bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Histone Deacetylases Induces Bovine Leukemia Virus Expression In Vitro and In Vivo
Authors: Merezak C. , Reichert M. , Van Lint C. , Kerkhofs P. , Portetelle D. , Willems L. , Kettmann R. 
Published: July 2002

ABSTRACT
Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only ∼2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.
Induction of the Bovine Papillomavirus Origin “Onion Skin”-Type DNA Replication at High E1 Protein Concentrations In Vivo
Authors: Männik Andres , Rünkorg Kertu , Jaanson Nele , Ustav Mart , Ustav Ene 
Published: July 2002

ABSTRACT
We have studied the replication of plasmids composed of bovine papillomavirus type 1 (BPV1) origin of replication and expression cartridges for viral proteins E1 and E2 in hamster and mouse cells. We found that the replication mode changed dramatically at different expression levels of the E1 protein. At high levels of the E1 protein, overreplication of the origin region of the plasmid was observed. Analysis of the replication products by one-dimensional and two-dimensional gel electrophoresis suggested that initially “onion skin”-type replication intermediates were generated, presumably resulting from initiation of the new replication forks before the leading fork completed the synthesis of the DNA on the episomal plasmid. These replication intermediates served as templates for generation of a heterogeneous set of origin region-containing linear fragments by displacement synthesis at the partially replicated plasmid. Additionally, the linear fragments may have been generated by DNA break-up of the onion skin-type intermediates. Analysis of replication products indicated that generated linear fragments recombined and formed concatemers or circular molecules, which presumably were able to replicate in an E1- and E2-dependent fashion. At moderate and low levels of E1, generated by transcription of the E1 open reading frame using weaker promoters, DNA replication was initiated at much lower levels, which allowed elongation of the replication fork starting from the origin to be more balanced and resulted in the generation of full-sized replication products.
Development and Characterization of Bovine × Human Hybrid Cell Lines That Efficiently Support the Replication of both Wild-Type Bovine and Human Adenoviruses and Those with E1 Deleted
Authors: van Olphen Alberto L. , Mittal Suresh K. 
Published: July 2002

ABSTRACT
The 293 cell line that was generated by transforming human embryonic kidney cells with human adenovirus type 5 (HAV5) early region 1 (E1) sequences is an excellent host for generating and growing HAV5 recombinants with E1 deleted, but it does not support the replication of bovine adenovirus type 3 (BAV3). Madin-Darby bovine kidney (MDBK), an established bovine cell line, is an excellent host for growing and plaquing BAV3. For the purpose of combining the unique characteristics of these two cell lines (293 and MDBK), we generated a number of bovine × human hybrid (BHH) cell lines. Comparison of three BHH hybrid clones—BHH3, BHH8, and BHH2C—with 293-Puro (puromycin-resistant 293 cells) and MDBK-Neo (G418-resistant MDBK cells) cell lines for total cellular DNA content, species-specific surface markers, isoenzyme analysis, and karyotyping indicate that they are hybrid in nature. BHH clones constitutively expressed the E1 proteins (E1A, E1B-21kDa, and E1B-55kDa) of HAV5 and efficiently supported the replication of both wild-type and replication-incompetent bovine or human adenoviruses. Transient gene expression experiments with a plasmid encoding the bacterial β-galactosidase gene demonstrated that BHH cell hybrids seem to have better transfection efficiencies than either of the parental cell lines. These cell lines will be useful for isolating and growing replication-competent human or bovine adenovirus recombinants with E1 deleted and for the study of cellular or viral factors important for viral replication. The development of somatic cell hybrids appears to be a simple way of combining some of the desirable characteristics present separately in two parental cell lines.
Virulence and Reduced Fitness of Simian Immunodeficiency Virus with the M184V Mutation in Reverse Transcriptase
Authors: Van Rompay Koen K. A. , Matthews Tim B. , Higgins Joanne , Canfield Don R. , Tarara Ross P. , Wainberg Mark A. , Schinazi Raymond F. , Pedersen Niels C. , North Thomas W. 
Published: July 2002

