bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of N2 addition on density and temperature of radicals in 60MHz capacitively coupled c-C4F8 gas plasma
Authors: Nagai M. , Hori M.
Published: September 2006

Behaviors of electrons and fluorocarbon radicals were investigated in 60MHz capacitively coupled plasma employing mixture gases of c-C4F8, Ar, and N2. Optical emission spectroscopy was applied to measurements of the rotational temperatures of CF radical and N2 molecule, and infrared diode laser absorption spectroscopy was applied to that of the densities of CF and CF2 radicals. The electron density was decreased from 7.3×1010to3.0×1010cm−3, and electron temperature was increased from 3.0to5.1eV with increasing N2 flow rate. The addition of N2 to Ar∕C4F8 plasma decreased the density of CF radical dominantly as compared with CF2 radical. The rotational temperatures of CF radical were estimated from the (1-2) band of BΔ2–XΠ2 electronic transition of CF radicals. It was found that the rotational temperatures of CF radicals were increased with increasing N2 flow rate. The temperatures of radical indicated the different behaviors from the temperatures of N2 molecule. The rotational temperature of CF radical has a good relation with the electron temperature.
Tantalum carbide etch characterization in inductively coupled Ar∕Cl2∕HBr plasmas
Authors: Kawai H. , Rauf S. , Luckowski E. , Ventzek P. L. G.
Published: September 2006

The etching properties of tantalum carbide (TaC) in inductively coupled Ar∕HBr∕Cl2 plasmas are investigated in this article. Both etching experiments on patterned and blanket wafers and an integrated plasma equipment-feature scale computational model are utilized in this investigation. Results show that TaC etching is adequately described by the classical reactive ion etching mechanism, whereby etching occurs due to the synergistic effect of Cl or Br atoms and energetic ions. TaC etches faster in the presence of Cl relative to Br. The TaC etch rate is small in gas mixtures containing 5% of Cl2 or HBr and 95% of Ar, and it increases considerably as Cl2 or HBr concentration is increased. Although this etch rate increase is partially due to the availability of more Cl or Br, the chemical nature of chlorine (Cl2+,Cl+) or bromine (Br+) ions also plays a strong role. The TaC etch rate increases little if Cl2 or HBr fraction in Ar∕Cl2 or Ar∕HBr gas mixture, respectively, is increased beyond 25%. The TaC etch rate increases with rf bias power under all conditions. Scanning electron micrographs of TaC films etched using a patterned mask show that TaC sidewalls are tapered at about 77°±3° and the angle does not change appreciably with gas mixture or rf bias power. It is determined that an angle dependent ion etching yield captures well the observed trends in TaC sidewall slope.
Corrosion resistance and antithrombogenic behavior of La and Nd ion implanted stainless steels
Authors: Jing F. J. , Jin F. Y. , Liu Y. W. , Wan G. J. , Liu X. M. , Zhao X. B. , Fu R. K. Y. , Leng Y. X. , Huang N. , Chu Paul K.
Published: September 2006

Lanthanide ions such as lanthanum (La) and neodymium (Nd) were implanted into 316 stainless steel samples using metal vapor vacuum arc to improve the surface corrosion resistance and antithrombogenic properties. X-ray photoelectron spectroscopy shows that lanthanum and neodymium exist in the +3 oxidation state in the surface layer. The corrosion properties of the implanted and untreated control samples were investigated utilizing electrochemical tests and our results show that La and Nd implantations enhance the surface corrosion resistance. In vitro activated partial thromboplastin time (APTT) tests were used to evaluate the antithrombogenic properties. The APTT time of the implanted samples was observed to be prolonged compared to that of the unimplanted stainless steel control. La and Nd ion implantations can be used to improve the surface corrosion resistance and biomedical properties of 316 stainless steels.
In vacuo substrate pretreatments for enhancing nanodiamond formation in electron cyclotron resonance plasma
Authors: Teii Kungen , Kouzuma Yutaka , Uchino Kiichiro
Published: September 2006

Substrate pretreatment conditions at low pressures have been examined for enhancing nanocrystalline diamond formation on silicon in electron cyclotron resonance (ECR) plasma. Three kinds of pretreatments, (I) exposure to an ECR H2 plasma with application of a substrate bias from −100to+30V, (II) hot-filament heating in H2 gas, and (III) hot-filament heating in vacuum, were used alone or followed by carburization prior to a two-step process of ion-enhanced nucleation in an ECR plasma and subsequent growth in a hot-filament system. The number density of diamond particles after the final growth step was greatly increased up to the order of 107–108cm−2 when applying pretreatment (I) at the bias of 0V corresponding to the ion-bombardment energy of around 10eV. In this treatment, a clean and smooth surface with minimal damage was made by the dominance of anisotropic etching by hydrogen ions over isotropic etching by hydrogen atoms. The number density of diamond particles was still more increased when applying pretreatment (II), but the treated surface was unfavorably contaminated and roughened.
Gas-phase and sample characterizations of multiwall carbon nanotube growth using an atmospheric pressure plasma
Authors: Chandrashekar Anand , Lee Jeong-Soo , Lee Gil S. , Goeckner Matthew J. , Overzet Lawrence J.
Published: September 2006

