bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma-surface interactions of nanoporous silica during plasma-based pattern transfer using C4F8 and C4F8∕Ar gas mixtures
Authors: Hua Xuefeng , Stolz Christian , Oehrlein G. S. , Lazzeri P. , Coghe N. , Anderle M. , Inoki C. K. , Kuan T. S. , Jiang P.
Published: January 2005

We have investigated plasma surface interactions of nanoporous silica (NPS) films with porosities up to 50%, and SiO2 with C4F8∕Ar discharges used for plasma etching. The pore size was about 2–3nm for all films. In highly polymerizing plasmas (e.g., pure C4F8 discharges), the porous structure of NPS material favors surface polymerization over etching and porosity-corrected etching rates (CER) were suppressed and lower than SiO2 etching rate for the same conditions. The etching rates of NPS were dramatically enhanced in ion rich discharges (e.g., C4F8∕90%Ar) and the CER in this case is greater than the SiO2 etching rate. Both x-ray photoelectron spectroscopy (XPS) and static secondary ion mass spectroscopy (static SIMS) show that fairly thick (∼2–3nm) fluorocarbon layers exist on the NPS surface during C4F8 etching. This layer blocks the direct interaction of ions with the NPS surface and results in a low etching rate. For C4F8∕90%Ar discharges, little fluorocarbon coverage is observed for NPS surfaces and the direct ion surface interaction is significantly enhanced, explaining the enhancement of CER. We can deduce from analysis of angular resolved XPS data that the surface of NPS materials and SiO2 remain smooth during C4F8 etching. For C4F8∕90%Ar etching, the NPS surfaces became rough. The surface roughening is due to angle-dependent ion etching effects. These surface models were directly verified by the transmission electron microscopy. Depth profiling study of NPS partially etched using C4F8 or C4F8∕90%Ar discharges using dynamic SIMS indicates that the plasma induced modification of NPS was enhanced significantly compared with SiO2 due to the porous structure, which allows the plasma attack of the subsurface region. The modified layer thickness is related to the overall porosity and dramatically increases for NPS with an overall porosity of 50%. The distinct etching behavior of high porosity NPS (∼50%) in fluorocarbon-based discharges relative to NPS material with lower overall porosity is possibly due to interconnected pores, which allow plasma species to more easily penetrate into the subsurface region.
Surface chemistry and infrared absorbance changes during ZnO atomic layer deposition on ZrO2 and BaTiO3 particles
Authors: Ferguson J. D. , Weimer A. W. , George S. M.
Published: January 2005

ZnO atomic layer deposition (ALD) was achieved using sequential exposures of Zn(CH2CH3)2 and H2O on ZrO2 and BaTiO3 particles at 450K. The surface chemistry of ZnO ALD was monitored in vacuum using Fourier transform infrared spectroscopy. The BaTiO3 and ZrO2 particles initially displayed vibrational features consistent with surface hydroxyl (-OH) groups. Zn(CH2CH3)2 exposure removed the surface hydroxyl groups and created Zn(CH2CH3)* surface species. The subsequent H2O exposure removed the Zn(CH2CH3)* surface species and produced ZnOH* surface species. Repeating the Zn(CH2CH3)2 and H2O exposures in an ABAB… reaction sequence at 450K progressively deposited ZnO. Because ZnO is a semiconductor, the background infrared absorbance increased with the number of AB cycles during the deposition of the ZnO film. The increasing background infrared absorbance during long Zn(CH2CH3)2 exposures also revealed that the Zn(CH2CH3)2 reaction is not self-limiting. The background absorbance was modulated dramatically by the presence of ethyl (–CH2CH3) or hydroxyl (-OH) groups on the surface of the growing ZnO film. The infrared absorbance was higher with hydroxyl (-OH) groups and lower with ethyl (–CH2CH3) groups on the ZnO surface. The background absorbance changes were not linear with surface coverage. The large changes in absorbance after low reactant exposure suggested that the most reactive surface sites may be most influential in affecting the film conductance. Transmission electron microscopy (TEM) was used to examine the ZnO films deposited on the ZrO2 and BaTiO3 particles. The TEM images revealed ZrO2 and BaTiO3 particles encapsulated by conformal ZnO films. The ZnO films had a thickness of ∼43Å after 20 AB reaction cycles and ∼65Å after 30 AB reaction cycles, respectively. These TEM images are consistent with a ZnO ALD growth rate at 450K of ∼2.2Å∕AB cycle.
Suppression of nanoscopic shadowing during physical vapor deposition by biased diffusion
Authors: Kools J. C. S.
Published: January 2005