ABSTRACTDrug-resistant mutants with a methionine-to-valine substitution at position 184 of reverse transcriptase (M184V) emerged within 5 weeks of initiation of therapy in four newborn macaques infected with simian immunodeficiency virus (SIVmac251) and treated with lamivudine (3TC) or emtricitabine [(−)-FTC] (two animals per drug). Thus, this animal model mimics the rapid emergence of M184V mutants of HIV-1 during 3TC therapy of human patients. One animal of each treatment group developed fatal immunodeficiency at 12 weeks of age, which is similar to the rapid disease course seen in most untreated SIVmac251-infected infant macaques. To further evaluate the effect of the M184V mutation on viral fitness and virulence, groups of juvenile macaques were inoculated with the molecular clone SIVmac239 with either the wild-type sequence (group A [n= 5]) or the M184V sequence (SIVmac239-184V; group B [n= 5] and group C [n= 2]). The two SIVmac239-184V-infected animals of group C did not receive any drug treatment, and in both animals the virus population reverted to predominantly wild type (184M) by 8 weeks after inoculation. The other five SIVmac239-184V-infected animals (group B) were treated with (−)-FTC to prevent reversion. Although virus levels 1 week after inoculation were lower in the SIVmac239-184V-infected macaques than in the SIVmac239-infected animals, no significant differences were observed from week 2 onwards. Two animals in each group developed AIDS and were euthanized, while all other animals were clinically stable at 46 weeks of infection. These data demonstrate that the M184V mutation in SIV conferred a slightly reduced fitness but did not affect disease outcome.
Subcellular Localization and Integration Activities of Rous Sarcoma Virus Reverse Transcriptase
Authors: Werner Susanne , Hindmarsh Patrick , Napirei Markus , Vogel-Bachmayr Karin , Wöhrl Birgitta M. 
Published: July 2002

ABSTRACT

Reverse transcriptases (RTs) αβ and β from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT αβ and the β subunit, when expressed independently, can be detected in the nucleus, whereas the separate α subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of β, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT αβ with an oligodeoxynucleotide-based assay system and with a donor substrate containing the
supF
gene flanked by the viral long terminal repeats. Our data show that RSV RT αβ is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT αβ could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).
Charting Latency Transcripts in Kaposi's Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time Quantitative PCR
Authors: Fakhari Farnaz D. , Dittmer Dirk P. 
Published: July 2002

ABSTRACT
The division into a latent or lytic life cycle is fundamental to all herpesviridae. In the case of Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8), latent genes have been implicated in cell autonomous transformation, while certain lytic genes procure a tumor friendly milieu through paracrine mechanism. To query KSHV transcription, we devised and validated a high-throughput, high-specificity, high-sensitivity, real-time quantitative reverse transcription-PCR array. This novel methodology is applicable to many human pathogens. Its first use demonstrated that the mRNA levels for KSHV LANA, v-cyclin, and v-FLIP do not increase at any time after viral reactivation. The mRNA for LANA-2/vIRF-3 is similarly resistant to viral reactivation. In contrast, every other latent or lytic message was induced. Hence, LANA, v-FLIP, v-cyclin, and LANA-2 constitute a group of uniquely regulated transcripts in the KSHV genome.
Dissection of Human Immunodeficiency Virus Type 1 Entry with Neutralizing Antibodies to gp41 Fusion Intermediates
Authors: Golding Hana , Zaitseva Marina , de Rosny Eve , King Lisa R. , Manischewitz Jody , Sidorov Igor , Gorny Miroslaw K. , Zolla-Pazner Susan , Dimitrov Dimiter S. , Weiss Carol D. 
Published: July 2002

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5°C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37°C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37°C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37°C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37°C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.
Mapping the Transcription and Replication Promoters of Respiratory Syncytial Virus
Authors: Fearns Rachel , Peeples Mark E. , Collins Peter L. 
Published: July 2002