Multiwall carbon nanotubes (CNTs) are synthesized using an atmospheric pressure rf plasma jet, with helium feed gas and acetylene gas as the precursor. The nanotubes are grown on a substrate with a thin catalyst (iron) film, with the substrate placed downstream from the plasma on a copper hot plate. In situ Fourier transformed infrared spectroscopy indicates an increase in gas temperature and a decrease in the density of the acetylene molecules at higher plasma powers. The helium metastables in the plasma break the C–H bonds in acetylene, causing molecular dissociation. It is apparent that the resultant formation of unsaturated carbon bonds causes taller and more graphitized CNT films to grow, as evident from scanning electron microscopy and Raman analyses of the samples. However, at higher substrate temperatures, taller and better quality films are obtained due to enhanced catalytic activity on the substrate surface.
Measurement uncertainties for vacuum standards at Korea Research Institute of Standards and Science
Authors: Hong S. S. , Shin Y. H. , Chung K. H.
Published: September 2006

The Korea Research Institute of Standards and Science has three major vacuum systems: an ultrasonic interferometer manometer (UIM) (Sec. II, Figs. 1 and 2) for low vacuum, a static expansion system (SES) (Sec. III, Figs. 3 and 4) for medium vacuum, and an orifice-type dynamic expansion system (DES) (Sec. IV, Figs. 5 and 6) for high and ultrahigh vacuum. For each system explicit measurement model equations with multiple variables are, respectively, given. According to ISO standards, all these system variable errors were used to calculate the expanded uncertainty (U). For each system the expanded uncertainties (k=1, confidence level=95%) and relative expanded uncertainty (expanded uncertainty/generated pressure) are summarized in Table IV and are estimated to be as follows. For UIM, at 2.5–300Pa generated pressure, the expanded uncertainty is <4.17×10−2Pa and the relative expanded uncertainty is <1.18×10−2; at 1–100kPa generated pressure, the expanded uncertainty is <7.87Pa and the relative expanded uncertainty is <7.84×10−5. For SES, at 3–100Pa generated pressure, the expanded uncertainty is <1.77×10−1Pa and the relative expanded uncertainty is <1.81×10−3. For DES, at 4.6×10−3–1.3×10−2Pa generated pressure, the expanded uncertainty is <1.04×10−4Pa and the relative expanded uncertainty is <8.37×10−3; at 3.0×10−6–9.0×10−4Pa generated pressure, the expanded uncertainty is <8.21×10−6Pa and the relative expanded uncertainty is <1.37×10−2. Within uncertainty limits our bilateral and key comparisons [CCM.P-K4 (10Pa–1kPa)] are extensive and in good agreement with those of other nations (Fig. 8 and Table V).
Growth and characterization of chromium oxide thin films prepared by reactive ac magnetron sputtering
Authors: Khanna Atul , Bhat Deepak G. , Payzant E. A.
Published: September 2006

Cr O x thin films were prepared on single crystal silicon (111 orientation) and glass substrates by midfrequency (41kHz) ac sputtering technique in an Isoflux ICM-10 sputter deposition system consisting of two hollow cylindrical targets of Cr in an argon-oxygen plasma at a discharge power of 5kW and without any deliberate substrate heating. X-ray diffraction studies showed that the coatings were of hcp rhombohedral α-phase chromium oxide. CrOx coating samples were found to grow preferentially towards the (110) crystal orientation of α-Cr2O3. Texturing was found to depend on the orientation of the substrates relative to the targets and also on the nature of substrates. One coating sample grown on Si substrate was annealed in air up to 1173K. Whereas heat treatment increased the crystallite size, CrOx coating was thermally stable and did not show any structural transformations. Scanning electron microscopy studies showed differences in the surface morphology of the coatings grown on glass and silicon substrates. Dynamic secondary ion mass spectrometry measurements performed on one CrOx coating deposited on silicon showed that the O∕Cr ratio in the films was 1.38. Ar and H impurity concentrations were also measured in this coating as a function of film thickness. Alumina coatings were prepared on Si substrates by reactive sputtering technique using CrOx coating as template layers. X-ray diffraction studies showed that CrOx template layers improved the crystallinity of alumina coatings grown on top of it and also facilitated the formation of the thermodynamically stable α-alumina phase.
Rotational and translational temperature equilibrium in an inductively coupled plasma
Authors: Shimada Masashi , Tynan George R. , Cattolica Robert
Published: September 2006