In this article, we study the interaction between two nanoscopic growth mechanisms during physical vapor deposition, namely, biased diffusion and atomic level shadowing by defects such as islands and ledges, by molecular dynamics simulations. The material system described here is homoepitaxy of Cu on Cu(111). We find that the phenomenon of biased diffusion has a strong effect on the details of the shadowing process at the atomic scale. In the particular case of deposition by high energy particles at off-normal angles, biased diffusion suppresses the shadowing effect that dominates at low energy and off-normal angles. This can be understood from an analysis of the potential energy landscape as seen by the arriving adatom.
Mechanical properties and thermal stability of TiN∕TiB2 nanolayered thin films
Authors: Martin K. J. , Madan A. , Hoffman D. , Ji J. , Barnett S. A.
Published: January 2005

This article describes a study of TiN∕TiB2 nanolayered coatings on sapphire and M2 tool steel substrates. Residual stress in as-deposited TiN∕TiB2 varied from tensile to compressive with increasing substrate bias. Increasing the density of nanolayer interfaces (i.e., decreasing bi-layer period) decreased the compressive stress; this effect was explained by diffusion of point defects to interfaces and/or an indirect effect of interfaces on stress via film structure. A thin TiN buffer layer substantially reduced the stress and improved adhesion on steel. Nanolayer film adhesion on steel was generally intermediate between that of monolithic TiN coatings, which was good, and TiB2 coatings, which was poor. As-deposited nanolayers showed no hardness enhancement relative to rule of mixtures. X-ray diffraction results showed that the boride layers tended to be amorphous, especially for small layer thicknesses. After annealing at 1000°C, nanolayer structure was retained, thin boride layers were at least partially crystallized, and hardnesses increased to as high as 49GPa due to crystallization of the TiB2 layers.
Pulsed dc operation of a Penning-type opposed target magnetron
Authors: Moiseev T. , Cameron D. C.
Published: January 2005

This article describes the medium frequency pulsed dc power operation of a Penning-type (opposed target) magnetron to sputter copper in an argon atmosphere. The current–voltage characteristics of the magnetron are detailed in dc and pulsed dc conditions. The variations in optical emission from the excited species in the plasma are measured as a function of time during the applied pulses. The intensity of the argon emissions closely follows the variations of the target voltage but the different behavior of the copper emissions shows some evidence of self-sputtering of the target by copper ions. The energy and flux of the ions arriving at the substrate are measured as a function of time and it is shown that during pulsed operation, as the pulse off-period is extended, there are large increases in ion energy and ion flux. The initial overshoots in the target voltage cause a rapid expansion of the plasma sheath at the analyzer probe and wide variations in plasma potential leading to an initial pulse of high energy ions during the first 500ns. The plasma potential then settles to ∼40eV and there is a second peak in ion flux at this energy with a peak at ∼4000ns due to the increased ion drift resulting from electron extraction from the target.
Temperature-dependent morphology evolution of the submonolayer clusters grown on fcc metal (110) surfaces
Authors: Liu Z.-J. , Shen Y. G.
Published: January 2005

The temperature dependent morphology transitions of epitaxial submonolayers grown on fcc metal (110) surfaces are studied by kinetic Monte Carlo simulation and rate-equation analysis. For a suitable system, there exists a complete morphology evolution: from small anisotropic clusters with preferential orientation in the [001] direction to anisotropic islands elongated to the [11¯0] channel at low temperatures, then to nearly one-dimensional (1D) monoatomic chains at intermediate temperatures, and finally to two-dimensional (2D) islands at high temperatures. The first transition in island orientation is controlled by the anisotropy in terrace diffusion and the strength of cross-channel bonding. The second transition of 1D chain to 2D island can be generated by the anisotropy in detachment for fully reversible aggregation in the presence of cross-channel atomic interaction or by the strong anisotropy in corner rounding for aggregation without detachment. However, using an approximation of bond-breaking model to the activation energies of adatom hopping, the anisotropic detachment or anisotropic corner rounding is actually related to the anisotropy in bonding. Based on the simulated results, we also derive general conditions for the occurrences of these transitions under our simulation conditions.
Laser-induced damage threshold of ZrO2 thin films prepared at different oxygen partial pressures by electron-beam evaporation
Authors: Zhang Dongping , Shao Jianda , Zhao Yuanan , Fan Shuhai , Hong Ruijing , Fan Zhengxiu
Published: January 2005

