bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron cyclotron resonance plasma sputtering growth of textured films of c-axis-oriented LiNbO3 on Si(100) and Si(111) surfaces
Authors: Akazawa H. , Shimada M.
Published: July 2004

Textured films of c-axis-oriented lithium niobate (LN) in a single Li:Nb=1:1 phase have been grown on Si(100) and Si(111) substrates by electron cyclotron resonance (ECR) plasma sputtering. The O2 partial pressure is critical in determining the composition of the film, whereas the substrate temperature mainly affects crystallinity. At O2 flow rates between 1 and 1.5 sccm and growth temperature between 510 and 550 °C, the refractive indices of the films are within their minimum range. A crystal image of the LN film revealed columnar textured domains, about 40% of which were c-axis oriented. The plasma-enhanced migration of surface atoms is responsible for the polarized grain growth. The amorphous interlayer formed between the LN film and the Si substrate consisted of either a single layer (2 nm) or double component layers (4 nm). The orientation of the LN crystalline overlayer has some correlation with the structure of the underlying amorphous interlayer.
Photochemical reaction of organosilane self-assembled monolayer as studied by scanning probe microscopy
Authors: Sugimura H. , Saito N. , Ishida Y. , Ikeda I. , Hayashi K. , Takai O.
Published: July 2004

A photopatterning process of an alkylsilane self-assembled monolayer (SAM) using vacuum ultraviolet (VUV) light at 172 nm has been studied by characterizing the VUV-irradiated SAM surfaces through lateral force microscopy (LFM) and Kelvin-probe force microscopy (KFM). The SAM was formed from n-octadecyltrimethoxysilane on a Si substrate covered with a 2-nm thick oxide layer. Due to VUV irradiation in the presence of atmospheric oxygen, polar functional groups, e.g., COOH, were generated through photochemical oxidation of the SAM which was gradually etched. Finally, the SAM was completely removed from the VUV-irradiated region. Relative frictional forces and surface potentials of the VUV-irradiated regions at various irradiation dose rates were measured by LFM and KFM using the unirradiated regions on each sample as a reference. Changes in the surface chemical composition of the SAM could be measured by these scanning probe microscopic methods. The LFM contrast increased monotonically with the increase in the dose rate, while the surface potential contrast measured by KFM showed a characteristic feature. At a certain dose rate of 10–15 J/cm2, VUV-irradiated SAMs showed particularly lower surface potentials than the other SAM surfaces with different dose rates. This is most likely because COOH groups, which were considered negatively charged in part, had been accumulated on the surfaces. LFM and KFM measurements are promising in order to probe chemistries on organic thin films proceeding in small domains with a scale.
Atomic layer deposition of hafnium silicate films using hafnium tetrachloride and tetra-n-butyl orthosilicate
Authors: Kim Won-Kyu , Rhee Shi-Woo , Lee Nae-In , Lee Jong-Ho , Kang Ho-Kyu
Published: July 2004

Atomic layer chemical vapor deposition (ALCVD) of hafnium silicate films with a precursor combination of HfCl4 and TBOS (tetra-n-butyl orthosilicate) was studied for high dielectric gate insulators. The effect of deposition conditions, such as deposition temperature and pulse time for precursor injection on the deposition rate per cycle and composition of the film (fraction of hafnia phase in the silicate film) were studied. The growth rate and composition ratio were saturated with the increase of the injection time of HfCl4 and TBOS and decreased with the increased deposition temperature from 300 to 500 °C. The growth rate was 1.4 Å/cycle and the fraction of hafnium phase in the Hf silicate thin films was 0.19 at the deposition temperature of 500 °C. Impurity content, such as carbon and chlorine was below the detection limit of XPS (x-ray photoelectron spectroscopy) and the impurity level detected by SIMS decreased with increasing deposition temperature. It was found that the incorporation rate of metal from halide source was lower than alkoxide source.
Incorporation of fluorine in hydrogenated silicon carbide films deposited by pulsed glow discharge
Authors: Jacobsohn L. G. , Afanasyev-Charkin I. V. , Cooke D. W. , Schulze R. K. , Averitt R. D. , Nastasi M.
Published: July 2004

The incorporation of fluorine in hydrogenated silicon carbide films was carried out by pulsed glow discharge deposition, and its effects on the structure, mechanical, optical, and surface properties were investigated. Fluorine incorporation occurs mostly in the form of single Si–F bonds and no C–F bonds were revealed by infrared and x-ray photoelectron spectroscopies. Films with higher F contents have lower Si/C ratios, and the excess of C favors the formation of graphitic agglomerates which decrease the hardness of the films. Films with 16 at. % F exhibit 10 GPa hardness. Our results indicate that the chemical states in which F is incorporated into a-SiC:H films play important roles in the surface and optical properties. C–F bonds, in opposition to Si–F bonds, control the wettability and optical gap of this material.
Integrated feature scale modeling of plasma processing of porous and solid SiO2. I. Fluorocarbon etching
Authors: Sankaran Arvind , Kushner Mark J.
Published: July 2004