Rotational temperature has been used widely as neutral gas temperature measurement in different types of plasmas (electron cyclotron resonance, inductively coupled plasma, helicon, hollow cathode, etc.), and has been assumed to be in equilibrium with translational temperature. The direct experimental comparison of rotational and translational temperature in low-temperature plasmas has not been reported. In this research, optical emission spectroscopy is used to measure the neutral gas rotational temperature, Trot, from the second-positive band of a nitrogen molecule (380nm). The results are compared with the Doppler-broadened translational temperature, Ttrans, of Ar (750nm) and He (587nm), determined with a high-resolution spectrometer at various partial pressures of N2 in Ar∕N2 and He∕N2 plasmas. The results demonstrated that Trot and Ttrans are in equilibrium in the conditions [1010<ne(cm−3)<5×1012, 1<Te(eV)<5, 1<pfill(mTorr)<50] of our plasma experiment.
Properties of a newly developed concave nickel gasket for the ConFlat-type sealing system
Authors: Kurokouchi Satoshi , Shinoda Satsuo , Morita Shinsaku
Published: September 2006

Nickel gaskets for large-bored vacuum flange systems remain virtually nonexistent in practical use, despite strong demand for a gasket invulnerable to both oxidation and reduction atmospheres in vacuum application processes. Nickel gaskets would seem preferable in these processes, which are characterized by sensitivity to copper contamination and in which highly corrosive gases could wear away copper gaskets. In working to develop a nickel gasket for the ConFlat-type sealing system with a seal reliability and handling impression comparable to conventional copper gaskets, the authors found that nickel-plated copper gaskets could not attain secure sealing because the nickel layer was too hard to form a tight fit with the flange face. In earlier developmental stages, seven types of prototype nickel gaskets were produced and subjected to a tightening test. These prototype gaskets varied in their respective annealings and/or applied surface finishes. In the tightening test, deep penetration of the flange knife edge and a wide seal area were obtained on all the annealed prototypes, but their seal ability was nevertheless inferior to copper gaskets, even in the best case. Traced surface profiles of the tightened prototype gaskets with relatively roughly finished surfaces revealed that the original outlines of many protuberances and dimples remained, even after considerable seal pressure was applied on them. This observation suggests that the actual contact area between the gasket face and flange face was narrower than the apparent seal area, and too small to achieve secure sealing. Such poor accommodation of the flange surface was rarely observed on tightened gaskets with smoothly finished surfaces, although fine scratches remaining on the surface frequently functioned as leak paths after the tightening was completed. This insufficient fine deformation ability of the surface was not observed in commercially available copper gaskets. Results of tensile tests on prototype nickel gaskets versus commercially available copper gaskets made it clear that undesirable stiffness is an intrinsic characteristic of nickel gasket surfaces. This arises from the high hardening rate of the nickel material, and therefore cannot be improved by annealing alone. On further evaluation of the prototypes, the authors concluded that three factors are indispensable in order for nickel gaskets to be put into practical use: annealing to reduce hardness, a proper surface finish to obtain a tight fit on the sealing interface, and some sort of protection against scratching the gasket surface. This last requirement can be met by machining to reduce the sealing face region of the gasket surface. The final prototype nickel gasket, a concave version annealed and finished with fine lathing, showed basic sealing and flange knife edge damage characteristics comparable to copper gaskets, with superior seal stability at elevated temperature.
Investigation of reactive ion etching of dielectrics and Si in CHF3∕O2 or CHF3∕Ar for photovoltaic applications
Authors: Gatzert C. , Blakers A. W. , Deenapanray Prakash N. K. , Macdonald D. , Auret F. D.
Published: September 2006

