bluuebunny/crossref_metadata_2025_split huggingface dataset:
Half-Life of the Duck Hepatitis B Virus Covalently Closed Circular DNA Pool In Vivo following Inhibition of Viral Replication
Authors: Addison William R. , Walters Kathie-Anne , Wong Winnie W. S. , Wilson John S. , Madej Danuta , Jewell Lawrence D. , Tyrrell D. Lorne J. 
Published: July 2002

ABSTRACT
Covalently closed circular DNA (cccDNA) is a crucial intermediate in the replication of hepadnaviruses. We inhibited the replication of duck hepatitis B virus in congenitally infected ducks with a combination of lamivudine and a dideoxyguanosine prodrug. Inhibition of viral replication should prevent renewal of the cccDNA pool, and its decay was measured in liver biopsy samples collected over a 5-month period. In three ducks, the cccDNA pools declined exponentially, with half-lives ranging from 35 to 57 days. In two others, the pools declined exponentially for about 70 days but then stabilized at about 6 copies/diploid genome. The selection of drug-resistant virus mutants is an unlikely explanation for this unexpected stabilization of cccDNA levels. Liver sections stained for the cell division marker PCNA showed that animals in which cccDNA loss was continuous had significantly greater numbers of PCNA-positive nuclei than did those animals in which cccDNA levels had plateaued.
Role of the Varicella-Zoster Virus gB Cytoplasmic Domain in gB Transport and Viral Egress
Authors: Heineman Thomas C. , Hall Susan L. 
Published: July 2002

ABSTRACT
To study the function of the varicella-zoster virus (VZV) gB cytoplasmic domain during viral infection, we produced a VZV recombinant virus that expresses a truncated form of gB lacking the C-terminal 36 amino acids of its cytoplasmic domain (VZV gB-36). VZV gB-36 replicates in noncomplementing cells and grows at a rate similar to that of native VZV. However, cells infected with VZVgB-36 form extensive syncytia compared to the relatively small syncytia formed during native VZV infection. In addition, electron microscopy shows that very little virus is present on the surfaces of cells infected with VZV gB-36, while cells infected with native VZV exhibit abundant virions on the cell surface. The C-terminal 36 amino acids of the gB cytoplasmic domain have been shown in transfection-based experiments to contain both an endoplasmic reticulum-to-Golgi transport signal (the C-terminal 17 amino acids) and a consensus YXXφ (where Y is tyrosine, X is any amino acid, and φ is any bulky hydrophobic amino acid) signal sequence (YSRV) that mediates the internalization of gB from the plasma membrane. As predicted based on these data, gB-36 expressed during the infection of cultured cells is transported inefficiently to the Golgi. Despite lacking the YSRV signal sequence, gB-36 is internalized from the plasma membrane; however, in contrast to native gB, it fails to localize to the Golgi. Therefore, the C-terminal 36 amino acids of the VZV gB cytoplasmic domain are required for normal viral egress and for both the pre- and post-Golgi transport of gB.
Regions of the Herpes Simplex Virus Scaffolding Protein That Are Important for Intermolecular Self-Interaction
Authors: Preston Valerie G. , McDougall Iris M. 
Published: July 2002

ABSTRACT
The herpes simplex virus type 1 (HSV-1) scaffolding protein encoded by gene UL26.5 promotes the formation of the icosahedral capsid shell through its association with the major capsid protein VP5 and through intermolecular interactions with itself. Inside the capsid shell, the UL26.5 product together with the maturational protease, a minor protein, form a spherical structure which is broken down and released from the capsid during packaging of the viral genome. Selected residues from four internal regions of the HSV-1 scaffolding protein that have significant conservation of amino acids within the scaffolding proteins of alphaherpesviruses were mutated, and the properties of the proteins were examined. Only the HSV-1 scaffolding protein with mutations in the conserved N-terminal domain showed reduced interaction with the varicella-zoster virus homologue in a cell-based immunofluorescence assay, providing the first evidence that this domain in the HSV-1 protein is likely to be involved in intermolecular self-interaction. Scaffolding protein with mutations in this domain or in either of two other domains failed to assemble into scaffold-like particles but retained the ability to self-interact, although the aggregates were significant smaller than most of the aggregates formed by the wild-type protein. These results suggest that there are multiple domains involved in the intermolecular self-association of the HSV-1 scaffolding protein that can act independently of one another. This conclusion was supported by the observation that none of the mutant proteins with lesions in an individual domain, including a protein with mutations in a central region previously implicated in self-interaction (A. Pelletier, F. Dô, J. J. Brisebois, L. Lagacé, and M. G. Cordingley, J. Virol. 71:5197–5208, 1997), interfered with capsid assembly in a baculovirus expression system. A protein mutated in the central region and another conserved domain, both of which had been predicted to form coiled coils, was impaired for capsid formation but still retained the capacity to interact with VP5.
Nef-Mediated Resistance of Human Immunodeficiency Virus Type 1 to Antiviral Cytotoxic T Lymphocytes
Authors: Yang Otto O. , Nguyen Phuong Thi , Kalams Spyros A. , Dorfman Tanya , Göttlinger Heinrich G. , Stewart Sheila , Chen Irvin S. Y. , Threlkeld Steven , Walker Bruce D. 
Published: July 2002