Zr O 2 films were deposited by electron-beam evaporation with the oxygen partial pressure varying from 3×10−3Pa to 11×10−3Pa. The phase structure of the samples was characterized by x-ray diffraction (XRD). The thermal absorption of the films was measured by the surface thermal lensing technique. A spectrophotometer was employed to measure the refractive indices of the samples. The laser-induced damage threshold (LIDT) was assessed using a 1064 nm Nd: yttritium–aluminum–garnet pulsed laser at pulse width of 12 ns. The influence of oxygen partial pressure on the microstructure and LIDT of ZrO2 films was investigated. XRD data revealed that the films changed from polycrystalline to amorphous as the oxygen partial pressure increased. The variation of refractive index at 550 nm wavelength indicated that the packing density of the films decreased gradually with increasing oxygen partial pressure. The absorptance of the samples decreased monotonically from 125.2 to 84.5 ppm with increasing oxygen partial pressure. The damage threshold values increased from 18.5 to 26.7J∕cm2 for oxygen partial pressures varying from 3×10−3Pa to 9×10−3Pa, but decreased to 17.3J∕cm2 in the case of 11×10−3Pa.
Study on the initial deposition of ZrO2 on hydrogen terminated silicon and native silicon oxide surfaces by high vacuum chemical vapor deposition
Authors: Song Zhe , Sullivan Lisa M. , Rogers Bridget R.
Published: January 2005

We used in situ spectroscopic ellipsometry to study the deposition process of ZrO2 from zirconium t-butoxide (ZTB) on both native silicon oxide and H-terminated silicon (H–Si) surfaces. The ZrO2 films deposited on native silicon oxide surfaces have higher refractive indexes and film densities. The properties of films deposited on different surfaces are affected by different nucleation and coalescence processes during the initial stage deposition. Due to the lack of reactive surface hydroxyl groups and high surface diffusivity of ZTB molecules, a three-dimensional nucleation process is predominant on H–Si surfaces. The resulting films have high surface roughness, and are inappropriate for gate dielectric applications. The highly reactive hydroxyl groups on native silicon oxide surface react with ZTB molecules to form a high-density film. At temperatures higher than the decomposition temperature of the t-butoxy group, further nucleation of ZrO2 is suppressed. The resulting films have high film density and low topology development, and are more suitable for gate dielectric applications. Additionally, we discuss the factors that influence the deposition process and film properties, and show that the deposition temperature can be used to control film density.
Einstein oscillators that impede thermal transport
Authors: Hermann Raphaël P. , Grandjean Fernande , Long Gary J.
Published: January 2005

The Einstein model of a solid usually lacks a clear illustration in introductory solid-state physics courses because most solids are much better described by the Debye model. Filled antimony skutterudites, materials that have recently attracted much attention because of their potential for thermoelectric applications, provide a canonical illustration of the Einstein model. The filling atoms are loosely bound in the atomic cage formed by their neighbors, and hence their description as independent harmonic oscillators is adequate. Simple models for the heat capacity and thermal conductivity of a solid are introduced, with emphasis on the density of vibrational states. These models are used in conjunction with experimental results obtained from heat capacity and inelastic neutron scattering measurements to demonstrate the applicability of the concept of the Einstein oscillator to the filling guests in antimony skutterudites. The importance of these Einstein oscillators for impeding thermal transport is discussed and some simple problems involving the heat capacity, thermal conductivity, and inelastic neutron scattering are proposed.
Paradoxical games and a minimal model for a Brownian motor
Authors: Astumian R. Dean
Published: January 2005