Using a combination of etch rate, photoconductance, and deep level transient spectroscopy (DLTS) measurements, the authors have investigated the use of reactive ion etching (RIE) of dielectrics and Si in CHF3∕O2 and CHF3∕Ar plasmas for photovoltaic applications. The radio frequency power (rf-power) and gas flow rate dependencies have shown that the addition of either O2 or Ar to CHF3 can be used effectively to change the etch selectivity between SiO2 and Si3N4. The effective carrier lifetime of samples degraded upon exposure to a CHF3-based plasma, reflecting the introduction of recombination centers in the near-surface region. The extent of minority carrier lifetime degradation was similar in both types of plasmas, suggesting that the same defects were responsible for the increased recombination. However, the rf-power dependence of lifetime degradation in n- and p-type Si was different. Moreover, the lifetime degradation did not exhibit a linear rf-power dependence, suggesting that primary defects were not the dominant recombination centers responsible for the decrease in lifetime. Indeed, DLTS measurements have shown that secondary defects were formed in samples exposed to the plasma after annealing at 400°C, the temperature at which a SiN:H layer is deposited on samples to passivate their surfaces. The minority carrier lifetime degradation in RIE processed samples could be partially avoided using post-RIE chemical treatments.
Development of novel tungsten-doped high mobility transparent conductive In2O3 thin films
Authors: Li Xifeng , Zhang Qun , Miao Weina , Huang Li , Zhang Zhuangjian , Hua Zhongyi
Published: September 2006

A novel high mobility transparent conductive oxide thin film, tungsten-doped indium oxide (IWO), has been successfully grown on conventional glass substrates by reactive direct current magnetron sputtering technique from a metallic target. Analyses of x-ray photoelectron spectroscopy and x-ray diffraction reveal that tetravalent and hexavalent tungsten ions substitute for trivalent host indium ions without changing the crystalline structure of In2O3. IWO thin films were grown with resistivity of 4.4×10−4Ωcm, carrier mobility of 52.8cm2V−1S−1; transmittance exceeding 80% at wavelengths between 380 and 900nm, and average roughness of 7.5nm.
Interfacial interaction of in situ Cu growth on tetrasulfide self-assembled monolayer on plasma treated parylene surface
Authors: Pimanpang S. , Wang Pei-I , Juneja Jasbir S. , Wang G.-C. , Lu T.-M.
Published: September 2006

The interfacial reactions of in situ grown Cu and functionalized parylene surfaces were examined using the x-ray photoelectron spectroscopy (XPS). It is observed that bis[3(triethoxysilane)propyl]tetrasulfide (tetrasulfide) forms a self-assembled monolayer (SAM) only on a parylene surface treated with either He plasma or N2 plasma, but not on the as-deposited parylene surface due to its lack of functional groups. The functional groups on the plasma treated parylene surface facilitate the formation of tetrasulfide SAM that subsequently improves the reactivity of parylene to Cu. The XPS spectra show a strong shift (∼1.1eV) of the S 2p peaks from a higher binding energy to a lower binding energy, which suggests the existence of an interaction between sulfide and Cu. In addition, the result of a higher XPS intensity ratio of Cu 2p3∕2∕C 1s for Cu growth on tetrasulfide SAM/plasma treated parylene surface than that of an as-deposited parylene surface indicates that the formation of tetrasulfide moiety (–SSSS–) on functionalized parylene surface leads to the increase of Cu growth rate.
Scaling of hollow cathode magnetrons for ionized metal physical vapor deposition
Authors: Vyas Vivek , Kushner Mark J.
Published: September 2006

Ionized metal physical vapor deposition is being increasingly used to deposit diffusion barriers and Cu seed layers into high aspect ratio trenches for microelectronics fabrication. Hollow cathode magnetrons (HCMs) represent a technology capable of depositing metal over large areas at pressures of a few millitorrs. The fundamental mechanisms of these devices are not well understood and so their optimization is difficult. In this article, results from a two-dimensional computational investigation of HCMs are discussed to illuminate scaling issues. The hybrid model incorporates algorithms whereby transport coefficients for use in fluid equations are derived using a kinetic simulation. The goal is to enable the fluid algorithms in the model to be able to more accurately represent low pressure operation. The consequences of power, pressure, and magnitude and orientation of applied magnetic fields were investigated. The authors found that the magnetic field configuration significantly affects the magnitude and distribution of fluxes incident on the substrate. A study of the Cu seed layer deposition process, carried out using a feature scale model, correlates changes in plasma properties with conformal deposition into trenches.
Surface kinetics modeling of silicon and silicon oxide plasma etching. I. Effect of neutral and ion fluxes on etching yield of silicon oxide in fluorocarbon plasmas
Authors: Kwon Ohseung , Sawin Herbert H.
Published: September 2006