ABSTRACTAlthough Nef has been proposed to effect the escape of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTL) through downmodulation of major histocompatibility complex class I molecules, little direct data have been presented previously to support this hypothesis. By comparingnef-competent andnef-deleted HIV-1 strains in an in vitro coculture system, we demonstrate that the presence of this viral accessory gene leads to impairment of the ability of HIV-1-specific CTL clones to suppress viral replication. Furthermore, inhibition by genetically modified CTL that do not require major histocompatibility complex class I-presented antigen (expressing the CD4 T-cell receptor [TCR] ζ-chain hybrid receptor) is similar for bothnef-competent and -deleted strains, indicating that Nef does not impair the effector functions of CTL but acts at the level of TCR triggering. In contrast, we note that another accessory gene,vpr, does not induce resistance of HIV-1 to suppression by CTL clones. We conclude that Nef (and not Vpr) contributes to functional HIV-1 immune evasion and that this effect is mediated by diminished antigen presentation to CTL.
Regulated Gene Expression in the Chicken Embryo by Using Replication-Competent Retroviral Vectors
Authors: Sato Noboru , Matsuda Kenji , Sakuma Chie , Foster Douglas N. , Oppenheim Ronald W. , Yaginuma Hiroyuki 
Published: July 2002

ABSTRACT

Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the
tet
regulatory elements also revealed doxycycline-dependent inducible GFP expression in the chicken embryo in ovo.
Construction of a Self-Excisable Bacterial Artificial Chromosome Containing the Human Cytomegalovirus Genome and Mutagenesis of the Diploid TRL/IRL13 Gene
Authors: Yu Dong , Smith Gregory A. , Enquist Lynn W. , Shenk Thomas 
Published: July 2002

ABSTRACT
The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.
Chronic Subclinical Prion Disease Induced by Low-Dose Inoculum
Authors: Thackray Alana M. , Klein Michael A. , Aguzzi Adriano , Bujdoso Raymond 
Published: July 2002

ABSTRACT

We have compared the transmission characteristics of the two mouse-adapted scrapie isolates, ME7 and Rocky Mountain Laboratory (RML), in tga20 mice. These mice express elevated levels of PrP protein compared to wild-type mice and display a relatively short disease incubation period following intracerebral prion inoculation. Terminal prion disease in tga20 mice induced by ME7 or RML was characterized by a distinct pattern of clinical signs and different incubation times. High-dose RML inoculated intracerebrally into tga20 mice induced the most rapid onset of clinical signs, with mice succumbing to terminal disease after only 58 ± 3 days. In contrast, high-dose ME7 gave a mean time to terminal disease of 74 ± 0 days. Histological examination of brain sections from prion-inoculated tga20 mice at terminal disease showed that ME7 gave rise to a more general and extensive pattern of vacuolation than RML. Low-dose inoculum failed to induce terminal disease but did cause preclinical symptoms, including the appearance of reversible clinical signs. Some mice oscillated between showing no clinical signs and early clinical signs for many months but never progressed to terminal disease. Brain tissue from these mice with chronic subclinical prion disease, sacrificed at >200 days postinoculation, contained high levels of infectivity and showed the presence of PrP
Sc
. Parallel analysis of brain tissue from mice with terminal disease showed similar levels of infectivity and detectable PrP
Sc
. These results show that high levels of infectivity and the presence of the abnormal isomer of PrP can be detected in mice with subclinical disease following low-dose prion inoculation.
Abrogation of Ref1 Retrovirus Restriction in Human Cells
Authors: Towers Greg , Collins Mary , Takeuchi Yasuhiro 
Published: July 2002

