bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Deletion or Stringent Repression of the H3L Envelope Gene on Vaccinia Virus Replication
Authors: da Fonseca Flávio G. , Wolffe Elizabeth J. , Weisberg Andrea , Moss Bernard
Published: July 2002

The C-terminal membrane anchor protein encoded by the H3L open reading frame of vaccinia virus is located on the surfaces of intracellular mature virions. To investigate the role of the H3L protein, we constructed deletion (vH3Δ) and inducible (vH3i) null mutants. The H3L protein was not detected in lysates of cells infected with vH3Δ or vH3i in the absence of inducer. Under these conditions, plaques were small and round instead of large and comet shaped, indicative of decreased virus replication or cell-to-cell spread. The mutant phenotype was correlated with reduced yields of infectious intra- and extracellular virus in one-step growth experiments. The defect in vH3i replication could not be attributed to a role of the H3L protein in virus binding, internalization, or any event prior to late gene expression. Electron microscopic examination of cells infected with vH3Δ or vH3i in the absence of inducer revealed that virion assembly was impaired, resulting in a high ratio of immature to mature virus forms with an accumulation of crescent membranes adjacent to granular material and DNA crystalloids. The absence of the H3L protein did not impair the membrane localization of virion surface proteins encoded by the A27L, D8L, and L1R genes. The wrapping of virions and actin tail formation were not specifically blocked, but there was an apparent defect in low-pH-mediated syncytium formation that could be attributed to decreased virus particle production. The phenotypes of the H3L deletion and repression mutants were identical to each other but differed from those produced by null mutations of genes encoding other vaccinia virus membrane components.
Alphaherpesvirus Proteins Related to Herpes Simplex Virus Type 1 ICP0 Affect Cellular Structures and Proteins
Authors: Parkinson Jane , Everett Roger D.
Published: July 2002

The herpes simplex virus type 1 (HSV-1) immediate-early protein ICP0 interacts with several cellular proteins and induces the proteasome-dependent degradation of others during infection. In this study we show that ICP0 is required for the proteasome-dependent degradation of the ND10 protein Sp100 and, as with the other target proteins, the ICP0 RING finger domain is essential. Further, comparison of the kinetics and ICP0 domain requirements for the degradation of PMI and Sp100 suggests that a common mechanism is involved. Homologues of ICP0 are encoded by other members of the alphaherpesvirus family. These proteins show strong sequence homology to ICP0 within the RING finger domain but limited similarity elsewhere. Using transfection assays, we have shown that all the ICP0 homologues that we tested have significant effects on the immunofluorescence staining character of at least one of the proteins destabilized by ICP0, and by using a recombinant virus, we found that the equine herpesvirus ICP0 homologue induced the proteasome-dependent degradation of endogenous CENP-C and modified forms of PML and Sp100. However, in contrast to ICP0, the homologue proteins had no effect on the distribution of the ubiquitin-specific protease USP7 within the cell, consistent with their lack of a USP7 binding domain. We also found that ICP0 by itself could induce the abrogation of SUMO-1 conjugation and then the proteasome-dependent degradation of unmodified exogenous PML in transfected cells, thus demonstrating that other HSV-1 proteins are not required. Surprisingly, the ICP0 homologues were unable to cause these effects. Overall, these data suggest that the members of the ICP0 family of proteins may act via a similar mechanism or pathway involving their RING finger domain but that their intrinsic activities and effects on endogenous and exogenous proteins differ in detail.
Lymphatic Dissemination and Comparative Pathology of Recombinant Measles Viruses in Genetically Modified Mice
Authors: Mrkic Branka , Odermatt Bernhard , Klein Michael A. , Billeter Martin A. , Pavlovic Jovan , Cattaneo Roberto
Published: July 2002