Increases in RC delay times in interconnect wiring for microelectronics as feature sizes decrease have motivated investigations into the use of low-dielectric constant insulators, and in particular, porous silicon-dioxide (PS). Profile evolution and maintenance of critical dimensions during plasma etching of PS are problematic due to the exposure of open pores. To investigate these issues, reaction mechanisms for fluorocarbon plasma etching of SiO2 in C2F6, CHF3, and C4F8 chemistries have been developed and incorporated into the Monte Carlo Feature Profile Model which was modified to address these two-phase systems. The reaction mechanism was validated by comparison to experiments by others for etching of PS and solid SiO2 (SS). We found that the etch rates for PS are generally higher than that of SS due to the inherently lower mass fraction. Mass corrected etch rates of PS can be larger or smaller than those for SS depending on the degree of pore filling by polymer and the degree of ion activated chemical sputtering. Pore filling is particularly important for PS having open networks with large pores and high porosities. We found little dependence of the taper of high aspect ratio profiles on the average pore radius and porosity. However, the profile changes from tapered to bowed as the interconnectivity of the porous network increases. Scaling laws for profile shapes are otherwise similar for both SS and PS.
Integrated feature scale modeling of plasma processing of porous and solid SiO2. II. Residual fluorocarbon polymer stripping and barrier layer deposition
Authors: Sankaran Arvind , Kushner Mark J.
Published: July 2004

The adoption of low dielectric constant materials as inter-level dielectrics in microelectronics fabrication will ultimately depend on process integration. Porous SiO2 (PS) is one candidate material. Cleaning of residual polymer from trenches following etching using fluorocarbon plasmas and the deposition of a continuous barrier layer are critical processes for integration of PS as inter-level dielectrics. To investigate these issues, reactions mechanisms for plasma stripping of fluorocarbon polymer using oxygen containing plasmas and deposition of metal barrier coatings into PS trenches were developed, and incorporated into a feature profile model. The reaction mechanism was validated by comparison to experiments for blanket plasma etching of polytetrafluoroethylene using Ar–O2 chemistries. Plasma stripping of fluorocarbon polymers from solid SiO2 (SS) trenches was found to be less efficient at higher aspect ratios. Stripping was also less efficient from PS trenches having large average pore radius and high interconnectivity. Cu ionized metal physical vapor deposition was investigated as a surrogate for barrier coating in SS and PS trenches. Compared to SS, thin film deposition was less conformal for PS having closed pore networks. Thicker films were required for interconnected PS to avoid pin-hole formation.
Displacive phase transition in SrTiO3 thin films grown on Si(001)
Authors: Aguirre-Tostado F. S. , Herrera-Gómez A. , Woicik J. C. , Droopad R. , Yu Z. , Schlom D. G. , Karapetrova J. , Zschack P. , Pianetta P.
Published: July 2004

Polarization dependent near and extended x-ray absorption fine-structure (XANES and EXAFS), in combination with x-ray diffraction, has been used to study the structure of SrTiO3 (STO) ultra thin films grown on Si(001). For the in-plane direction (200), the x-ray diffraction data indicate that all films (from 40 to 200 Å) are equally expanded. This is in contradiction to previous reports claiming that the growth is pseudomorphic and epitaxial (coherent), which would predict an inplane contraction. Even the thinnest films (40 Å) grow in a relaxed mode (not coherent) at the deposition temperature (700 °C). As the system is brought to room temperature, the films (now anchored to the substrate) are not allowed to compress as much as bulk STO. The residual film expansion is quantitatively explained by the differential thermal expansion of Si and STO. For the out-of-plane direction (002), the x-ray diffraction data indicate that STO films are expanded for the thinnest films, and relaxed for a thickness of 200 Å. The in-plane and out-of-plane EXAFS and XANES data show that the perpendicular expansion of the thinner films is accompanied by a displacive phase transition of SrTiO3 where the Ti atom moves toward the (002) direction. This ferroelectric-type behavior of the thinner films implies important potential applications in electronics.
Herpes Simplex Virus 1 Has Multiple Mechanisms for Blocking Virus-Induced Interferon Production
Authors: Melroe Gregory T. , DeLuca Neal A. , Knipe David M.
Published: July 2004

In response to viral infection, host cells elicit a number of responses, including the expression of alpha/beta interferon (IFN-α/β). In these cells, IFN regulatory factor-3 (IRF-3) undergoes a sequence of posttranslational modifications that allow it to act as a potent transcriptional coactivator of specific IFN genes, including IFN-β. We investigated the mechanisms by which herpes simplex virus 1 (HSV-1) inhibits the production of IFN-β mediated by the IRF-3 signaling pathway. Here, we show that HSV-1 infection can block the accumulation of IFN-β triggered by Sendai virus (SeV) infection. Our results indicate that HSV-1 infection blocks the nuclear accumulation of activated IRF-3 but does not block the initial virus-induced phosphorylation of IRF-3. The former effect was at least partly mediated by increased turnover of IRF-3 in HSV-1-infected cells. Using mutant viruses, we determined that the immediate-early protein ICP0 was necessary for the inhibition of IRF-3 nuclear accumulation. Expression of ICP0 also had the ability to reduce IFN-β production induced by SeV infection. ICP0 has been shown previously to play a role in HSV-1 sensitivity to IFN and in the inhibition of antiviral gene production. However, we observed that an ICP0 mutant virus still retained the ability to inhibit the production of IFN-β. These results argue that HSV-1 has multiple mechanisms to inhibit the production of IFN-β, providing additional ways in which HSV-1 can block the IFN-mediated host response.
Integrated Self-Inactivating Lentiviral Vectors Produce Full-Length Genomic Transcripts Competent for Encapsidation and Integration
Authors: Logan Aaron C. , Haas Dennis L. , Kafri Tal , Kohn Donald B.
Published: July 2004