I give an extended analysis of the very simple game that I previously published that shows the paradoxical behavior whereby two losing games randomly combine to form a winning game. The game, modeled on a random walk, requires only two states and is described by a first-order Markov process.
Einstein’s boxes
Authors: Norsen Travis
Published: January 2005

At the 1927 Solvay conference, Albert Einstein presented a thought experiment intended to demonstrate the incompleteness of the quantum mechanical description of reality. In the following years, the experiment was modified by Einstein, de Broglie, and several other commentators into a simple scenario involving the splitting in half of the wave function of a single particle in a box. This paper collects together several formulations of this thought experiment from the literature, analyzes and assesses it from the point of view of the Einstein–Bohr debates, the EPR dilemma, and Bell’s theorem, and argues for “Einstein’s Boxes” taking its rightful place alongside similar but historically better known quantum mechanical thought experiments such as EPR and Schrödinger’s Cat.
Energy transfer in electrical circuits: A qualitative account
Authors: Galili Igal , Goihbarg Elisabetta
Published: January 2005

We demonstrate that the use of the Poynting vector for a model of the surface charge of a current carrying conductor can help qualitatively explain the transfer of energy in a dc closed circuit. The application of the surface charge model to a simple circuit shows that electromagnetic energy flows from both terminals of the battery, mainly in the vicinity of the wires (and not inside them) to the load where it enters and is converted into heat at a rate obtained from Ohm’s law.
The drop in the pot: Waves from a point source in a circular enclosure
Authors: Kaplan Harvey
Published: January 2005

We present the results of a computational study of the waves that leave a point source in a circular enclosure. We work in the geometric optics approximation. A video results from the instantaneous source, and a single picture from the effectively steady source. We thus find Green’s function for the scalar wave equation and for steady waves, respectively. We illustrate and explain some unexpected qualitative features of the waves, including the dependence of the focusing properties of the enclosure on the position of the point source. We compare our work with previous work on the natural focusing of light. The MATHEMATICA code that produces the video discussed in the paper is available on EPAPS.
Retinoic Acid Signaling Restricts the Cardiac Progenitor Pool
Authors: Keegan Brian R. , Feldman Jessica L. , Begemann Gerrit , Ingham Philip W. , Yelon Deborah
Published: January 2005

Organogenesis begins with specification of a progenitor cell population, the size of which provides a foundation for the organ's final dimensions. Here, we present a new mechanism for regulating the number of progenitor cells by limiting their density within a competent region. We demonstrate that retinoic acid signaling restricts cardiac specification in the zebrafish embryo. Reduction of retinoic acid signaling causes formation of an excess of cardiomyocytes, via fate transformations that increase cardiac progenitor density within a multipotential zone. Thus, retinoic acid signaling creates a balance between cardiac and noncardiac identities, thereby refining the dimensions of the cardiac progenitor pool.
CX
3
CR1-Mediated Dendritic Cell Access to the Intestinal Lumen and Bacterial Clearance
Authors: Niess Jan Hendrik , Brand Stephan , Gu Xiubin , Landsman Limor , Jung Steffen , McCormick Beth A. , Vyas Jatin M. , Boes Marianne , Ploegh Hidde L. , Fox James G. , Littman Dan R. , Reinecker Hans-Christian
Published: January 2005

Dendritic cells (DCs) and macrophages are critical to innate and adaptive immunity to the intestinal bacterial microbiota. Here, we identify a myeloid-derived mucosal DC in mice, which populates the entire lamina propria of the small intestine. Lamina propria DCs were found to depend on the chemokine receptor CX
3
CR1 to form transepithelial dendrites, which enable the cells to directly sample luminal antigens. CX
3
CR1 was also found to control the clearance of entero-invasive pathogens by DCs. Thus, CX
3
CR1-dependent processes, which control host interactions of specialized DCs with commensal and pathogenic bacteria, may regulate immunological tolerance and inflammation.
Testing Hypotheses: Prediction and Prejudice
Authors: Lipton Peter
Published: January 2005

Observations that fit a hypothesis may be made before or after the hypothesis is formulated. Can that difference be relevant to the amount of support that the observations provide for the hypothesis? Philosophers of science and statisticians are both divided on this question, but there is an argument that predictions ought to count more than accommodations, because of the risk of “fudging” that accommodations run and predictions avoid.
Stat3 Dimerization Regulated by Reversible Acetylation of a Single Lysine Residue
Authors: Yuan Zheng-long , Guan Ying-jie , Chatterjee Devasis , Chin Y. Eugene
Published: January 2005