The dissemination of the Edmonston measles virus (Ed-MV) vaccine strain was studied with genetically modified mice defective for the alpha/beta interferon receptor and expressing human CD46 with human-like tissue specificity and efficiency. A few days after intranasal infection, macrophages expressing Ed-MV RNA were detected in the lungs, in draining lymph nodes, and in the thymus. In lymph nodes, large syncytia which stained positive for viral RNA and for macrophage surface marker proteins were found and apoptotic cell death was monitored. In the thymus, smaller syncytia which stained positive for macrophage and dendritic cell markers were detected. Thus, macrophages appear to be the main vectors for dissemination of MV infection in these mice; human macrophages may have a similar function in the natural host. We then compared the pathogenicities of two recombinant viruses lacking the C or V nonstructural proteins to that of the parental strain, Ed-MV. These viruses were less effective in spreading through the lymphatic system and, unlike Ed-MV, were not detected in the liver. After intracerebral inoculation the recombinant viruses caused lethal disease less often than Ed-MV and induced distinctive patterns of gliosis and inflammation. Ed-MV was reisolated from brain tissue, but its derivatives were not. C- and V-defective viruses should be considered as more-attenuated MV vaccine candidates.
Replication of Lengthened Moloney Murine Leukemia Virus Genomes Is Impaired at Multiple Stages
Authors: Shin Nam-Hee , Hartigan-O'Connor Dennis , Pfeiffer Julie K. , Telesnitsky Alice
Published: July 2002

It has been assumed that RNA packaging constraints limit the size of retroviral genomes. This notion of a retroviral “headful” was tested by examining the ability of Moloney murine leukemia virus genomes lengthened by 4, 8, or 11 kb to participate in a single replication cycle. Overall, replication of these lengthened genomes was 5- to 10-fold less efficient than that of native-length genomes. When RNA expression and virion formation, RNA packaging, and early stages of replication were compared, long genomes were found to complete each step less efficiently than did normal-length genomes. To test whether short RNAs might facilitate the packaging of lengthy RNAs by heterodimerization, some experiments involved coexpression of a short packageable RNA. However, enhancement of neither long vector RNA packaging nor long vector DNA synthesis was observed in the presence of the short RNA. Most of the proviruses templated by 12 and 16 kb vectors appeared to be full length. Most products of a 19.2-kb vector contained deletions, but some integrated proviruses were around twice the native genome length. These results demonstrate that lengthy retroviral genomes can be packaged and that genome length is not strictly limited at any individual replication step. These observations also suggest that the lengthy read-through RNAs postulated to be intermediates in retroviral transduction can be packaged directly without further processing.
Basic Residues in Human Immunodeficiency Virus Type 1 Nucleocapsid Promote Virion Assembly via Interaction with RNA
Authors: Cimarelli Andrea , Sandin Sara , Höglund Stefan , Luban Jeremy
Published: July 2002

Retroviral Gag polyproteins drive virion assembly by polymerizing to form a spherical shell that lines the inner membrane of nascent virions. Deletion of the nucleocapsid (NC) domain of the Gag polyprotein disrupts assembly, presumably because NC is required for polymerization. Human immunodeficiency virus type 1 NC possesses two zinc finger motifs that are required for specific recognition and packaging of viral genomic RNA. Though essential, zinc fingers and genomic RNA are not required for virion assembly. NC promiscuously associates with cellular RNAs, many of which are incorporated into virions. It has been hypothesized that Gag polymerization and virion assembly are promoted by nonspecific interaction of NC with RNA. Consistent with this model, we found an inverse relationship between the number of NC basic residues replaced with alanine and NC's nonspecific RNA-binding activity, Gag's ability to polymerize in vitro and in vivo, and Gag's capacity to assemble virions. In contrast, mutation of NC's zinc fingers had only minor effects on these properties.
Biology of E1-Deleted Adenovirus Vectors in Nonhuman Primate Muscle
Authors: Zoltick Philip W. , Chirmule Narendra , Schnell Michael A. , Gao Guang-ping , Hughes Joseph V. , Wilson James M.
Published: July 2002

Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.
Spindle Cell Conversion by Kaposi's Sarcoma-Associated Herpesvirus: Formation of Colonies and Plaques with Mixed Lytic and Latent Gene Expression in Infected Primary Dermal Microvascular Endothelial Cell Cultures
Authors: Ciufo Dolores M. , Cannon Jennifer S. , Poole Lynn J. , Wu Frederick Y. , Murray Paul , Ambinder Richard F. , Hayward Gary S.
Published: July 2002

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that
de novo
KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.
Accumulation and Intranuclear Distribution of Unintegrated Human Immunodeficiency Virus Type 1 DNA
Authors: Bell Peter , Montaner Luis J. , Maul Gerd G.
Published: July 2002

The RNA genome of human immunodeficiency virus type 1 (HIV-1) is converted into DNA after infection in order to integrate into the host cell DNA. However, a large number of these reverse-transcribed genomes remain unintegrated in the nucleus of infected cells. Currently, there are no data available about the intranuclear distribution pattern of unintegrated HIV-1 DNA in relation to nuclear structures as observed on the single-cell level. In the present study, we investigated the intranuclear fate of unintegrated viral DNA in cell lines expressing CD4 and coreceptors (HOS-CD4.CCR5 and U373-MAGI-CXCR4
CEM
) infected with HIV-1 (strain 89.6). We used a novel approach to distinguish in situ unintegrated from integrated viral DNA by performing fluorescent in situ hybridization on cells in which stress-induced chromosome condensation had been induced, a procedure that contracts chromosomes independent of the cell cycle. Cells infected for 15 h accumulated large amounts of HIV-1 DNA which was located between the condensed chromosome strands, allowing the identification of this viral DNA as unintegrated. In contrast, in HeLa/LAV, a cell line carrying integrated HIV-1 genomes, the great majority of viral DNA colocalized with the cellular DNA. We show that unintegrated HIV-1 DNA does not evenly distribute within the host cell nucleus but tends to aggregate into clusters containing many copies of the viral genomes. The formation of these DNA clusters was independent of viral DNA replication and thus appeared to result solely from multiple infections. The DNA aggregates remained in the nuclei of infected cells for at least 25 h after the infection was stopped. The emergence of transcription sites, which most likely denote sites of the integrated provirus, lagged clearly behind the accumulation of viral DNA. These transcription foci could not be linked to unintegrated DNA molecules, suggesting that this DNA type is unable to transcribe, at least at levels comparable to those of integrated DNA. Neither unintegrated HIV-1 DNA nor transcription foci nor integrated DNA was observed to associate with nuclear domain 10 (ND10), a nuclear structure known to represent the site where several DNA viruses replicate and transcribe. Also, HIV-1 does not modify ND10 at early or late times of infection. There was no specific association of HIV-1 transcripts with splicing factor SC35 domains, in contrast to what has been reported for a number of both cellular and viral genes. Surprisingly, unintegrated HIV-1 DNA was found to accumulate within or in close association with SC35 domains, demonstrating a specific distribution of the viral DNA within the host cell nucleus. Taken together, our results demonstrate that unintegrated proviral HIV-1 DNA does not randomly localize within infected cells but preferentially aggregates in the nucleus within SC35 domains.
DNA Vaccination with the Hantaan Virus M Gene Protects Hamsters against Three of Four HFRS Hantaviruses and Elicits a High-Titer Neutralizing Antibody Response in Rhesus Monkeys
Authors: Hooper J. W. , Custer D. M. , Thompson E. , Schmaljohn C. S.
Published: July 2002