To make human immunodeficiency virus type 1 (HIV-1)-based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of promoter and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this deletion are thought to have no LTR-directed transcription and are called self-inactivating (SIN) lentivectors. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5′ LTR, which is responsible for the production of full-length vector transcripts. To verify that transcripts derived from the LTR in SIN lentivectors are competent for encapsidation and integration, we transduced a lentiviral packaging cell line with a SIN lentivector and then observed the production of viable vector particles containing full-length SIN lentivector genomes. We have also attempted to identify sequences in the SIN lentivector which are responsible for transcriptional activation at the 5′ LTR. Using different segments of the vector LTR and leader region in a promoter assay, we have determined that the residual promoter activity is contained entirely within the leader region and that, although this element is downstream of the transcription initiation site, it is capable of initiating transcription from the 5′ end of R in the LTR. Mutation of leader region binding sites for the transcriptional activators downstream binding factor 1 (DBF1) and SP1 reduces transcription from the SIN LTR by up to 80%. Knowledge of the potential for mobilization of HIV-1-derived SIN lentivectors will be important for the design of future gene therapy trials with such vectors.
Frequent Transmission of Cytotoxic-T-Lymphocyte Escape Mutants of Human Immunodeficiency Virus Type 1 in the Highly HLA-A24-Positive Japanese Population
Authors: Furutsuki Tae , Hosoya Noriaki , Kawana-Tachikawa Ai , Tomizawa Mariko , Odawara Takashi , Goto Mieko , Kitamura Yoshihiro , Nakamura Tetsuya , Kelleher Anthony D. , Cooper David A. , Iwamoto Aikichi
Published: July 2004

Although Japan is classified as a country with a low prevalence of human immunodeficiency virus type 1 (HIV-1), domestic sexual transmission has been increasing steadily. Because 70% of the Japanese population expresses HLA-A24 (genotype HLA-A*2402), we wished to assess the effect of the dominant HLA type on the evolution and transmission of HIV-1 among the Japanese population. Twenty-three out of 25 A24-positive Japanese patients had a Y-to-F substitution at the second position [Nef138-10(2F)] in an immunodominant A24-restricted CTL epitope in their HIV-1nefgene (Nef138-10). None of 12 A24-negative Japanese hemophiliacs but 9 out of 16 patients infected through unprotected sexual intercourse had Nef138-10(2F) (P< 0.01). Two of two A24-positive but none of six A24-negative Australians had Nef138-10(2F). Nef138-10(2F) peptides bound well to the HLA-A*2402 heavy chain; however, Nef138-10(2F) was expressed poorly on the cell surface from the native protein. Thus, HIV-1 with Nef138-10(2F) appears to be a cytotoxic-T-lymphocyte escape mutant and has been transmitted frequently by sexual contact among the highly A24-positive Japanese population.
Characterization of Recombinant Respiratory Syncytial Viruses with the Region Responsible for Type 2 T-Cell Responses and Pulmonary Eosinophilia Deleted from the Attachment (G) Protein
Authors: Elliott Matthew B. , Pryharski Karin S. , Yu Qingzhong , Boutilier L. A. , Campeol N. , Melville K. , Laughlin Todd S. , Gupta C. K. , Lerch Robert A. , Randolph Valerie B. , LaPierre Natisha A. , Dack Kristen M. Heers , Hancock Gerald E.
Published: July 2004

It is essential that preventative vaccines for respiratory syncytial virus (RSV) elicit balanced T-cell responses. Immune responses dominated by type 2 T cells against RSV antigens are believed to cause exaggerated respiratory tract disease and may also contribute to unwanted inflammation in the airways that predisposes infants to wheeze through adolescence. Here we report on the construction and characterization of recombinant RSV (rRSV) strains with amino acids 151 to 221 or 178 to 219 of the attachment (G) glycoprotein deleted (rA2cpΔG150-222 or rA2cpΔG177-220, respectively). The central ectodomain was chosen for modification because a peptide spanning amino acids 149 to 200 of G protein has recently been shown to prime several strains of naïve inbred mice for polarized type 2 T-cell responses, and peripheral blood T cells from most human donors recognize epitopes within this region. Quantitative PCR demonstrated that synthesis of nascent rRSV genomes in human lung epithelial cell lines was similar to that for the parent virus (cp-RSV). Plaque assays further indicated that rRSV replication was not sensitive to 37°C, but pinpoint morphology was observed at 39°C. Both rRSV strains replicated in the respiratory tracts of BALB/c mice and elicited serum neutralization and anti-F-protein immunoglobulin G titers that were equivalent to those elicited by cp-RSV and contributed to a 3.9-log
10
-unit reduction in RSV A2 levels 4 days after challenge. Importantly, pulmonary eosinophilia was significantly diminished in BALB/c mice primed with native G protein and challenged with either rA2cpΔG150-222 or rA2cpΔG177-220. These findings are important for the development of attenuated RSV vaccines.
PKR-Dependent and -Independent Mechanisms Are Involved in Translational Shutoff during Sindbis Virus Infection
Authors: Gorchakov Rodion , Frolova Elena , Williams Bryan R. G. , Rice Charles M. , Frolov Ilya
Published: July 2004

