-SCID-spl mice) together with autologous mature DCs pulsed with either inactivated HIV-1 (strain R5 or X4) or ovalbumin (OVA), followed by a booster injection 5 days later with autologous DCs pulsed with the same respective antigens. Five days later, these mice were challenged intraperitoneally with R5 HIV-1JR-CSF. Analysis of infection at 7 days postinfection showed that the DC-HIV-1-immunized hu-PBL-SCID-spl mice, irrespective of the HIV-1 isolate used for immunization, were protected against HIV-1 infection. In contrast, none of the DC-OVA-immunized mice were protected. Sera from the DC-HIV-1- but not the DC-OVA-immunized mice inhibited the in vitro infection of activated PBMC and macrophages with R5, but not X4, HIV-1. Upon restimulation with HIV-1 in vitro, the human CD4+T cells derived from the DC-HIV-1-immunized mice produced a similar R5 HIV-1 suppressor factor. Neutralizing antibodies against human RANTES, MIP-1α, MIP-1β, alpha interferon (IFN-α), IFN-β, IFN-γ, interleukin-4 (IL-4), IL-10, IL-13, IL-16, MCP-1, MCP-3, tumor necrosis factor alpha (TNF-α), or TNF-β failed to reverse the HIV-1-suppressive activity. These results show that inactivated HIV-1-pulsed autologous DCs can stimulate splenic resident human CD4+T cells in hu-PBL-SCID-spl mice to produce a yet-to-be-defined, novel soluble factor(s) with protective properties against R5 HIV-1 infection.
Plasmid Chemokines and Colony-Stimulating Factors Enhance the Immunogenicity of DNA Priming-Viral Vector Boosting Human Immunodeficiency Virus Type 1 Vaccines
Authors: Barouch Dan H. , McKay Paul F. , Sumida Shawn M. , Santra Sampa , Jackson Shawn S. , Gorgone Darci A. , Lifton Michelle A. , Chakrabarti Bimal K. , Xu Ling , Nabel Gary J. , Letvin Norman L.
Published: July 2003

Heterologous “prime-boost” regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.
Identification of the E9^E2C cDNA and Functional Characterization of the Gene Product Reveal a New Repressor of Transcription and Replication in Cottontail Rabbit Papillomavirus
Authors: Jeckel Sonja , Loetzsch Ekaterina , Huber Evamaria , Stubenrauch Frank , Iftner Thomas
Published: July 2003

Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9^E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9^E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9^E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9^E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9^E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9^E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.