bluuebunny/crossref_metadata_2025_split huggingface dataset:
De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus
Authors: Luo Guangxiang , Hamatake Robert K. , Mathis Danielle M. , Racela Jason , Rigat Karen L. , Lemm Julie , Colonno Richard J. 
Published: July 2002

ABSTRACT

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn
2+
than in the presence of Mg
2+
. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.
Human Immunodeficiency Virus Type 1 Vpr Induces Apoptosis in Human Neuronal Cells
Authors: Patel Charvi A. , Mukhtar Muhammad , Pomerantz Roger J. 
Published: July 2002

ABSTRACT
Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC.
Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys
Authors: Yamaguchi T. , Kaplan S. L. , Wakenell P. , Schat K. A. 
Published: July 2002

ABSTRACTThe QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16,meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisensemeq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies.
The Exceptionally Large Genome of Hendra Virus: Support for Creation of a New Genus within the Family
Paramyxoviridae
Authors: Wang Lin-Fa , Yu Meng , Hansson Eric , Pritchard L. Ian , Shiell Brian , Michalski Wojtek P. , Eaton Bryan T. 
Published: July 2002

ABSTRACT

An outbreak of acute respiratory disease in Hendra, a suburb of Brisbane, Australia, in September 1994 resulted in the deaths of 14 racing horses and a horse trainer. The causative agent was a new member of the family
Paramyxoviridae
. The virus was originally called
Equine morbillivirus
but was renamed
Hendra virus
(HeV) when molecular characterization highlighted differences between it and members of the genus
Morbillivirus
. Less than 5 years later, the closely related
Nipah virus
(NiV) emerged in Malaysia, spread rapidly through the pig population, and caused the deaths of over 100 people. We report the characterization of the HeV L gene and protein, the genome termini, and gene boundary sequences, thus completing the HeV genome sequence. In the highly conserved region of the L protein, the HeV sequence GDNE differs from the GDNQ found in almost all other nonsegmented negative-strand (NNS) RNA viruses. HeV has an absolutely conserved intergenic trinucleotide sequence, 3′-GAA-5′, and highly conserved transcription initiation and termination sequences similar to those of respiroviruses and morbilliviruses. The large genome size (18,234 nucleotides), the unique complementary genome terminal sequences of HeV, and the limited homology with other members of the
Paramyxoviridae
suggest that HeV, together with NiV, should be classified in a new genus in this family. The large genome of HeV also fills a gap in the spectrum of genome sizes observed with NNS RNA virus genomes. As such, it provides a further piece in the puzzle of NNS RNA virus evolution.
Borna Disease Virus Persistence Causes Inhibition of Glutamate Uptake by Feline Primary Cortical Astrocytes
Authors: Billaud Jean-Noel , Ly Calvin , Phillips Tom R. , de la Torre Juan Carlos 
Published: July 2002

ABSTRACT
Borna disease virus (BDV), a nonsegmented, negative-stranded (NNS) RNA virus, causes central nervous system (CNS) disease in a broad range of vertebrate species, including felines. Both viral and host factors contribute to very diverse clinical and pathological manifestations associated with BDV infection. BDV persistence in the CNS can cause neurobehavioral and neurodevelopmental abnormalities in the absence of encephalitis. These BDV-induced CNS disturbances are associated with altered cytokine and neurotrophin expression, as well as cell damage that is very restricted to specific brain regions and neuronal subpopulations. BDV also targets astrocytes, resulting in the development of prominent astrocytosis. Astrocytes play essential roles in maintaining CNS homeostasis, and disruption of their normal activities can contribute to altered brain function. Therefore, we have examined the effect of BDV infection on the astrocyte's physiology. We present here evidence that BDV can establish a nonlytic chronic infection in primary cortical feline astrocytes that is associated with a severe impairment in the astrocytes' ability to uptake glutamate. In contrast, the astrocytes' ability to uptake glucose, as well as their protein synthesis, viability, and rate of proliferation, was not affected by BDV infection. These findings suggest that, in vivo, BDV could also affect an important astrocyte function required to prevent neuronal excitotoxicity. This, in turn, might contribute to the neuropathogenesis of BDV.
Development of Multigene and Regulated Lentivirus Vectors
Authors: Reiser Jakob , Lai Zhennan , Zhang Xian-Yang , Brady Roscoe O. 
Published: July 2002