The replication of Sindbis virus (SIN) profoundly affects the metabolism of infected vertebrate cells. One of the main events during SIN infection is the strong inhibition of translation of cellular mRNAs. In this study, we used a combination of approaches, including the study of SIN replication in PKR−/−mouse embryo fibroblasts or in the presence of an excess of catalytically inactive PKR. We show that the PKR-dependent inhibition of translation is not the only and most likely not the major pathway mediating translational shutoff during SIN infection. The PKR-independent mechanism strongly affects the translation of cellular templates, whereas translation of SIN subgenomic RNA is resistant to inhibition, and this leads to a benefit for viral replication. Our findings suggest that both PKR-dependent and non-PKR-dependent mechanisms of SIN-induced translational shutoff can be manipulated by using SIN replicons expressing mutated SIN nsP2 or kinase-defective PKR. Specifically, we show that expression of heterologous genes from SIN-based and most likely other alphavirus-based replicons can be increased by downregulating both the PKR-dependent and PKR-independent translational shutoffs.
Development of a DNA Vaccine Targeting Human Papillomavirus Type 16 Oncoprotein E6
Authors: Peng Shiwen , Ji Hongxiu , Trimble Cornelia , He Liangmei , Tsai Ya-Chea , Yeatermeyer Jessica , Boyd David A. K. , Hung Chien-Fu , Wu T.-C.
Published: July 2004

Human papillomavirus (HPV), particularly type 16 (HPV-16), is present in more than 99% of cervical cancers. The HPV oncoproteins E6 and E7 are constantly expressed and therefore represent ideal targets for HPV vaccine development. We previously developed DNA vaccines encoding calreticulin (CRT) linked to HPV-16 E7 and generated potent E7-specific CD8+T-cell immune responses and antitumor effects against an E7-expressing tumor. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a DNA vaccine encoding CRT linked to E6 (CRT/E6). Our results indicated that the CRT/E6 DNA vaccine, but not a wild-type E6 DNA vaccine, generated significant E6-specific CD8+T-cell immune responses in vaccinated mice. Mapping of the immunodominant epitope of E6 revealed that an E6 peptide comprising amino acids (aa) 48 to 57 (E6 aa48-57), presented by H-2Kb, is the optimal peptide and that the region of E6 comprising aa 50 to 57 represents the minimal core sequence required for activating E6-specific CD8+T lymphocytes. We also demonstrated that E6 aa48-57 contains cytotoxic T-lymphocyte epitopes naturally presented by E6-expressing TC-1 cells. Vaccination with a CRT/E6 but not a CRT/mtE6 (lacking aa 50 to 57 of E6) DNA vaccine could protect vaccinated mice from challenge with E6-expressing TC-1 tumors. Thus, our data indicate that E6 aa48-57 contains the immunodominant epitope and that a CRT/E6 DNA vaccine may be useful for control of HPV infection and HPV-associated lesions.
Initial Cleavage of the Human Immunodeficiency Virus Type 1 GagPol Precursor by Its Activated Protease Occurs by an Intramolecular Mechanism
Authors: Pettit Steven C. , Everitt Lorraine E. , Choudhury Sumana , Dunn Ben M. , Kaplan Andrew H.
Published: July 2004

Processing of the GagPol polyprotein precursor of human immunodeficiency virus type 1 (HIV-1) is a critical step in viral assembly and replication. The HIV-1 protease (PR) is translated as part of GagPol and is both necessary and sufficient for precursor processing. The PR is active only as a dimer; enzyme activation is initiated when the PR domains in two GagPol precursors dimerize. The precise mechanism by which the PR becomes activated and the subsequent initial steps in precursor processing are not well understood. However, it is clear that processing is initiated by the PR domain that is embedded within the precursor itself. We have examined the earliest events in precursor processing using an in vitro assay in which full-length GagPol is cleaved by its embedded PR. We demonstrate that the embedded, immature PR is as much as 10,000-fold less sensitive to inhibition by an active-site PR inhibitor than is the mature, free enzyme. Further, we find that different concentrations of the active-site inhibitor are required to inhibit the processing of different cleavage sites within GagPol. Finally, our results indicate that the first cleavages carried out by the activated PR within GagPol are intramolecular. Overall, our data support a model of virus assembly in which the first cleavages occur in GagPol upstream of the PR. These intramolecular cleavages produce an extended form of PR that completes the final processing steps accompanying the final stages of particle assembly by an intermolecular mechanism.
Mutations in Multiple Domains Activate Paramyxovirus F Protein-Induced Fusion
Authors: Seth Shaguna , Goodman Andrew L. , Compans Richard W.
Published: July 2004