Silicon oxide etching processes in C2F6 and C4F8+80% Ar plasmas were investigated. Neutral and ion compositions in the plasma were measured using quadrupole mass spectrometry and etching yield was measured by a quartz-crystal microbalance. In C2F6 plasma, the concentration of atomic fluorine in the neutral flux was 5%–25%, whereas there was less than 0.5% of atomic fluorine in C4F8+80% Ar plasma. A surface plot representing the etching yield as a function of neutral and ion fluxes was constructed and used to qualitatively explain the etching characteristics of silicon oxide in fluorocarbon plasmas. In C2F6 chemistry, etching yield decreases slightly with increasing rf coil power. This is attributed to the decrease in both F/ion and CFx/ion, which is caused by an increase in ion flux, with a more significant effect due to a decrease in F/ion. In C4F8+80% Ar chemistry, however, etching yield increases with increasing rf coil power. This is attributed to the decrease in CFx, without the effect of F/ion due to the low atomic fluorine concentration. With increased operating pressure, etching yield decreases for both chemistries because as the pressure increases, ion current decreases, and CFx neutral concentration increases to have more deposition and less etching.
Surface kinetics modeling of silicon and silicon oxide plasma etching. II. Plasma etching surface kinetics modeling using translating mixed-layer representation
Authors: Kwon Ohseung , Sawin Herbert H.
Published: September 2006

A novel surface kinetic model using translating mixed-layer representation was proposed and demonstrated. In this model, a translating mixed layer was constructed where the total number of atoms is conserved as the etching or deposition proceeds, to have a convection or movement flux from or to the substrate volume (the volume under the translating mixed layer) that corresponds to the difference between the adsorption flux and removal flux. The model is demonstrated for silicon etching with chlorine chemistry and silicon oxide etching with fluorine chemistry, with results agreeing well with measured data, as well as with previously developed Monte Carlo simulation results. The computation speed of the translating mixed-layer model was 100–10 000 times faster than that of Monte Carlo simulation.
Surface kinetics modeling of silicon and silicon oxide plasma etching. III. Modeling of silicon oxide etching in fluorocarbon chemistry using translating mixed-layer representation
Authors: Kwon Ohseung , Bai Bo , Sawin Herbert H.
Published: September 2006

Silicon oxide etching was modeled using a translating mixed-layer model, a novel surface kinetic modeling technique, and the model showed good agreement with measured data. Carbon and fluorine were identified as the primary contributors to deposition and etching, respectively. Atomic fluorine flux is a major factor that determines the etching behavior. With a chemistry having a small amount of atomic fluorine (such as the C4F8 chemistry), etching yield shows stronger dependence on the composition change in the gas flux.
Study of nonisothermal crystallization in amorphous Se90In9.3Cu0.7 alloy
Authors: Abo-Ghazala M. S. , Farag El-Sayed M. , Ammar A. H.
Published: September 2006

Amorphous alloy of Se90In9.3Cu0.7 has been prepared by the usual melt-quench technique under vacuum. Data from differential thermal analysis (DTA) at various heating rates and x-ray analysis of this alloy were investigated and reported. The activation energies of glass transition, Eg, and crystallization transition, Ec, were evaluated from the dependence of the glass temperature Tg and the crystallization temperature Tc on the heating rate. The crystalline phases resulting from DTA were identified using x-ray diffraction. The results reveal that the prepared alloy is structurally heterogeneous at a molecular level, in which In6Se7, Cu5Se4, and Cu2Se dispersed in Se–Se matrix.
ORF18 Is a Transfactor That Is Essential for Late Gene Transcription of a Gammaherpesvirus
Authors: Arumugaswami Vaithilingaraja , Wu Ting-Ting , Martinez-Guzman DeeAnn , Jia Qingmei , Deng Hongyu , Reyes Nichole , Sun Ren
Published: September 2006

Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene,
ORF18
, essential for viral replication.
ORF18
is conserved in both beta- and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis.
The Pathogenic NY-1 Hantavirus G1 Cytoplasmic Tail Inhibits RIG-I- and TBK-1-Directed Interferon Responses
Authors: Alff Peter J. , Gavrilovskaya Irina N. , Gorbunova Elena , Endriss Karen , Chong YuSon , Geimonen Erika , Sen Nandini , Reich Nancy C. , Mackow Erich R.
Published: September 2006