ABSTRACT
We have previously described postentry restriction of murine leukemia virus in mammals. Here we characterize the block in human cells. Restricted infection kinetics are multiple hit at high virus dose, and restriction is abrogated by preexposure to restricted virus. We hypothesize that restricted capsid can titrate out the restriction factor.
Specific Inactivation of Inhibitory Sequences in the 5′ End of the Human Papillomavirus Type 16 L1 Open Reading Frame Results in Production of High Levels of L1 Protein in Human Epithelial Cells
Authors: Collier Brian , Öberg Daniel , Zhao Xiaomin , Schwartz Stefan 
Published: July 2002

ABSTRACT
The expression of human papillomavirus type 16 late genes encoding virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We wish to understand the molecular mechanisms that determine the levels of expression of the human papillomavirus type 16 late genes. We have previously shown that the L1 coding region contains inhibitory sequences. Here we extend previous findings to show that the 5′ end of the L1 gene contains strong inhibitory sequences but that the 3′ end does not. We show that the first 514 nucleotides of the L1 coding region contain multiple inhibitory elements that act independently of one another and that the major inhibitory element is located within the first 129 nucleotides of the L1 gene. Introduction of point mutations in the inhibitory elements in the 5′ end of the L1 gene which altered the RNA sequence without affecting the protein sequence specifically inactivated the inhibitory elements and resulted in production of high levels of human papillomavirus type 16 L1 mRNA and protein in human epithelial cells. Furthermore, we show that inhibitory sequences are present in the L1 coding regions of multiple human papillomavirus types, demonstrating that these elements are conserved among the human papillomaviruses, and suggest that they have an important function in the viral life cycle.
Structural Analysis of the Hepatitis C Virus RNA Polymerase in Complex with Ribonucleotides
Authors: Bressanelli Stéphane , Tomei Licia , Rey Félix A. , De Francesco Raffaele 
Published: July 2002

ABSTRACT
We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 Å away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-Å resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage φ6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the φ6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis.
Human Immunodeficiency Virus cDNA Metabolism: Notable Stability of Two-Long Terminal Repeat Circles
Authors: Butler Scott L. , Johnson Erik P. , Bushman Frederic D. 
Published: July 2002

ABSTRACT
Early steps of retroviral replication involve reverse transcription of the viral RNA to yield a linear double-stranded cDNA copy and then integration of the viral cDNA into a chromosome of the host cell. A portion of the viral cDNA can also follow nonproductive pathways in which it becomes circularized. In one pathway, the ends of the linear cDNA become joined together by the cellular nonhomologous DNA end-joining system to form two-long terminal repeat (2-LTR) circles. It has been argued that 2-LTR circles are quickly degraded in human immunodeficiency virus (HIV)-infected cells, allowing the presence of 2-LTR circles to be used as a marker for ongoing de novo infection in patients. Following this idea, detection of 2-LTR circles in patients undergoing successful highly active antiretroviral therapy has led to the proposal that viral replication persists despite treatment. We have used fluorescence-monitored PCR (Taqman) to quantitate the metabolism of HIV cDNA early after infection. Contrary to previous work, we find that 2-LTR circles are actually quite stable in experiments where confounding variables are controlled. Thus, studies relying on the lability of 2-LTR circles are open to reinterpretation. We also used the quantitative PCR methods to analyze the effects of MG132, a proteasome inhibitor, which revealed that viral complexes containing mostly completed cDNAs are the primary substrates for proteasome-mediated degradation.
Improved Primate Foamy Virus Vectors and Packaging Constructs
Authors: Heinkelein Martin , Dressler Marco , Jármy Gergely , Rammling Matthias , Imrich Horst , Thurow Jana , Lindemann Dirk , Rethwilm Axel 
Published: July 2002

ABSTRACT

Foamy virus (FV) vectors that have minimal
cis
-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV
gag
gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5′ splice donor (SD) site located in the R region identified this sequence element as responsible for regulating
gag
gene expression by an unidentified mechanism. Replacement of the FV 5′ SD with heterologous splice sites enabled expression of the
gag
and
pol
genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5′ SD or an unrelated intron sequence was substituted for the FV 5′ untranslated region and in which
gag
expression and
pol
expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.
Fusion-Defective Gibbon Ape Leukemia Virus Vectors Can Be Rescued by Homologous but Not Heterologous Soluble Envelope Proteins
Authors: Farrell Karen B. , Ting Yuan-Tsang , Eiden Maribeth V. 
Published: July 2002