SER virus, a paramyxovirus that is closely related to simian virus 5 (SV5), is unusual in that it fails to induce syncytium formation. The SER virus F protein has an unusually long cytoplasmic tail (CT), and it was previously observed that truncations or specific mutations of this domain result in enhanced syncytium formation. In addition to the long CT, the SER F protein has nine amino acid differences from the F protein of SV5. We previously observed only a partial suppression of fusion in a chimeric SV5 F protein with a CT derived from SER virus, indicating that these other amino acid differences between the SER and SV5 F proteins also play a role in regulating the fusion phenotype. To examine the effects of individual amino acid differences, we mutated the nine SER residues individually to the respective residues of the SV5 F protein. We found that most of the mutants were expressed well and were transported to the cell surface at levels comparable to that of the wild-type SER F protein. Many of the mutants showed enhanced lipid mixing, calcein transfer, and syncytium formation even in the presence of the long SER F protein CT. Some mutants, such as the I310 M, T438S, M489I, T516V, and N529K mutants, also showed fusion at lower temperatures of 32, 25, and 18°C. The residue Asn529 plays a critical role in the suppression of fusion activity, as the mutation of this residue to lysine caused a marked enhancement of fusion. The effect of the N529K mutation on the enhancement of fusion by a previously described mutant, L539,548A, as well as by chimeric SV5/SER F proteins was also dramatic. These results indicate that activation to a fusogenic conformation is dependent on the interplay of residues in the ectodomain, the transmembrane domain, and the CT domain of paramyxovirus F proteins.
Basal Core Promoter and Precore Mutations in the Hepatitis B Virus Genome Enhance Replication Efficacy of Lamivudine-Resistant Mutants
Authors: Tacke Frank , Gehrke Christina , Luedde Tom , Heim Albert , Manns Michael P. , Trautwein Christian
Published: July 2004

During chronic hepatitis B virus (HBV) infection, mutations in the precore (PC) or basal core promoter (BCP) region affecting HBV e antigen (HBeAg) expression occur commonly and represent the predominant virus species in patients with HBeAg-negative chronic hepatitis B. The PC mutation (G1896A+C1858T) creates a translational stop codon resulting in absent HBeAg expression, whereas BCP mutations (A1762T/G1764A) reduce HBeAg expression by transcriptional mechanisms. Treatment of chronic HBV infection with lamivudine (LMV) often selects drug-resistant strains with single (rtM204I) or double (rtL180M+rtM204V) point mutations in the YMDD motif of HBV reverse transcriptase. We cloned replication-competent HBV vectors (genotype A, adw2) combining mutations in the core (wild type [wt], PC, and BCP) and polymerase gene (wt, rtM204I, and rtL180M/M204V) and analyzed virus replication and drug sensitivity in vitro. Resistance to LMV (rtM204I/rtL180M+rtM204V) was accompanied by a reduced replication efficacy as evidenced by reduced pregenomic RNA, encapsidated progeny DNA, polymerase activity, and virion release. PC mutations alone did not alter virus replication but restored replication efficacy of the LMV-resistant mutants without affecting drug resistance. BCP mutants had higher replication capacities than did the wt, also in combination with LMV resistance mutations. All nine HBV constructs showed similar sensitivities to adefovir. In conclusion, BCP-PC mutations directly impact the replication capacity of LMV-resistant mutants. PC mutations compensated for replication inefficiency of LMV-resistant mutants, whereas BCP mutations increased viral replication levels to above the wt baseline values, even in LMV-resistant mutants, without affecting drug sensitivity in vitro. Adefovir may be an effective treatment when combinations of core and polymerase mutations occur.
Identification of Protein Tyrosine Kinases Required for B-Cell- Receptor-Mediated Activation of an Epstein-Barr Virus Immediate-Early Gene Promoter
Authors: Lavens Sandra , Faust Emmanuel A. , Lu Fang , Jacob Michele , Leta Messele , Lieberman Paul M. , Puré Ellen
Published: July 2004