Hantaviruses cause two diseases with prominent vascular permeability defects, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. All hantaviruses infect human endothelial cells, although it is unclear what differentiates pathogenic from nonpathogenic hantaviruses. We observed dramatic differences in interferon-specific transcriptional responses between pathogenic and nonpathogenic hantaviruses at 1 day postinfection, suggesting that hantavirus pathogenesis may in part be determined by viral regulation of cellular interferon responses. In contrast to pathogenic NY-1 virus (NY-1V) and Hantaan virus (HTNV), nonpathogenic Prospect Hill virus (PHV) elicits early interferon responses following infection of human endothelial cells. We determined that PHV replication is blocked in human endothelial cells and that RNA and protein synthesis by PHV, but not NY-1V or HTNV, is inhibited at 2 to 4 days postinfection. The addition of antibodies to beta interferon (IFN-β) blocked interferon-directed MxA induction by >90% and demonstrated that hantavirus infection induces the secretion of IFN-β from endothelial cells. Coinfecting endothelial cells with NY-1V and PHV resulted in a 60% decrease in the induction of interferon-responsive MxA transcripts by PHV and further suggested the potential for NY-1V to regulate early IFN responses. Expression of the NY-1V G1 cytoplasmic tail inhibited by >90% RIG-I- and downstream TBK-1-directed transcription from interferon-stimulated response elements or β-interferon promoters in a dose-dependent manner. In contrast, expression of the NY-1V nucleocapsid or PHV G1 tail had no effect on RIG-I- or TBK-1-directed transcriptional responses. Further, neither the NY-1V nor PHV G1 tails inhibited transcriptional responses directed by a constitutively active form of interferon regulatory factor 3 (IRF-3 5D), and IRF-3 is a direct target of TBK-1 phosphorylation. These findings indicate that the pathogenic NY-1V G1 protein regulates cellular IFN responses upstream of IRF-3 phosphorylation at the level of the TBK-1 complex. These findings further suggest that the G1 cytoplasmic tail contains a virulence element which determines the ability of hantaviruses to bypass innate cellular immune responses and delineates a mechanism for pathogenic hantaviruses to successfully replicate within human endothelial cells.
Essential Functions of the Unique N-Terminal Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain in Viral Replication and in the Pathogenesis of Skin Infection
Authors: Berarducci Barbara , Ikoma Minako , Stamatis Shaye , Sommer Marvin , Grose Charles , Arvin Ann M.
Published: September 2006

Varicella-zoster virus (VZV) glycoprotein E (gE) is a multifunctional protein important for cell-cell spread, envelopment, and possibly entry. In contrast to other alphaherpesviruses, gE is essential for VZV replication. Interestingly, the N-terminal region of gE, comprised of amino acids 1 to 188, was shown not to be conserved in the other alphaherpesviruses by bioinformatics analysis. Mutational analysis was performed to investigate the functions associated with this unique gE N-terminal region. Linker insertions, serine-to-alanine mutations, and deletions were introduced in the gE N-terminal region in the VZV genome, and the effects of these mutations on virus replication and cell-cell spread, gE trafficking and localization, virion formation, and replication in vivo in the skin were analyzed. In summary, mutagenesis of the gE N-terminal region identified a new functional region in the VZV gE ectodomain essential for cell-cell spread and the pathogenesis of VZV skin tropism and demonstrated that different subdomains of the unique N-terminal region had specific roles in viral replication, cell-cell spread, and secondary envelopment.
Role of RNA Structure and RNA Binding Activity of Foot-and-Mouth Disease Virus 3C Protein in VPg Uridylylation and Virus Replication
Authors: Nayak Arabinda , Goodfellow Ian G. , Woolaway Kathryn E. , Birtley James , Curry Stephen , Belsham Graham J.
Published: September 2006

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3D

pol

), the precursor 3CD, and an RNA template containing the
cre
/
bus
. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5′ untranslated region but outside of the
cre/bus
can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B
3
3C and 3B
123
3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the
cre/bus
RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.
Persistent High Frequencies of Varicella-Zoster Virus ORF4 Protein-Specific CD4+T Cells after Primary Infection
Authors: Jones Louise , Black Antony P. , Malavige Gathsaurie N. , Ogg Graham S.
Published: September 2006

Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4+T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4+T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-γ) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4+T cells. Indeed, CD4+T cells were found to dominate the overall virus-specific IFN-γ cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4+T cells, with implications for disease pathogenesis and future vaccine development.
Soluble Epstein-Barr Virus Glycoproteins gH, gL, and gp42 Form a 1:1:1 Stable Complex That Acts Like Soluble gp42 in B-Cell Fusion but Not in Epithelial Cell Fusion
Authors: Kirschner Austin N. , Omerović Jasmina , Popov Boris , Longnecker Richard , Jardetzky Theodore S.
Published: September 2006

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.
Selection on the Human Immunodeficiency Virus Type 1 Proteome following Primary Infection
Authors: Liu Yi , McNevin John , Cao Jianhong , Zhao Hong , Genowati Indira , Wong Kim , McLaughlin Sherry , McSweyn Matthew D. , Diem Kurt , Stevens Claire E. , Maenza Janine , He Hongxia , Nickle David C. , Shriner Daniel , Holte Sarah E. , Collier Ann C. , Corey Lawrence , McElrath M. Juliana , Mullins James I.
Published: September 2006

Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at ≤17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.
Specificity of the VP1 GH Loop of Foot-and-Mouth Disease Virus for αv Integrins
Authors: Burman Alison , Clark Stuart , Abrescia Nicola G. A. , Fry Elizabeth E. , Stuart David I. , Jackson Terry
Published: September 2006

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by αvβ6 or αvβ8 but not for inhibition of virus binding to αvβ3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for αvβ6. In contrast, although RGDM-containing peptides were effective for αvβ8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses αvβ8 less efficiently than αvβ6 as a receptor for infection. Finally, evidence is presented that shows αvβ3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to αvβ6 with a higher affinity than to αvβ3 and αvβ8.
Regulation of the Transcription and Replication Cycle of Human Cytomegalovirus Is Insensitive to Genetic Elimination of the Cognate NF-κB Binding Sites in the Enhancer
Authors: Gustems Montse , Borst Eva , Benedict Chris A. , Pérez Carmen , Messerle Martin , Ghazal Peter , Angulo Ana
Published: September 2006

The role of NF-κB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-κB response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEP-enhancer that, although this region is essential for HCMV growth, none of the four NF-κB response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.
Cellular Target Genes of Epstein-Barr Virus Nuclear Antigen 2
Authors: Maier Sabine , Staffler Gabriele , Hartmann Andrea , Höck Julia , Henning Karen , Grabusic Kristina , Mailhammer Reinhard , Hoffmann Reinhard , Wilmanns Matthias , Lang Roland , Mages Jörg , Kempkes Bettina
Published: September 2006

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.
Delayed Biosynthesis of Varicella-Zoster Virus Glycoprotein C: Upregulation by Hexamethylene Bisacetamide and Retinoic Acid Treatment of Infected Cells
Authors: Storlie Johnathan , Jackson Wallen , Hutchinson Jennifer , Grose Charles
Published: September 2006

In the course of examining the trafficking pathways of varicella-zoster virus (VZV) glycoproteins gE, gI, gH, and gB, we discovered that all four are synthesized within 4 to 6 h postinfection (hpi) in cultured cells. Thereafter, they travel via the trans-Golgi network to the outer cell membrane. When we carried out a similar analysis on VZV gC, we observed little gC biosynthesis in the first 72 hpi. Further examination disclosed that gC was present in the inocula of infected cells, but no new gC biosynthesis occurred during the first 24 to 48 h thereafter, during which time new synthesis of gE, gH, and major capsid protein was easily detectable. Similarly, delayed gC biosynthesis was confirmed with three different VZV strains and two different cell lines. Bioinformatics analyses disclosed the presence of PBX/HOX consensus binding domains in the promoter/enhancer regions of the genes for VZV gC and ORF4 protein (whose orthologs transactivate gC in other herpesviruses). Bioinformatics analysis also identified two HOXA9 activation regions on ORF4 protein. Treatment of infected cultures with chemicals known to induce the production of PBX/HOX transcription proteins, namely, hexamethylene bisacetamide (HMBA) and retinoic acid, led to more rapid gC biosynthesis. Immunoblotting demonstrated a fivefold increase in the HOXA9 protein after HMBA treatment. In summary, these results documented that gC was not produced during early VZV replication cycles, presumably related to a deficiency in the PBX/HOX transcription factors. Furthermore, these results explain the apparent spontaneous loss of VZV gC in some passaged viruses, as well as other anomalous gC results.
Reduction of the Rate of Poliovirus Protein Synthesis through Large-Scale Codon Deoptimization Causes Attenuation of Viral Virulence by Lowering Specific Infectivity
Authors: Mueller Steffen , Papamichail Dimitris , Coleman J. Robert , Skiena Steven , Wimmer Eckard
Published: September 2006

