bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stress singularities in classical elasticity–I: Removal, interpretation, and analysis
Authors: Sinclair GB 
Published: October 2004

AbstractThis review article has two parts, published in separate issues of this journal, which consider the stress singularities that occur in linear elastostatics. In the present Part I, after a brief review of the singularities that attend concentrated loads, attention is focused on the singularities that occur away from such loading, and primarily on 2D configurations. A number of examples of these singularities are given in the Introduction. For all of these examples, it is absolutely essential that the presence of singularities at least be recognized if the stress fields are to be used in attempts to ensure structural integrity. Given an appreciation of a stress singularity’s occurrence, there are two options open to the stress analyst if the stress analysis is to actually be used. First, to try and improve the modeling so that the singularity is removed and physically sensible stresses result. Second, to try and interpret singularities that persist in a physically meaningful way. Section 2 of the paper reviews avenues available for the removal of stress singularities. At this time, further research is needed to effect the removal of all singularities. Section 3 of the paper reviews possible interpretations of singularities. At this time, interpretations using the singularity coefficient, or stress intensity factor, would appear to be the best available. To implement an approach using stress intensity factors in a general context, two types of companion analysis are usually required: analytical asymptotics to characterize local singular fields; and numerical analysis to capture participation in global configurations. Section 4 of the paper reviews both types of analysis. At this time, methods for both are fairly well developed. Studies in the literature which actually effect asymptotic analyses of specific singular configurations will be considered in Part II of this review article. The present Part I has 182 references.
The Amino Terminus of Epstein-Barr Virus (EBV) Nuclear Antigen 1 Contains AT Hooks That Facilitate the Replication and Partitioning of Latent EBV Genomes by Tethering Them to Cellular Chromosomes
Authors: Sears John , Ujihara Maki , Wong Samantha , Ott Christopher , Middeldorp Jaap , Aiyar Ashok 
Published: October 2004

ABSTRACT

During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin,
oriP
. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of
oriP
plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition
oriP
plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of
oriP
plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.
The N- and C-Terminal Domains of the NS1 Protein of Influenza B Virus Can Independently Inhibit IRF-3 and Beta Interferon Promoter Activation
Authors: Donelan Nicola R. , Dauber Bianca , Wang Xiuyan , Basler Christopher F. , Wolff Thorsten , García-Sastre Adolfo 
Published: October 2004

ABSTRACT
The NS1 proteins of influenza A and B viruses (A/NS1 and B/NS1 proteins) have only ∼20% amino acid sequence identity. Nevertheless, these proteins show several functional similarities, such as their ability to bind to the same RNA targets and to inhibit the activation of protein kinase R in vitro. A critical function of the A/NS1 protein is the inhibition of synthesis of alpha/beta interferon (IFN-α/β) during viral infection. Recently, it was also found that the B/NS1 protein inhibits IFN-α/β synthesis in virus-infected cells. We have now found that the expression of the B/NS1 protein complements the growth of an influenza A virus with A/NS1 deleted. Expression of the full-length B/NS1 protein (281 amino acids), as well as either its N-terminal RNA-binding domain (amino acids 1 to 93) or C-terminal domain (amino acids 94 to 281), in the absence of any other influenza B virus proteins resulted in the inhibition of IRF-3 nuclear translocation and IFN-β promoter activation. A mutational analysis of the truncated B/NS1(1-93) protein showed that RNA-binding activity correlated with IFN-β promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation, as determined by its nuclear translocation, and of IFN-α/β synthesis than wild-type influenza B virus. Our results support the hypothesis that the NS1 protein of influenza B virus plays an important role in antagonizing the IRF-3- and IFN-induced antiviral host responses to virus infection.
Viral Evolution and Interferon Resistance of Hepatitis C Virus RNA Replication in a Cell Culture Model
Authors: Sumpter Rhea , Wang Chunfu , Foy Eileen , Loo Yueh-Ming , Gale Michael 
Published: October 2004

ABSTRACT
Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-β production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-α treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.
Differential Effects on Human Immunodeficiency Virus Type 1 Replication by α-Defensins with Comparable Bactericidal Activities
Authors: Tanabe Hiroki , Ouellette Andre J. , Cocco Melanie J. , Robinson W. Edward 
Published: October 2004