Epstein-Barr Virus (EBV) is a potentially oncogenic herpesvirus that infects >90% of the world's population. EBV exists predominantly as a latent infection in B lymphocytes, with periodic lytic-cycle reactivation essential for cellular and host transmission. Viral reactivation can be stimulated by ligand-induced activation of B-cell-receptor (BCR)-coupled signaling pathways. The critical first step in the transition from latency to the lytic cycle is the expression of the viral immediate-early gene BZLF1 through the transcription activation of its promoter, Zp. However, the BCR-coupled signal transduction cascade(s) leading to the induction of Zp and the expression of the BZLF1 gene product, Zta, is currently unclear. A major obstacle to delineating the relevant signal transduction events has been the lack of a model of EBV infection that is amenable to genetic manipulation. The use of the avian B-cell line DT40 has proven to be a powerful tool for delineating BCR-mediated signal transduction pathways that appear to be highly conserved between avian and mammalian systems. We demonstrate that the DT40 cell line is a robust and genetically tractable system for the study of BCR-mediated signaling pathways leading to transcriptional activation of BZLF1. Using this system, we demonstrate that activation of Zp requires the BCR-coupled protein tyrosine kinases Syk and Btk and that it is positively regulated by Lyn. Thus, the use of DT40 cells has allowed us to delineate the early signaling components required for BCR-dependent reactivation of latent EBV, and this system is likely to prove useful for further dissection of the downstream signaling cascades involved.
Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination
Authors: Wierzchoslawski Rafal , Dzianott Aleksandra , Bujarski Jozef
Published: July 2004

Previously, we and others mapped an increased homologous recombination activity within the subgenomic promoter (sgp) region in brome mosaic virus (BMV) RNA3 (A. Bruyere et al., J. Virol. 74:4214-4219, 2000; R. Wierzchoslawski et al., J. Virol. 77:6769-6776, 2003). In order to correlate sgp-mediated recombination and transcription, in the present work we used BMV RNA3 constructs that carried altered sgp repeats. We observed that the removal or extension of the poly(U) tract reduced or increased recombination, respectively. Deletion of the sgp core hairpin or its replacement by a different stem-loop structure inhibited recombination activity. Nucleotide substitutions at the +1 or +2 transcription initiation position reduced recombination. The sgp core alone supported only basal recombination activity. The sites of crossovers mapped to the poly(U) region and to the core hairpin. The observed effects on recombination did not parallel those observed for transcription. To explain how both activities operate within the sgp sequence, we propose a dual mechanism whereby recombination is primed at the poly(U) tract by the predetached nascent plus strand, whereas transcription is initiated de novo at the sgp core.
Pulmonary Collectins Modulate Strain-Specific Influenza A Virus Infection and Host Responses
Authors: Hawgood Samuel , Brown Cynthia , Edmondson Jess , Stumbaugh Amber , Allen Lennell , Goerke Jon , Clark Howard , Poulain Francis
Published: July 2004

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A
−/−
mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A
−/−
mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.
Evidence that Stable Retroviral Transduction and Cell Survival following DNA Integration Depend on Components of the Nonhomologous End Joining Repair Pathway
Authors: Daniel René , Greger James G. , Katz Richard A. , Taganov Konstantin D. , Wu Xiaoyun , Kappes John C. , Skalka Anna Marie
Published: July 2004

We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PK
CS
) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PK
CS
-deficient
scid
fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in
scid
cells that are complemented with a functional DNA-PK
CS
gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.
Herpes Simplex Virus 1 Induces Cytoplasmic Accumulation of TIA-1/TIAR and both Synthesis and Cytoplasmic Accumulation of Tristetraprolin, Two Cellular Proteins That Bind and Destabilize AU-Rich RNAs
Authors: Esclatine Audrey , Taddeo Brunella , Roizman Bernard
Published: July 2004

Herpes simplex virus 1 causes a shutoff of cellular protein synthesis through the degradation of RNA that is mediated by the virion host shutoff (Vhs) protein encoded by the UL41 gene. We reported elsewhere that the Vhs-dependent degradation of RNA is selective, and we identified RNAs containing AU-rich elements (AREs) that were upregulated after infection but degraded by deadenylation and progressive 3′-to-5′ degradation. We also identified upregulated RNAs that were not subject to Vhs-dependent degradation (A. Esclatine, B. Taddeo, L. Evans, and B. Roizman, Proc. Natl. Acad. Sci. USA 101:3603-3608, 2004). Among the latter was the RNA encoding tristetraprolin, a protein that binds AREs and is known to be associated with the degradation of RNAs containing AREs. Prompted by this observation, we examined the status of the ARE binding proteins tristetraprolin and TIA-1/TIAR in infected cells. We report that tristetraprolin was made and accumulated in the cytoplasm of wild-type virus-infected human foreskin fibroblasts as early as 2 h and in HEp-2 cells as early as 6 h after infection. The amounts of tristetraprolin that accumulated in the cytoplasm of cells infected with a mutant virus lacking UL41 were significantly lower than those in wild-type virus-infected cells. The localization of tristetraprolin was not modified in cells infected with a mutant lacking the gene encoding infected cell protein 4 (ICP4). TIA-1 and TIAR are two other proteins that are associated with the regulation of ARE-containing RNAs and that normally reside in nuclei. In infected cells, they started to accumulate in the cytoplasm after 6 h of infection. In cells infected with the mutant virus lacking UL41, TIA-1/TIAR accumulated in the cytoplasm in granular structures reminiscent of stress granules in a significant percentage of the cells. In addition, an antibody to tristetraprolin coprecipitated the Vhs protein from lysates of cells late in infection. The results indicate that the Vhs-dependent degradation of ARE-containing RNAs correlates with the transactivation, cytoplasmic accumulation, and persistence of tristetraprolin in infected cells.
Role of JC Virus Agnoprotein in DNA Repair
Authors: Darbinyan Armine , Siddiqui Khwaja M. , Slonina Dorota , Darbinian Nune , Amini Shohreh , White Martyn K. , Khalili Kamel
Published: July 2004