Upon cytokine treatment, members of the signal transducers and activators of transcription (STAT) family of proteins are phosphorylated on tyrosine and serine sites within the carboxyl-terminal region in cells. We show that in response to cytokine treatment, Stat3 is also acetylated on a single lysine residue, Lys
685
. Histone acetyltransferase p300–mediated Stat3 acetylation on Lys
685
was reversible by type I histone deacetylase (HDAC). Use of a prostate cancer cell line (PC3) that lacks Stat3 and PC3 cells expressing wild-type Stat3 or a Stat3 mutant containing a Lys
685
-to-Arg substitution revealed that Lys
685
acetylation was critical for Stat3 to form stable dimers required for cytokine-stimulated DNA binding and transcriptional regulation, to enhance transcription of cell growth–related genes, and to promote cell cycle progression in response to treatment with oncostatin M.
Al Cluster Superatoms as Halogens in Polyhalides and as Alkaline Earths in Iodide Salts
Authors: Bergeron D. E. , Roach P. J. , Castleman A. W. , Jones N. O. , Khanna S. N.
Published: January 2005

Two classes of gas-phase aluminum-iodine clusters have been identified whose stability and reactivity can be understood in terms of the spherical shell jellium model. Experimental reactivity studies show that the Al
13
I
–

x

clusters exhibit pronounced stability for even numbers of I atoms. Theoretical investigations reveal that the enhanced stability is associated with complementary pairs of I atoms occupying the on-top sites on the opposing Al atoms of the Al
13
–
core. We also report the existence of another series, Al
14
I
–

x

, that exhibits stability for odd numbers of I atoms. This series can be described as consisting of an Al
14
I
–
3
core upon which the I atoms occupy on-top locations around the Al atoms. The potential synthetic utility of superatom chemistry built upon these motifs is addressed.
Self-Propagating, Molecular-Level Polymorphism in Alzheimer's ß-Amyloid Fibrils
Authors: Petkova Aneta T. , Leapman Richard D. , Guo Zhihong , Yau Wai-Ming , Mattson Mark P. , Tycko Robert
Published: January 2005

Amyloid fibrils commonly exhibit multiple distinct morphologies in electron microscope and atomic force microscope images, often within a single image field. By using electron microscopy and solid-state nuclear magnetic resonance measurements on fibrils formed by the 40-residue β-amyloid peptide of Alzheimer's disease (Aβ
1–40
), we show that different fibril morphologies have different underlying molecular structures, that the predominant structure can be controlled by subtle variations in fibril growth conditions, and that both morphology and molecular structure are self-propagating when fibrils grow from preformed seeds. Different Aβ
1–40
fibril morphologies also have significantly different toxicities in neuronal cell cultures. These results have implications for the mechanism of amyloid formation, the phenomenon of strains in prion diseases, the role of amyloid fibrils in amyloid diseases, and the development of amyloid-based nano-materials.
West Nile Virus Inhibits the Signal Transduction Pathway of Alpha Interferon
Authors: Guo Ju-Tao , Hayashi Junpei , Seeger Christoph
Published: January 2005

West Nile virus (WNV) is a human pathogen that can cause neurological disorders, including meningoencephalitis. Experiments with mice and mammalian cell cultures revealed that WNV exhibited resistance to the innate immune program induced by alpha interferon (IFN-α). We have investigated the nature of this inhibition and have found that WNV replication inhibited the activation of many known IFN-inducible genes, because it prevented the phosphorylation and activation of the Janus kinases JAK1 and Tyk2. As a consequence, activation of the transcription factors STAT1 and STAT2 did not occur in WNV-infected cells. Moreover, we demonstrated that the viral nonstructural proteins are responsible for this effect. Thus, our results provided an explanation for the observed resistance of WNV to IFN-α in cells of vertebrate origin.
Resistance to Apo2 Ligand (Apo2L)/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Mediated Apoptosis and Constitutive Expression of Apo2L/TRAIL in Human T-Cell Leukemia Virus Type 1-Infected T-Cell Lines
Authors: Matsuda Takehiro , Almasan Alex , Tomita Mariko , Uchihara Jun-nosuke , Masuda Masato , Ohshiro Kazuiku , Takasu Nobuyuki , Yagita Hideo , Ohta Takao , Mori Naoki
Published: January 2005