ABSTRACT

Previously we described safe and efficient three-component human immunodeficiency virus type 1 (HIV-1)-based gene transfer systems for delivery of genes into nondividing cells (H. Mochizuki, J. P. Schwartz, K. Tanaka, R. O. Brady, and J. Reiser, J. Virol. 72:8873–8883, 1998). To apply such vectors in anti-HIV gene therapy strategies and to express multiple proteins in single target cells, we have engineered HIV-1 vectors for the concurrent expression of multiple transgenes. Single-gene vectors, bicistronic vectors, and multigene vectors expressing up to three exogenous genes under the control of two or three different transcriptional units, placed within the viral
gag-pol
coding region and/or the viral
nef
and
env
genes, were designed. The genes encoding the enhanced version of green fluorescent protein (EGFP), mouse heat-stable antigen (HSA), and bacterial neomycin phosphotransferase were used as models whose expression was detected by fluorescence-activated cell sorting, fluorescence microscopy, and G418 selection. Coexpression of these reporter genes in contact-inhibited primary human skin fibroblasts (HSFs) persisted for at least 6 weeks in culture. Coexpression of the
HSA
and
EGFP
reporter genes was also achieved following cotransduction of target cells using two separate lentivirus vectors encoding HSA and EGFP, respectively. For the regulated expression of transgenes, tetracycline (Tet)-regulatable lentivirus vectors encoding the reverse Tet transactivator (rtTA) and EGFP controlled by a Tet-responsive element (TRE) were constructed. A binary HIV-1-based vector system consisting of a lentivirus encoding rtTA and a second lentivirus harboring a TRE driving the
EGFP
reporter gene was also designed. Doxycycline-modulated expression of the
EGFP
transgene was confirmed in transduced primary HSFs. These versatile vectors can potentially be used in a wide range of gene therapy applications.
Replication of Wild-Type and Mutant Human Cytomegalovirus in Life-Extended Human Diploid Fibroblasts
Authors: Bresnahan Wade A. , Hultman Gretchen E. , Shenk Thomas 
Published: July 2002

ABSTRACTA cDNA encoding the catalytic subunit of human telomerase was used to generate life-extended derivatives of primary human diploid fibroblasts. The life-extended cells supported efficient human cytomegalovirus (HCMV) replication. A subclone of the life-extended cells was generated containing the HCMV UL82 gene and used to isolate and propagate a virus that exhibited a profound growth defect after infection at a low input multiplicity.
ATP Binding and ATPase Activities Associated with Recombinant Rabbit Hemorrhagic Disease Virus 2C-Like Polypeptide
Authors: Marín M. Soledad , Casais Rosa , Alonso José M. Martín , Parra Francisco 
Published: July 2002

ABSTRACT

The carboxy-terminal region of the rabbit hemorrhagic disease virus p37 polyprotein cleavage product has been expressed in
Escherichia coli
as a glutathione
S
-transferase (GST) fusion protein. The recombinant GST-Δ2C protein showed in vitro ATP-binding and ATPase activities. Site-directed mutagenesis studies of the conserved residues G
522
and T
529
in motif A, D
566
and E
567
in motif B, and K
600
in motif C were also performed. These results provide the first experimental characterization of a 2C-like ATPase activity in a member of the
Caliciviridae.
Herpesvirus Saimiri vFLIP Provides an Antiapoptotic Function but Is Not Essential for Viral Replication, Transformation, or Pathogenicity
Authors: Glykofrydes Diana , Niphuis Henk , Kuhn Eva M. , Rosenwirth Brigitte , Heeney Jonathan L. , Bruder Joseph , Niedobitek Gerald , Müller-Fleckenstein Ingrid , Fleckenstein Bernhard , Ensser Armin 
Published: July 2002