The late region of human neurotropic JC virus encodes a small 71-amino-acid agnoprotein that is also found in the polyomaviruses simian virus 40 and BK virus. Several functions of agnoprotein have been identified, including roles in regulating viral transcription and virion maturation. Earlier studies showed that agnoprotein expressed alone induced p21/WAF-1 expression and caused cells to accumulate in the G2/M stage of the cell cycle. Here we report that agnoprotein expression sensitized cells to the cytotoxic effects of the DNA-damaging agent cisplatin. Agnoprotein reduced the viability of cisplatin-treated cells and increased chromosome fragmentation and micronucleus formation. Whereas cisplatin-treated control cells accumulated in S phase, cells expressing agnoprotein did not, instead becoming aneuploid. Agnoprotein expression correlated with impaired double-strand-break repair activity in cellular extracts and reduced expression of the Ku70 and Ku80 DNA repair proteins. After agnoprotein expression, much of the Ku70 protein was located in the perinuclear space, where agnoprotein was also found. Results from binding studies showed an interaction of agnoprotein with Ku70 which was mediated by the N terminus. The ability of agnoprotein to inhibit double-strand break repair activity when it was added to cellular extracts was also mediated by the N terminus. We conclude that agnoprotein inhibits DNA repair after DNA damage and interferes with DNA damage-induced cell cycle regulation. Since Ku70 is a subunit of the DNA-dependent protein kinase that is responsible both for double-strand break repair and for signaling damage-induced cell cycle arrest, the modulation of Ku70 and/or Ku80 by agnoprotein may represent an important event in the polyomavirus life cycle and in cell transformation.
Continuing Evolution of H9N2 Influenza Viruses in Southeastern China
Authors: Choi Y. K. , Ozaki H. , Webby R. J. , Webster R. G. , Peiris J. S. , Poon L. , Butt C. , Leung Y. H. C. , Guan Y.
Published: July 2004

H9N2 influenza viruses are panzootic in domestic poultry in Eurasia and since 1999 have caused transient infections in humans and pigs. To investigate the zoonotic potential of H9N2 viruses, we studied the evolution of the viruses in live-poultry markets in Hong Kong in 2003. H9N2 was the most prevalent influenza virus subtype in the live-poultry markets between 2001 and 2003. Antigenic and phylogenetic analysis of hemagglutinin (HA) showed that all of the 19 isolates found except one belonged to the lineage represented by A/Duck/Hong Kong/Y280/97 (H9N2). The exception was A/Guinea fowl/NT184/03 (H9N2), whose HA is most closely related to that of the human isolate A/Guangzhou/333/99 (H9N2), a virus belonging to the A/Chicken/Beijing/1/94-like (H9N2) lineage. At least six different genotypes were recognized. The majority of the viruses had nonstructural (and HA) genes derived from the A/Duck/Hong Kong/Y280/97-like virus lineage but had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses isolated in 2001. Viruses of all six genotypes of H9N2 found were able to replicate in chickens and mice without adaptation. The infected chickens showed no signs of disease, but representatives of two viral genotypes were lethal to mice. Three genotypes of virus replicated in the respiratory tracts of swine, which shed virus for at least 5 days. These results show an increasing genetic and biologic diversity of H9N2 viruses in Hong Kong and support their potential role as pandemic influenza agents.
Kaposi's Sarcoma-Associated Herpesvirus
ori
-
Lyt
-Dependent DNA Replication:
cis
-Acting Requirements for Replication and
ori
-
Lyt
-Associated RNA Transcription
Authors: Wang Yan , Li Hong , Chan Man Yee , Zhu Fan Xiu , Lukac David M. , Yuan Yan
Published: July 2004