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-κB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-κB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed byN-acetyl-l-leucinyl-l-leucinyl-l-norleucinal (LLnL), an NF-κB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-κB is essential forApo2Lgene induction and protection against Apo2L-induced apoptosis and that suppression of NF-κB may be a useful adjunct in clinical use of Apo2L against ATL.
Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus
Authors: Hema M. , Gopinath K. , Kao C.
Published: January 2005

The
3′ portions of plus-strand brome mosaic virus (BMV) RNAs mimic
cellular tRNAs. Nucleotide substitutions or deletions in the 3′
CCA of the tRNA-like sequence (TLS) affect minus-strand initiation
unless repaired. We observed that 2-nucleotide deletions involving the
CCA 3′ sequence in one or all BMV RNAs still allowed RNA
accumulation in barley protoplasts at significant levels. Alterations
of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at
wild-type levels. However, substitutions in all three BMV RNAs severely
reduced RNA accumulation, demonstrating that substitutions have
different repair requirements than do small deletions. Furthermore,
wild-type BMV RNA1 was required for the repair and replication of RNAs
with nucleotide substitutions. Results from sequencing of progeny viral
RNA from mutant input RNAs demonstrated that RNA1 did not contribute
its sequence to the mutant RNAs. Instead, the repaired ends were
heterogeneous, with one-third having a restored CCA and others having
sequences with the only commonality being the restoration of one
cytidylate. The role of BMV RNA1 in increased repair was
examined.
Vaccination of Rhesus Macaques with RecombinantMycobacterium bovisBacillus Calmette-Guérin Env V3 Elicits Neutralizing Antibody-Mediated Protection against Simian-Human Immunodeficiency Virus with a Homologous but Not a Heterologous V3 Motif
Authors: Someya Kenji , Cecilia Dayaraj , Ami Yasushi , Nakasone Tadashi , Matsuo Kazuhiro , Burda Sherri , Yamamoto Hiroshi , Yoshino Naoto , Kaizu Masahiko , Ando Shuji , Okuda Kenji , Zolla-Pazner Susan , Yamazaki Shudo , Yamamoto Naoki , Honda Mitsuo
Published: January 2005

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinantMycobacterium bovisbacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.
Virus Particle Core Defects Caused by Mutations in the Human Immunodeficiency Virus Capsid N-Terminal Domain
Authors: Scholz Isabel , Arvidson Brian , Huseby Doug , Barklis Eric
Published: January 2005

The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures.
Sindbis Virus Translation Is Inhibited by a PKR/RNase L-Independent Effector Induced by Alpha/Beta Interferon Priming of Dendritic Cells
Authors: Ryman K. D. , Meier K. C. , Nangle E. M. , Ragsdale S. L. , Korneeva N. L. , Rhoads R. E. , MacDonald M. R. , Klimstra W. B.
Published: January 2005

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-α/β)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol.15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-α/β treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-α/β priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1−/−, and TD mice following infection with SB or treatment with IFN-α/β. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-α/β treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-α/β-treated IFNAR1−/−cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.
Conditional Mutations in the Mitotic Chromosome Binding Function of the Bovine Papillomavirus Type 1 E2 Protein
Authors: Zheng Peng-Sheng , Brokaw Jane , McBride Alison A.
Published: January 2005