ABSTRACTMurine leukemia virus (MLV)-derived envelope proteins containing alterations in or adjacent to the highly conserved PHQ motif present at the N terminus of the envelope surface subunit (SU) are incorporated into vector particles but are not infectious due to a postbinding block to viral entry. These mutants can be rendered infectious by the addition of soluble receptor-binding domain (RBD) proteins in the culture medium. The RBD proteins that rescue the infectivity of these defective MLV vectors can be derived from the same MLV or from other MLVs that use distinct receptors to mediate entry. We have now constructed functional immunologically reactive gibbon ape leukemia virus (GALV) envelope proteins, tagged with a feline leukemia virus (FeLV)-derived epitope tag, which are efficiently incorporated into infectious particles. Tagged GALV envelope proteins bind specifically to cells expressing the phosphate transporter protein Pit1, demonstrating for the first time that Pit1 is the binding receptor for GALV and not a coreceptor or another type of GALV entry factor. We have also determined that GALV particles bearing SU proteins with an insertion C-terminal to the PHQ motif (GALV I10) bind Pit1 but fail to infect cells. Incubation with soluble GALV RBD renders GALV I10particles infectious, whereas incubation with soluble RBDs from MLV or FeLV-B does not. This finding is consistent with the results obtained by Lauring et al. using FeLV-T, a virus that employs Pit1 as a receptor but requires soluble FeLV RBD for entry. MLV and GALV RBDs are not able to render FeLV-T infectious (A. S. Lauring, M. M. Anderson, and J. Overbaugh, J. Virol. 75:8888-8898, 2001). Together, these results suggest that fusion-defective FeLV-T and GALV are restricted to homologous RBD rescue of infectivity.
Kinetics and Mechanism of Ligand Substitutionin Triglycinatovanadium(III) by EDTA and NTA in Aqueous Solutions
Authors: Ikeda Yasuhisa , Hassan Refat M. , Abd El-Fattah Moustafa M. , Park Yul Y. , Tomiyasu Hiroshi 
Published: July 2002

The kinetics of ligand substitution reactions in triglycinatovanadium(III) by EDTA and NTA as multidentate ligands were investigated at a constant ionic strength of 2.5 mol dm-3 spectrophotometrically. The substitutions were second-order reactions, first order in both reactants. Their rate could be expressed in the form r = k [V(gly)3] [EDTA(NTA)] where k = 2.62 .10-2 and 6.96 .10-2 mol-1 dm3 s-1 at 25 °C for EDTA and NTA, respectively. The results obtained at different pH indicated that [H2edta]2- and [Hnta]2- ions were the most reactive species in the substitution. Kinetic parameters have been evaluated and a tentative mechanism of the reaction has been proposed.
N6-Methoxyadenine-Pyrimidine Base Pairs as Substrates for the Mismatch Repair System of Escherichia coli
Authors: Jiricny Josef 
Published: July 2002

The availability of nucleoside analogues with ambiguous base-pairing properties would be of considerable value in molecular biology. We have incorporated deoxynebularine [9-(2-deoxy-β-D-ribofuranosyl)purine, P], deoxyinosine [9-(2-deoxy-β-D-ribofuranosyl)-6-hydroxypurine, I) and [9-(2-deoxy-β-D-ribofuranosyl)-6-methoxyaminopurine,MeOA] into hexadecamer oligodeoxyribonucleotides and tested their behaviour in DNA•DNA hybridisationsin vitro, as well as in oligonucleotide-directed mutagenesis experimentsin vivo. The results showed that P behaved as an adenine analogue in all assays. Oligonucleotide duplexes containing I/C or I/T base pairs displayed similar thermal stabilities in DNA•DNA hybridisation experiments, however, during DNA synthesisin vitroandin vivo, hypoxanthine behaved strictly as a guanine analogue. OnlyMeOA was truly ambiguous in all assays. The1H NMR spectrum of the nucleoside demonstrated the existence of two distinct tautomeric forms in a ratio ofca8 : 2, implying that the base might pair with both C and T. Indeed, within the context of synthetic hexadecamer duplexes,MeOA/C andMeOA/T pairs brought about a similar thermal destabilisation, with the former base pair being only marginally less favoured. When used as hybridisation probes on single-stranded M13 DNA, theMeOA-containing hexadecamer oligonucleotides were shown to bind with similar efficiencies to target sequences containing either C or T opposite the analogue. Interestingly, whenMeOA is in the template strand during DNA replication, the polymerase III holoenzyme ofE. colireads it predominantly as a G, which indicates thatMeOA exists in B-DNA mostly as theanti-imino tautomer.