ABSTRACT
In addition to their antibacterial activities, certain antimicrobial peptides inactivate enveloped viruses, including the human immunodeficiency virus (HIV). To determine whether peptide bactericidal activities are predictive of antiviral activity, the anti-HIV properties of recombinant human α-defensin 5, mouse α-defensins, cryptdins (Crp) 3 and 4, and rhesus macaque myeloid α-defensins (RMADs) 3 and 4 were determined in vitro. The peptides, purified to homogeneity, had equivalent bactericidal activities that were similar to those of the native molecules. Nuclear magnetic resonance spectroscopy showed RMAD-4 and Crp3 had characteristic α-defensin tridisulfide arrays. Of the peptides analyzed, only RMAD-4 inhibited HIV infectivity at 150 μg/ml, and Crp3 unexpectedly increased HIV replication. Quantitative real-time PCRs for minus-strand strong stop DNA and complete viral cDNA synthesis were used to distinguish between preentry and postentry anti-HIV effects by RMAD-4. Viral exposure to RMAD-4 for 1 h prior to infection reduced HIV minus-strand strong stop DNA and HIV cDNA by 4- to 20-fold during the first round of replication, showing that RMAD-4-exposed virions were not entering cells during the first 24 h. On the other hand, when RMAD-4 was added coincident with HIV inoculation, no anti-HIV activity was detected. Viral exposure to Crp3 resulted in a threefold increase in both HIV minus-strand strong stop DNA and HIV cDNA over the first round of replication. Therefore, two α-defensins, RMAD-4 and Crp3, inhibit or augment HIV replication, respectively, by mechanisms that precede reverse transcription.
Genome-Wide Analyses of Avian Sarcoma Virus Integration Sites
Authors: Narezkina Anna , Taganov Konstantin D. , Litwin Samuel , Stoyanova Radka , Hayashi Junpei , Seeger Christoph , Skalka Anna Marie , Katz Richard A. 
Published: October 2004

ABSTRACT
The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 avian sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.
A Novel Approach for Producing Lentiviruses That Are Limited to a Single Cycle of Infection
Authors: Evans David T. , Bricker Jennifer E. , Desrosiers Ronald C. 
Published: October 2004

ABSTRACT

We have devised a novel approach for producing simian immunodeficiency virus (SIV) strains and, potentially, human immunodeficiency virus type 1 (HIV-1) strains that are limited to a single cycle of infection. Unlike previous lentiviral vectors, our single-cycle SIV is capable of expressing eight of the nine viral gene products and infected cells release immature virus particles that are unable to complete subsequent rounds of infection. Single-cycle SIV (scSIV) was produced by using a two-plasmid system specifically designed to minimize the possibility of generating replication-competent virus by recombination or nucleotide reversion. One plasmid carried a full-length SIV genome with three nucleotide substitutions in the
gag-pol
frameshift site to inactivate Pol expression. To ensure inactivation of Pol and to prevent the recovery of wild-type virus by nucleotide reversion, deletions were also introduced into the viral
pol
gene. In order to provide Gag-Pol in
trans
, a Gag-Pol-complementing plasmid that included a single nucleotide insertion to permanently place
gag
and
pol
in the same reading frame was constructed. We also mutated the frameshift site of this Gag-Pol expression construct so that any recombinants between the two plasmids would remain defective for replication. Cotransfection of both plasmids into 293T cells resulted in the release of Gag-Pol-complemented virus that was capable of one round of infection and one round of viral gene expression but was unable to propagate a spreading infection. The infectivity of scSIV was limited by the amount of Gag-Pol provided in
trans
and was dependent on the incorporation of a functional integrase. Single-cycle SIV produced by this approach will be useful for addressing questions relating to viral dynamics and viral pathogenesis and for evaluation as an experimental AIDS vaccine in rhesus macaques.
Functional Analysis of the Noncoding Regions of the Uukuniemi Virus (
Bunyaviridae
) RNA Segments
Authors: Flick Kirsten , Katz Anna , Överby Anna , Feldmann Heinz , Pettersson Ralf F. , Flick Ramon 
Published: October 2004

ABSTRACT

The role of the variable portion of the noncoding regions (NCRs) of the three
Bunyaviridae
RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for
Uukuniemi
(UUK)
virus
, genus
Phlebovirus
(11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5′ and 3′ ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses.
Translation of Duck Hepatitis B Virus Reverse Transcriptase by Ribosomal Shunting
Authors: Sen Nandini , Cao Feng , Tavis John E. 
Published: October 2004

ABSTRACT
The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5′ end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5′ end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.
Influence of
gag
on Human Immunodeficiency Virus Type 1 Species-Specific Tropism
Authors: Ikeda Yasuhiro , Ylinen Laura M. J. , Kahar-Bador Maria , Towers Greg J. 
Published: October 2004

ABSTRACT

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is due in part to dominant acting restriction factors in humans (Ref1) and monkeys (Lv1). Here we show that
gag
encodes determinants of species-specific lentiviral infection, related in part to such restriction factors. Interaction between capsid and host cyclophilin A (CypA) protects HIV-1 from restriction in human cells but is essential for maximal restriction in simian cells. We show that sequence variation between HIV-1 isolates leads to variation in sensitivity to restriction factors in human and simian cells. We present further evidence for the importance of target cell CypA over CypA packaged in virions, specifically in the context of gp160 pseudotyped HIV-1 vectors. We also show that sensitivity to restriction is controlled by an H87Q mutation in the capsid, implicated in the immune control of HIV-1, possibly linking immune and innate control of HIV-1 infection.
The Putative Zinc Finger of the Human Cytomegalovirus IE2 86-Kilodalton Protein Is Dispensable for DNA Binding and Autorepression, Thereby Demarcating a Concise Core Domain in the C Terminus of the Protein
Authors: Asmar Jasmin , Wiebusch Lüder , Truss Matthias , Hagemeier Christian 
Published: October 2004