ABSTRACT

An important, unresolved issue in mononegavirus biology is whether or not transcription is initiated by the same promoter as RNA replication. In this study, residues important for respiratory syncytial virus (RSV) transcription and RNA replication were identified by subjecting the first 26 nucleotides of genome RNA to saturation mutagenesis. This analysis was performed using a genome analog that allowed transcription and RNA replication to be dissociated from each other and monitored as independent events in an intracellular assay. This analysis showed that nucleotides 3C, 5C, 8U, 9U, 10U, and 11U were important for transcription and RNA replication. Additional nucleotides (1U, 2G, 6U, and 7U) were important for RNA replication, but not transcription. At position 4, G versus C or U augmented transcription and decreased replication, showing that the naturally occurring assignments in the genomic (4G) and antigenomic (4U) promoters are optimal for transcription and RNA replication, respectively. These data show that RSV transcription and RNA replication each involve a
cis
-acting signal at the very 3" end of the genome. This signal appears to contain a minimum, common element that functions in both transcription and RNA replication, defined by those substitutions that had similar effects on the two processes. Apart from these common nucleotides, other positions were involved in RNA replication but not transcription or had different effects on the two processes. This indicates that the promoters for transcription and replication involve overlapping sets of nucleotides at the very 3" end of the genome and provides evidence that the nucleotide preferences for the two processes are not identical.
Evolution of a Human Immunodeficiency Virus Type 1 Variant with Enhanced Replication in Pig-Tailed Macaque Cells by DNA Shuffling
Authors: Pekrun Katja , Shibata Riri , Igarashi Tatsuhiko , Reed Margaret , Sheppard Liana , Patten Philip A. , Stemmer Willem P. C. , Martin Malcolm A. , Soong Nay-Wei 
Published: July 2002

ABSTRACT

DNA shuffling facilitated the evolution of a human immunodeficiency virus type 1 (HIV-1) variant with enhanced replication in pig-tailed macaque peripheral blood mononuclear cells (pt mPBMC). This variant consists exclusively of HIV-1-derived sequences with the exception of simian immunodeficiency virus (SIV)
nef
. Sequences spanning the
gag
-protease-reverse transcriptase (
gag
-
pro
-RT) region from several HIV-1 isolates were shuffled and cloned into a parental HIV-1 backbone containing SIV
nef
. Neither this full-length parent nor any of the unshuffled HIV-1 isolates replicated appreciably or sustainably in pt mPBMC. Upon selection of the shuffled viral libraries by serial passaging in pt mPBMC, a species emerged which replicated at substantially higher levels (50 to 100 ng/ml p24) than any of the HIV-1 parents and most importantly, could be continuously passaged in pt mPBMC. The parental HIV-1 isolates, when selected similarly, became extinct. Analyses of full-length improved proviral clones indicate that multiple recombination events in the shuffled region and adaptive changes in the rest of the genome contributed synergistically to the improved phenotype. This improved variant may prove useful in establishing a pig-tailed macaque model of HIV-1 infection.
CD28−/−Mice Show Defects in Cellular and Humoral Immunity but Are Able To Control Infection with Murine Gammaherpesvirus 68
Authors: Lee Bong Joo , Reiter Su Khoh , Anderson Mandy , Sarawar Sally R. 
Published: July 2002

ABSTRACTThe role of CD28-dependent costimulatory interactions in the development and maintenance of antiviral immune responses was investigated in a mouse model of gammaherpesvirus infection. CD28−/−mice could clear a productive infection with murine gammaherpesvirus 68 (MHV-68), although early lung viral titers were significantly increased. Both CD28−/−and CD28+/+mice maintained effective long-term control of MHV-68. Gamma interferon responses appeared to develop more slowly in CD28−/−mice, while cytotoxic T-cell activity was similar to that in wild-type mice. Splenomegaly developed normally in CD28−/−mice, whereas virus-specific antibody responses were significantly reduced and aberrant class switching was observed. This work demonstrates that costimulatory interactions involving CD28 are not an absolute requirement for the control of infection with MHV-68.
Open Reading Frame 50 Protein of Kaposi's Sarcoma-Associated Herpesvirus Directly Activates the Viral PAN and K12 Genes by Binding to Related Response Elements
Authors: Chang Pey-Jium , Shedd Duane , Gradoville Lyn , Cho Myung-Sam , Chen Lee-Wen , Chang Jimmy , Miller George 
Published: July 2002