Exploring the utility of de novo gene synthesis with the aim of designing stably attenuated polioviruses (PV), we followed two strategies to construct PV variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type (wt) poliovirus codons (PV-SD). Despite 934 nucleotide changes in the capsid coding region, PV-SD RNA produced virus with wild-type characteristics. In contrast, no viable virus was recovered from PV-AB RNA carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level. After subcloning of smaller portions of the AB capsid coding sequence into the wt background, several viable viruses were obtained with a wide range of phenotypes corresponding to their efficiency of directing genome translation. Surprisingly, when inoculated with equal infectious doses (PFU), even the most replication-deficient viruses appeared to be as pathogenic in PV-sensitive CD155tg (transgenic) mice as the PV(M) wild type. However, infection with equal amounts of virus particles revealed a neuroattenuated phenotype over 100-fold. Direct analysis indicated a striking reduction of the specific infectivity of PV-AB-type virus particles. Due to the distribution effect of many silent mutations over large genome segments, codon-deoptimized viruses should have genetically stable phenotypes, and they may prove suitable as attenuated substrates for the production of poliovirus vaccines.
Human T-Cell Lymphotropic Virus Type 3: Complete Nucleotide Sequence and Characterization of the Human Tax3 Protein
Authors: Calattini Sara , Chevalier Sébastien Alain , Duprez Renan , Afonso Philippe , Froment Alain , Gessain Antoine , Mahieux Renaud
Published: September 2006

We and others have recently uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3), the third member of the HTLV family. We have now sequenced the full-length HTLV-3
Pyl43
provirus. As expected, HTLV-3
Pyl43
contains open reading frames corresponding to the
gag
,
pol
,
env
,
tax
, and
rex
genes. Interestingly, its long terminal repeat (LTR) includes only two Tax-responsive elements, as is the case for type 3 simian T-cell lymphotropic viruses (STLV-3). Phylogenetic analyses reveal that HTLV-3
Pyl43
is closely related to central African STLV-3. Unexpectedly, the proximal pX region of HTLV-3
Pyl43
lacks 366 bp compared to its STLV-3 counterpart. Because of this deletion, the previously described RorfII sequence is lacking. At the amino acid level, Tax3
Pyl43
displays strong similarities with HTLV-1 Tax, including the sequence of a PDZ class I binding motif. In transient-transfection assays, Tax3
Pyl43
activates the transcriptions from HTLV-3, HTLV-1, and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF-κB pathway are similar but not identical in Tax1 and Tax3
Pyl43
. We also show that Tax3
Pyl43
transactivates the human interleukin-8 and Bcl-X
L
promoters through the induction of the NF-κB pathway. On the other hand, Tax3
Pyl43
represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter. Altogether, these results demonstrate that although HTLV-3 and HTLV-1 have only 60% identity, Tax3
Pyl43
is functionally closely related to the transforming protein Tax1 and suggest that HTLV-3, like HTLV-1, might be pathogenic in vivo.
Interaction of Bunyamwera Orthobunyavirus NSs Protein with Mediator Protein MED8: a Mechanism for Inhibiting the Interferon Response
Authors: Léonard Vincent H. J. , Kohl Alain , Hart Timothy J. , Elliott Richard M.
Published: September 2006

The NSs protein of Bunyamwera virus (
Bunyaviridae
) is an antiapoptotic interferon antagonist involved in silencing host protein expression by interfering with mRNA synthesis. Here, we show that the ability to inhibit both host transcription and the interferon response is linked to interaction of NSs with the MED8 component of Mediator, a protein complex necessary for mRNA production. The interacting domain on NSs was mapped to the C-terminal region, which contains amino acids conserved among orthobunyavirus NSs proteins. A recombinant virus in which the interacting domain in NSs was deleted had strongly reduced ability to inhibit host protein expression and was unable to inhibit the interferon response. This study provides further information on the mechanisms by which bunyavirus nonstructural proteins are involved in pathogenesis.
The Negative Regulator of Splicing Element of Rous Sarcoma Virus Promotes Polyadenylation
Authors: Wilusz Jeremy E. , Beemon Karen L.
Published: September 2006

The Rous sarcoma virus
gag
gene contains a
cis
-acting negative regulator of splicing (NRS) element that is implicated in viral polyadenylation regulation. To study the mechanism of polyadenylation promotion at the viral poly(A) site located over 8 kb downstream, we performed in vitro polyadenylation analysis. RNA containing only the poly(A) site and flanking sequences in the 3′ long terminal repeat (LTR) was not polyadenylated detectably in vitro; however, if the transcript contained the NRS upstream of the LTR, polyadenylation was observed. Insertion of the viral
env
3′ splice site sequence between the NRS and the LTR did not alter the level of polyadenylation appreciably. We conclude that the NRS promotes polyadenylation in vitro and can do so without formation of a splicing complex with a 3′ splice site. We then explored the roles of several cellular factors in NRS-mediated polyadenylation. Mutation of the binding sites of U1 and U11 snRNPs to the NRS did not affect polyadenylation, whereas hnRNP H strongly inhibited polyadenylation. We propose a model in which hnRNP H and SR proteins compete for binding to the NRS. Bound SR proteins may bridge between the NRS and the 3′ LTR and aid in the recruitment of the 3′-end processing machinery.