ABSTRACT
The IE2 86-kDa gene product is an essential regulatory protein of human cytomegalovirus (HCMV) with several functions, including transactivation, negative autoregulation, and cell cycle regulation. In order to understand the physiological significance of each of the IE2 functions, discriminating mutants of IE2 are required that can be tested in a viral background. However, no such mutants of IE2 are available, possibly reflecting structural peculiarities of the large and ill-defined C-terminal domain of IE2. Here, we revisited the C-terminal domain by analyzing IE2 mutants for transactivation, DNA binding, autoregulation, and cell cycle regulation in parallel. We found it to contain an unexpectedly concise core domain (amino acids 450 to 544) that is defined by its absolute sensitivity to any kind of mutation. In contrast, the region adjacent to the core (amino acids 290 to 449) generally tolerates mutations much better. Although it contributes more specific sequence information to distinct IE2 activities, none of the mutations analyzed abolished any particular function. The core is demarcated from the adjacent region by the putative zinc finger region (amino acids 428 to 452). Surprisingly, the deletion of the putative zinc finger region from IE2 revealed that this region is entirely dispensable for any of the IE2 functions tested here in transfection assays. Our work supports the view that the 100 amino acids of the core domain hold the key to most functions of IE2. A systematic, high-density mutational analysis of this region may identify informative mutants discriminating between various IE2 functions that can then be tested in a viral background.
Endocytic Host Cell Machinery Plays a Dominant Role in Intracellular Trafficking of Incoming Human Immunodeficiency Virus Type 1 in Human Placental Trophoblasts
Authors: Vidricaire Gaël , Imbeault Michael , Tremblay Michel J. 
Published: October 2004

ABSTRACT
Vertical transmission of human immunodeficiency virus type 1 (HIV-1) is the primary cause of infection by this retrovirus in infants. In this study, we report for the first time that there is a correlation between endocytic uptake of HIV-1 and virus gene expression in polarized trophoblasts. To shed light on the relationship between endocytosis and the fate of HIV-1 in polarized trophoblasts, the step-by-step movements of HIV-1 within the endocytic compartments were tracked by confocal imaging. Incoming virions were initially located in early endosomes. As time progressed, virions accumulated in late endosomes. HIV-1 was also found in apical recycling endosomes and at the basolateral pole. Experiments performed with indicator cells revealed that HIV-1 is recycled and transcytosed. These data indicate that the intracellular trafficking of HIV-1 upon entry into polarized human trophoblasts is a complex process which requires the active participation of the endocytic host cell machinery.
Transmission Study of Andes Hantavirus Infection in Wild Sigmodontine Rodents
Authors: Padula P. , Figueroa R. , Navarrete M. , Pizarro E. , Cadiz R. , Bellomo C. , Jofre C. , Zaror L. , Rodriguez E. , Murúa R. 
Published: October 2004

ABSTRACT

Our study was designed to contribute to an understanding of the timing and conditions under which transmission of Andes hantavirus in
Oligoryzomys longicaudatus
reservoir populations takes place. Mice were caged in test habitats consisting of steel drums containing holding cages, where seronegative rodents were exposed to wild seropositive individuals by freely sharing the same cage or being separated by a wire mesh. Tests were also performed for potential viral transmission to mice from excrement-tainted bedding in the cages. Andes virus transmitted efficiently; from 130 attempts with direct contact, 12.3% resulted in virus transmission. However, if we consider only those rodents that proved to be infectious, from 93 attempts we obtained 16 infected animals (17.2%). Twelve of them resulted from intraspecies
O. longicaudatus
encounters where male mice were differentially affected and 4 resulted from
O. longicaudatus
to
Abrothrix olivaceus
. Experiments using
Abrothrix longipilis
as receptors were not successful. Transmission was not observed between wire mesh-separated animals, and mice were not infected from excrement-tainted bedding. Bites seemed not to be a requisite for oral transmission. Genomic viral RNA was amplified in two out of three saliva samples from seropositive rodents, but it was not detected in urine samples obtained by vesicle puncture from two other infected rodents. Immunohistochemistry, using antibodies against Andes (AND) hantavirus proteins, revealed strong reactions in the lung and salivary glands, supporting the possibility of oral transmission. Our study suggests that AND hantavirus may be principally transmitted via saliva or saliva aerosols rather than via feces and urine.