ABSTRACT
Apoptosis of infected cells is an important host defense mechanism, and many viruses have exploited antiapoptotic proteins that interfere with crucial cellular pathways. Viral FLICE inhibitory proteins (vFLIPs) are encoded by rhadinoviruses like herpesvirus saimiri, the related Kaposi's sarcoma-associated herpesvirus-human herpesvirus 8 (KSHV/HHV8), and the poxvirus responsible for molluscum contagiosum. The vFLIPs can block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8), and this prevents the initiation of the downstream caspase cascade. KSHV/HHV8 vFLIP overexpression can confer resistance to T-cell-mediated apoptosis and acts as a tumor progression factor in a murine B-cell lymphoma model. To analyze the function of herpesvirus vFLIPs in the genetic background of the virus and in a model for viral pathogenesis, we deleted the vFLIP gene (open reading frame 71) from the genome of herpesvirus saimiri strain C488. The viral deletion mutant was viable and replicated like the wild-type virus. An antiapoptotic effect could be attributed to the vFLIP gene, but we also show that the vFLIP gene of herpesvirus saimiri is dispensable for viral transformation of T cells in vitro and for pathogenicity in cottontop tamarins in vivo.
Apelin, the Natural Ligand of the Orphan Seven-Transmembrane Receptor APJ, Inhibits Human Immunodeficiency Virus Type 1 Entry
Authors: Cayabyab Mark , Hinuma Shuji , Farzan Michael , Choe Hyeryun , Fukusumi Shoji , Kitada Chieko , Nishizawa Naoki , Hosoya Masaki , Nishimura Osamu , Messele Tsehaynesh , Pollakis Georgios , Goudsmit Jaap , Fujino Masahiko , Sodroski Joseph 
Published: July 2002

ABSTRACT
In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolates from different clades into cells expressing CD4 and APJ. Analysis of apelin analogues demonstrated that potent and specific antiviral activity was retained by a 13-residue, arginine-rich peptide. Antiviral potency was influenced by the integrity of methionine 75, which contributes to APJ-binding affinity, and by the retention of apelin residues 63 to 65. These studies demonstrate the ability of a small peptide ligand to block the function of APJ as an HIV-1 coreceptor, identify apelin sequences important for the inhibition, and provide new reagents for the investigation of the significance of APJ to HIV-1 infection and pathogenesis.
Differential Glycosylation of the Cas-Br-E Env Protein Is Associated with Retrovirus-Induced Spongiform Neurodegeneration
Authors: Lynch William P. , Sharpe Arlene H. 
Published: July 2002

ABSTRACT

The wild mouse ecotropic retrovirus, Cas-Br-E, induces progressive, noninflammatory spongiform neurodegenerative disease in susceptible mice. Functional genetic analysis of the Cas-Br-E genome indicates that neurovirulence maps to the
env
gene, which encodes the surface glycoprotein responsible for binding and fusion of virus to host cells. To understand how the envelope protein might be involved in the induction of disease, we examined the regional and temporal expression of Cas-Br-E Env protein in the central nervous systems (CNS) of mice infected with the highly neurovirulent chimeric virus FrCas
E
. We observed that multiple isoforms of Cas-Br-E Env were expressed in the CNS, with different brain regions exhibiting unique patterns of processed Env glycoprotein. Specifically, the expression of gp70 correlated with regions showing microglial infection and spongiform neurodegeneration. In contrast, regions high in neuronal infection and without neurodegenerative changes (the cerebellum and olfactory bulb) were characterized by a gp65 Env protein isoform. Sedimentation analysis of brain region extracts indicated that gp65 rather than gp70 was incorporated into virions. Biochemical analysis of the Cas-Br-E Env isoforms indicated that they result from differential processing of N-linked sugars. Taken together, these results indicate that differential posttranslational modification of the Cas-Br-E Env is associated with a failure to incorporate certain Env isoforms into virions in vivo, suggesting that defective viral assembly may be associated with the induction of spongiform neurodegeneration.
Diminished Human Immunodeficiency Virus Type 1 Reverse Transcription and Nuclear Transport in Primary Macrophages Arrested in Early G
1
Phase of the Cell Cycle
Authors: Kootstra Neeltje A. , Zwart Bianca M. , Schuitemaker Hanneke 
Published: July 2002