ABSTRACT
Open reading frame (ORF) 50 protein is capable of activating the entire lytic cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), but its mechanism of action is not well characterized. Here we demonstrate that ORF 50 protein activates two KSHV lytic cycle genes, PAN (polyadenylated nuclear RNA) and K12, by binding to closely related response elements located approximately 60 to 100 nucleotides (nt) upstream of the start of transcription of the two genes. The 25-nt sequence 5′ AAATGGGTGGCTAACCTGTCCAAAA from the PAN promoter (PANp) confers a response to ORF 50 protein in both epithelial cells and B cells in the absence of other KSHV proteins. The responsive region of DNA can be transferred to a heterologous minimal promoter. Extensive point mutagenesis showed that a span of at least 20 nt is essential for a response to ORF 50 protein. However, a minimum of six positions within this region were ambiguous. The related 26-nt responsive element in the K12 promoter (K12p), 5′ GGAAATGGGTGGCTAACCCCTACATA, shares 20 nt (underlined) with the comparable region of PANp. The divergence is primarily at the 3′ end. The DNA binding domain of ORF 50 protein, encompassing amino acids 1 to 490, fused to a heterologous activation domain from herpes simplex virus VP16 [ORF 50(1-490)+VP] can mediate activation of reporter constructs bearing these response elements. Most importantly, ORF 50(1-490)+VP can induce PAN RNA and K12 transcripts in transfected cells. ORF 50(1-490)+VP expressed in human cells binds specifically to duplex oligonucleotides containing the responsive regions from PANp and K12p. These DNA-protein complexes were supershifted by antibody to VP16. ORF 50(1-490) without a VP16 tag also bound to the response element. There was a strong correlation between DNA binding by ORF 50 and transcriptional activation. Mutations within PANp and K12p that impaired transactivation by ORF 50 or ORF 50(1-490)+VP also abolished DNA binding. Only one of eight related complexes formed on PANp and K12p oligonucleotides was due to ORF 50(1-490)+VP. The other complexes were due to cellular proteins. Two KSHV lytic-cycle promoters are activated by a similar mechanism that involves direct recognition of a homologous response element by the DNA binding domain of ORF 50 protein in the context of related cellular proteins.
Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production
Authors: Kaushik Neerja , Basu Amartya , Palumbo Paul , Myers Rene L. , Pandey Virendra N. 
Published: July 2002

ABSTRACT

The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNA
TAR
), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNA
TAR
-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNA
TAR
-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNA
TAR
-transportan as a potential anti-HIV agent.
Low Temperature Sintering Studies of Ammonium Molybdate, Nickel Hydroxide and Nickel Molybdate
Authors: Halawy Samih A. , Abd El-Salaam Khalaf Alla M. , Ragih Hesham M. 
Published: July 2002

The thermal decomposition of ammonium molybdate, nickel hydroxide and MoO3-NiO 50 mole % mixture were studied by means of TG and DTG non-isothermally, in a dynamic atmosphere of N2. The activation energy of the thermal process of such samples, also, were evaluated using Maple's theory, in isothermal condition. The variation of SBET with time, during heating ammonium molybdate, nickel hydroxide and MoO3-NiO 50 mole % mixture at different temperatures, in the range 200 - 400 °C, was monitored, and the activation energy for the sintering process of each compound were calculated from the Arrhenius plot. The final products of heating each sample at the sintering temperatures after 6 h, were characterized by IR and XRD analyses. MoO3 was formed clearly at 350 °C, while Ni forms a mixture of nickel(II) and -(III) oxides at the same temperature. NiMoO4 was produced, also, at 350 °C in MoO3-NiO 50 mole % mixture sample.
Removal of Anions from Solution by Calcined Hydrotalcite and Regeneration of Used Sorbent in Repeated Calcination-Rehydration-Anion Exchange Processes
Authors: Kovanda František , Kovácsová Eva , Koloušek David 
Published: July 2002

Synthetic hydrotalcite calcined at 350-550 °C was used for the removal of arsenate, chromate, and vanadate ions from water solutions. The initial anion concentrations were 0.002 mol l-1. The sorption isotherms were measured at 20 °C and at neutral pH. The Langmuir adsorption isotherm was used for the sorption capacity evaluation. The ability of the calcined hydrotalcite to remove the anions from solution decreased in the order of vanadate - arsenate - chromate. The hydrotalcite calcined at 450 °C exhibited the best sorption ability for all the anions. The sorbed anions were released by anion exchange in a carbonate-containing solution and the hydrotalcite after subsequent calcination was used again for the removal of anions. The repeating cycles calcination-rehydration-anion exchange gradually reduced the adsorption capacity of the hydrotalcite. The sorption capacity decreased by 50% after the first two cycles but it did not change significantly in the subsequent cycles. When chromate anions were adsorbed, the decrease in sorption capacity was not observed during repeated calcination-rehydration-anion exchange cycles. The change in the sorption capacity was influenced by the ability of calcined hydrotalcite to regenerate the layered crystal structure during the rehydration process.