The papillomavirus E2 protein is required for viral transcriptional regulation, DNA replication and genome segregation. We have previously shown that the E2 transactivator protein and BPV1 genomes are associated with mitotic chromosomes; E2 links the genomes to cellular chromosomes to ensure efficient segregation to daughter nuclei. The transactivation domain of the E2 protein is necessary and sufficient for association of the E2 protein with mitotic chromosomes. To determine which residues of this 200-amino-acid domain are important for chromosomal interaction, E2 proteins with amino acid substitutions in each conserved residue of the transactivation domain were tested for their ability to associate with mitotic chromosomes. Chromatin binding was assessed by using immunofluorescence on both spread and directly fixed mitotic chromosomes. E2 proteins defective in the transactivation and replication functions were unable to associate with chromosomes, and those that were competent in these functions were attached to mitotic chromosomes. However, several mutated proteins that were defective for chromosomal interaction could associate with chromosomes after treatment with agents that promote protein folding or when cells were incubated at lower temperatures. These results indicate that precise folding of the E2 transactivation domain is crucial for its interaction with mitotic chromosomes and that this association can be modulated.
Complex Formation between the UL16 and UL21 Tegument Proteins of Pseudorabies Virus
Authors: Klupp Barbara G. , Böttcher Sindy , Granzow Harald , Kopp Martina , Mettenleiter Thomas C.
Published: January 2005

The products of the UL16 and UL21 genes represent tegument proteins which are conserved throughout the mammalian herpesviruses. To identify and functionally characterize the respective proteins in the alphaherpesvirus pseudorabies virus, monospecific antisera against bacterially expressed fusion proteins were generated. In immunoblots the UL16 antiserum detected a ca. 40-kDa protein in infected cells and purified virion preparations, whereas the anti-UL21 serum recognized a protein of approximately 60 kDa. Interestingly, in immunoprecipitations using either antiserum, both proteins were coprecipitated, demonstrating the formation of a physical complex. To investigate protein function, viruses lacking either UL16, UL21, or both were constructed. Mutant viruses could be propagated on noncomplementing cells, indicating that these proteins, either alone or in combination, are not required for viral replication in cell culture. However, plaque sizes and viral titers were reduced. Electron microscopy showed only slight alterations in cytoplasmic virion morphogenesis, whereas intranuclear maturation stages were not affected. Similar results were obtained with a triple mutant simultaneously lacking the three conserved tegument proteins UL11, UL16, and UL21. In summary, our results uncover a novel interaction between conserved herpesvirus tegument proteins that increases the complexity of the intricate network of protein-protein interactions involved in herpesvirus morphogenesis.
Blockade of the Poliovirus-Induced Cytopathic Effect in Neural Cells by Monoclonal Antibody against Poliovirus or the Human Poliovirus Receptor
Authors: Yanagiya Akiko , Jia Qingmei , Ohka Seii , Horie Hitoshi , Nomoto Akio
Published: January 2005

The poliovirus (PV)-induced cytopathic effect (CPE) was blocked in neural cells but not in HeLa cells by the addition of monoclonal antibody (MAb) against PV or the human PV receptor (CD155) 2 h postinfection (hpi). Since each MAb has the ability to block viral infection, no CPE in PV-infected neural cells appeared to result from the blockade of multiple rounds of viral replication. Pulse-labeling experiments revealed that virus-specific protein synthesis proceeded 5 hpi with or without MAbs. However, in contrast to the results obtained without MAbs, virus-specific protein synthesis with MAbs was not detected 7 hpi. Shutoff of host translation was also not observed in the presence of MAbs. Western blot analysis showed that 2A
pro
, the viral protein which mediates the cleavage of eukaryotic translation initiation factor eIF4G, was still present 11 hpi. However, intact eIF4G appeared 11 hpi. An immunocytochemical study indicated that 2A
pro
was detected only in the nucleus 11 hpi. These results suggest that neural cells possess protective response mechanisms against PV infection as follows: (i) upon PV infection, neural cells produce a factor(s) to suppress PV internal ribosome entry site activity by 7 hpi, (ii) a factor which supports cap-dependent translation for eIF4G may exist in infected cells when no intact eIF4G is detected, and (iii) the remaining 2A
pro
is not effective in cleaving eIF4G because it is imported into the nucleus by 11 hpi.
Elimination of Retroviral Infectivity byN-Ethylmaleimide with Preservation of Functional Envelope Glycoproteins
Authors: Morcock David R. , Thomas James A. , Gagliardi Tracy D. , Gorelick Robert J. , Roser J. David , Chertova Elena N. , Bess Julian W. , Ott David E. , Sattentau Quentin J. , Frank Ines , Pope Melissa , Lifson Jeffrey D. , Henderson Louis E. , Crise Bruce J.
Published: January 2005