ABSTRACT

Previously, we and others have demonstrated that the process of reverse transcription of human immunodeficiency virus type 1 (HIV-1) is disturbed in nondividing macrophages and quiescent T lymphocytes. Here we analyzed which phase of the cell cycle in macrophages is crucial for early steps in the HIV-1 replication cycle. HIV-1 Ba-L-inoculated macrophages arrested early in the G
1
phase by
n
-butyrate contained incomplete products of reverse transcription. In gamma-irradiated macrophages, reverse transcription was successfully completed but proviral integration could not be detected. In these cells, nuclear import was disturbed as reflected by the absence of two-long-terminal-repeat circles. In macrophages arrested late in G
1
phase by aphidicolin or 5,6-dichloro-1-β-
d
-ribofuranosyl-benzimidazole (DRB), reverse transcription was unaffected. Proviral integration occurred efficiently in DRB-treated macrophages, whereas integrated proviral DNA could not be detected after aphidicolin treatment. Arrest at G
2
phase of the cell cycle by nocodazole did not affect reverse transcription or proviral integration. Treatment of macrophages with hydroxyurea (HU), which reduces the intracellular deoxynucleoside triphosphate (dNTP) pool by blocking the de novo synthesis of dNTP, resulted in a dose-dependent inhibition of HIV-1 reverse transcription. This could partially be restored by the addition of nucleoside precursors. Addition of nucleoside precursors enhanced both reverse transcription and cell proliferation. However, the disturbed reverse transcription observed in the nonproliferating and
n
-butyrate-treated macrophages could not be restored by addition of nucleoside precursors. Similar to observations in quiescent T lymphocytes, incomplete proviral DNA species were arrested in the cytoplasm of the macrophages. Our results indicate that also in primary macrophages the intracellular nucleotide pools and other cellular factors that coincide with late G
1
phase of the cell cycle may contribute to efficient reverse transcription and nuclear localization.
Most Retroviral Recombinations Occur during Minus-Strand DNA Synthesis
Authors: Zhang Jiayou , Tang Ling-Yun , Li Ting , Ma Yan , Sapp Christy M. 
Published: July 2002

ABSTRACT

Retroviral RNA molecules are plus, or sense in polarity, equivalent to mRNA. During reverse transcription, the first strand of the DNA molecule synthesized is minus-strand DNA. After the minus strand is polymerized, the plus-strand DNA is synthesized using the minus-strand DNA as the template. In this study, a helper cell line that contains two proviruses with two different mutated
gfp
genes was constructed. Recombination between the two frameshift mutant genes resulted in a functional
gfp
. If recombination occurs during minus-strand DNA synthesis, the plus-strand DNA will also contain the functional sequence. After the cell divides, all of its offspring will be green. However, if recombination occurs during plus-strand DNA synthesis, then only the plus-strand DNA will contain the wild-type
gfp
sequence and the minus-strand DNA will still carry the frameshift mutation. The double-stranded DNA containing this mismatch was subsequently integrated into the host chromosomal DNA of D17 cells, which were unable to repair the majority of mismatches within the retroviral double-strand DNA. After the cell divided, one daughter cell contained the wild-type
gfp
sequence and the other daughter cell contained the frameshift mutation in the
gfp
sequence. Under fluorescence microscopy, half the cells in the offspring were green and the other half of the cells were colorless or clear. Thus, we demonstrated that more than 98%, if not all, retroviral recombinations occurred during minus-strand DNA synthesis.