Herpesvirus lytic DNA replication requires both the
cis
-acting element, the origin, and
trans
-acting factors such as virally encoded origin-binding protein and DNA replication enzymes. Recently, the origins of lytic DNA replication (
ori
-
Lyt
) in Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and a virally encoded bZip protein, K8, has been shown to specifically bind to the origin. To map
cis
-acting elements within KSHV
ori
-
Lyt
that are required for DNA replication function and to define the nature of K8 bZip protein binding to the origin, we constructed consecutive internal deletion mutations across the core domain of a KSHV
ori
-
Lyt
and tested them for DNA replication function in a transient replication assay. This mutagenesis study allowed the identification of four components within the
ori
-
Lyt
, and all were indispensable for
ori
-
Lyt
function. The first component contains eight CCAAT/enhancer binding protein (C/EBP) binding motifs that organize as four spaced C/EBP palindromes. Each palindrome contains two head-to-head CCAAT consensus motifs that are separated by a 13- or 12-bp space sequence. Substitution mutagenesis of these C/EBP motifs showed that these C/EBP palindromes are required for both K8 binding and
ori
-
Lyt
-dependent DNA replication. The second component is an 18-bp AT palindrome, which is essential for
ori
-
Lyt
function. The third component was determined to be a 32-bp previously unidentified sequence and is required for DNA replication. The last component consists of an open reading frame 50 (ORF50)/Rta responsive element (RRE) and a TATA box. We showed that the binding of an ORF50/Rta protein to the RRE was essential for
ori
-
Lyt
-dependent DNA replication. The presence of a functional RRE and a downstream TATA box suggested that this region serves as an ORF50/Rta-dependent promoter and a transcription event may be necessary for
ori
-
Lyt
-dependent DNA replication. Using a luciferase reporter system, we demonstrated that the region of the RRE and TATA box constitutes an ORF50/Rta-dependent promoter. Furthermore, a polyadenylated RNA of 1.4 kb was identified downstream of the promoter.
Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins
Authors: Kingston Richard L. , Baase Walter A. , Gay Leslie S.
Published: July 2004

We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly α-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (

K
d

) was determined to be 13 μM at 20°C and 35 μM at 37°C. Our data are consistent with a model in which an α-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.
Suppression of Herpes Simplex Virus 1 in MDBK Cells via the Interferon Pathway
Authors: Barreca Cristina , O'Hare Peter
Published: July 2004

Herpes simplex virus (HSV) normally undergoes productive infection in culture, causing cell destruction and plaque formation. Here we characterize an unusual pattern of HSV type 1 (HSV-1) infection in MDBK cells which surprisingly results in suppression of replication, cell recovery, and maintenance of virus. Compared to Vero cells, MDBK cells supported a normal productive infection at a high multiplicity with complete cell destruction. At low multiplicity, HSV also showed an identical initial specific infectivity in the two cell types. Thereafter, the progression of infection was radically different. In contrast to the rapid plaque expansion and eventual destruction in Vero monolayers, in MDBK cells, after initial plaque formation, plaque size actually decreased and, with time, monolayers recovered. Using a green fluorescent protein (GFP)-VP16-expressing virus, we monitored infection in live individual plaques. After early stages of intense GFP-VP16 expression, expression regressed to a thin boundary at the edge of the plaques and was completely suppressed by 10 days. Cells lacking expression then began to grow into the plaque boundaries. Furthermore, following media replacement, individual cells expressing GFP-VP16 could be observed reinitiating infection. The results indicated the production of a potent inhibitory component during infection in MDBK cells, and we show the continued and prolonged presence of interferon in the medium, at times when there was no longer evidence of ongoing productive infection. We exploited the ability of V protein of simian virus 5 to degrade Stat1 and prevent interferon signaling. We established MDBK cells constitutively expressing the V protein with the resultant loss of Stat1. In comparison to the parental cells, infection in these cells now progressed at a rapid rate with expanding plaque formation. We believe the conclusions have significant implications for the study of HSV-1 and interferon signaling both in culture and in animal models.
Spirodiketopiperazine-Based CCR5 Inhibitor Which Preserves CC-Chemokine/CCR5 Interactions and Exerts Potent Activity against R5 Human Immunodeficiency Virus Type 1 In Vitro
Authors: Maeda Kenji , Nakata Hirotomo , Koh Yasuhiro , Miyakawa Toshikazu , Ogata Hiromi , Takaoka Yoshikazu , Shibayama Shiro , Sagawa Kenji , Fukushima Daikichi , Moravek Joseph , Koyanagi Yoshio , Mitsuya Hiroaki
Published: July 2004

We identified a novel spirodiketopiperazine (SDP) derivative, AK602/ONO4128/GW873140, which specifically blocked the binding of macrophage inflammatory protein 1α (MIP-1α) to CCR5 with a high affinity (

K
d

of ≈3 nM), potently blocked human immunodeficiency virus type 1 (HIV-1) gp120/CCR5 binding and exerted potent activity against a wide spectrum of laboratory and primary R5 HIV-1 isolates, including multidrug-resistant HIV-1 (HIV-1
MDR
) (50% inhibitory concentration values of 0.1 to 0.6 nM) in vitro. AK602 competitively blocked the binding to CCR5 expressed on Chinese hamster ovary cells of two monoclonal antibodies, 45523, directed against multidomain epitopes of CCR5, and 45531, specific against the C-terminal half of the second extracellular loop (ECL2B) of CCR5. AK602, despite its much greater anti-HIV-1 activity than other previously published CCR5 inhibitors, including TAK-779 and SCH-C, preserved RANTES (regulated on activation normal T-cell expressed and secreted) and MIP-1β binding to CCR5
+
cells and their functions, including CC-chemokine-induced chemotaxis and CCR5 internalization, while TAK-779 and SCH-C fully blocked the CC-chemokine/CCR5 interactions. Pharmacokinetic studies revealed favorable oral bioavailability in rodents. These data warrant further development of AK602 as a potential therapeutic for HIV-1 infection.