The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine ifN-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2′-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.
Involvement of Clathrin-Mediated Endocytosis in Human Immunodeficiency Virus Type 1 Entry
Authors: Daecke Jessica , Fackler Oliver T. , Dittmar Matthias T. , Kräusslich Hans-Georg
Published: January 2005

Productive entry of human immunodeficiency virus (HIV) is believed to occur by direct fusion at the plasma membrane. Endocytic uptake of HIV particles has been observed in several studies but is considered to be nonproductive, leading to virus degradation in the lysosome. We show here that endocytosis contributes significantly to productive HIV entry in HeLa cells by using
trans
dominant-negative mutants of dynamin and Eps15. Inducible expression of a dominant-negative mutant of dynamin in a CD4-positive HeLa cell line reduced HIV infection by 40 to 80%. This effect was independent of the infectious dose and was observed for three different isolates. Analysis of reverse transcription products by real-time PCR and of virus entry by delivery of a virion-associated Vpr-β-lactamase fusion protein revealed a similar reduction, indicating that the block occurred at the entry stage. A strong reduction of HIV entry was also observed upon transient transfection of a different
trans
dominant-negative variant of dynamin, and this reduction correlated with the relative inhibition of transferrin endocytosis. Expression of a dominant-negative variant of Eps15, which is specific for clathrin-dependent endocytosis, reduced HIV entry in HeLa cells by ca 95%, confirming the role of endocytosis for productive infection. In contrast, no effect was observed for a dominant-negative variant of caveolin. We conclude that dynamin-dependent, clathrin-mediated endocytosis can lead to productive entry of HIV in HeLa cells, suggesting this pathway as an alternative route of virus entry.
Herpes Simplex Virus Type 1 Infection via the Bloodstream with Apolipoprotein E Dependence in the Gonads Is Influenced by Gender
Authors: Burgos Javier S. , Ramirez Carlos , Sastre Isabel , Alfaro Juan M. , Valdivieso Fernando
Published: January 2005

Herpes simplex virus type 1 (HSV-1) causes disease in humans and animals. Infection usually occurs via the neural route and possibly occurs via the hematogenous route. The latter, however, is the main route by which immunosuppressed individuals and neonates are infected. Gender-dependent differences in the incidence and severity of some viral infections have been reported. To detect differences between the sexes with respect to HSV-1 colonization and disease, the characteristics of both acute and latent infections in hematogenously infected male and female mice were compared. In acute infection, the female mice had a poorer outcome: HSV-1 colonization was more effective, especially in the gonads and brain. In the encephalon, the midbrain had the highest viral load. In latent infection, brain viral loads were not significantly different with respect to sex. Significant differences were seen, however, in the blood and trigeminal ganglia: HSV-1 seroprevalence was observed in females, with no virus detected in males. In brain dissections, only the cerebral cortex of the females had viral loads statistically higher than those observed in the males. The spread of the virus to several organs of interest during acute infection was examined immunohistochemically. Female mice showed greater viral immunostaining, especially in the adrenal cortex, gonads, and midbrain. In male mice, HSV-1 was detected predominantly in the adrenal cortex. It was also found that apolipoprotein E promotes virus colonization of the ovaries, theAPOEgene dose being directly related to viral invasiveness.
Vaccinia Virus Penetration Requires Cholesterol and Results in Specific Viral Envelope Proteins Associated with Lipid Rafts
Authors: Chung Che-Sheng , Huang Cheng-Yen , Chang Wen
Published: January 2005

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-β-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4°C and was only observed when virion penetration occurred at 37°C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.
Molecular and Biological Characterization of Human Monoclonal Antibodies Binding to the Spike and Nucleocapsid Proteins of Severe Acute Respiratory Syndrome Coronavirus
Authors: van den Brink Edward N. , ter Meulen Jan , Cox Freek , Jongeneelen Mandy A. C. , Thijsse Alexandra , Throsby Mark , Marissen Wilfred E. , Rood Pauline M. L. , Bakker Alexander B. H. , Gelderblom Hans R. , Martina Byron E. , Osterhaus Albert D. M. E. , Preiser Wolfgang , Doerr Hans Wilhelm , de Kruif John , Goudsmit Jaap
Published: January 2005

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.