Four hantaviruses—Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus—are known to cause hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia. HTNV causes the most severe form of HFRS (5 to 15% case-fatality rate) and afflicts tens of thousands of people annually. Previously, we demonstrated that DNA vaccination with a plasmid expressing the SEOV M gene elicited neutralizing antibodies and protected hamsters against infection with SEOV and HTNV. Here, we report the construction and evaluation of a DNA vaccine that expresses the HTNV M gene products, G1 and G2. DNA vaccination of hamsters with the HTNV M gene conferred sterile protection against infection with HTNV, SEOV, and DOBV. DNA vaccination of rhesus monkeys with either the SEOV or HTNV M gene elicited high levels of neutralizing antibodies. These are the first immunogenicity data for hantavirus DNA vaccines in nonhuman primates. Because a neutralizing antibody response is considered a surrogate marker for protective immunity in humans, our protection data in hamsters combined with the immunogenicity data in monkeys suggest that hantavirus M gene-based DNA vaccines could protect humans against the most severe forms of HFRS.
Protective Immunity and Antibody-Secreting Cell Responses Elicited by Combined Oral Attenuated Wa Human Rotavirus and Intranasal Wa 2/6-VLPs with MutantEscherichia coliHeat-Labile Toxin in Gnotobiotic Pigs
Authors: Yuan Lijuan , Iosef Cristiana , Azevedo Marli S. P. , Kim Yunjeong , Qian Yuan , Geyer Annelise , Nguyen Trang Van , Chang Kyeong-Ok , Saif Linda J.
Published: July 2002

Two combined rotavirus vaccination regimens were evaluated in a gnotobiotic pig model of rotavirus infection and disease and were compared to previously tested rotavirus vaccination regimens. The first (AttHRV/VLP2×) involved oral inoculation with one dose of attenuated (Att) Wa human rotavirus (HRV), followed by two intranasal (i.n.) doses of a rotavirus-like particle (2/6-VLPs) vaccine derived from Wa (VP6) and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were coadministered with a mutantEscherichia coliheat-labile toxin, LT-R192G (mLT) adjuvant. For the second regimen (VLP2×/AttHRV), two i.n. doses of 2/6-VLPs+mLT were given, followed by one oral dose of attenuated Wa HRV. To compare the protective efficacy and immune responses induced by the combined vaccine regimens with individual rotavirus vaccine regimens, we included in the experiments the following vaccine groups: one oral dose of attenuated Wa HRV (AttHRV1× and Mock2×/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3×), three i.n. doses of 2/6-VLPs plus mLT (VLP3×), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3×), mLT alone, and mock-inoculated pigs. The isotype, magnitude, and tissue distribution of antibody-secreting cells (ASCs) in the intestinal and systemic lymphoid tissues were evaluated using an enzyme-linked immunospot assay. The AttHRV/VLP2× regimen stimulated the highest mean numbers of intestinal immunoglobulin A (IgA) ASCs prechallenge among all vaccine groups. This regimen induced partial protection against virus shedding (58%) and diarrhea (44%) upon challenge of pigs with virulent Wa HRV. The reverse VLP2×/AttHRV regimen was less efficacious than the AttHRV/VLP2× regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3× or InactHRV3× (no protection). In conclusion, the AttHRV/VLP2× vaccination regimen stimulated the strongest B-cell responses in the intestinal mucosal immune system at challenge and conferred a moderately high protection rate against rotavirus disease, indicating that priming of the mucosal inductive site at the portal of natural infection with a replicating vaccine, followed by boosting with a nonreplicating vaccine at a second mucosal inductive site, may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against various mucosal pathogens.
Biochemical Analyses of the Interactions between Human Immunodeficiency Virus Type 1 Vpr and p6
Gag
Authors: Jenkins Yonchu , Pornillos Owen , Rich Rebecca L. , Myszka David G. , Sundquist Wesley I. , Malim Michael H.
Published: July 2002

The nonstructural human immunodeficiency virus type 1 Vpr protein is packaged into progeny virions at significant levels (∼200 copies/virion). Genetic analyses have demonstrated that efficient Vpr packaging is dependent upon a leucine-X-X-leucine-phenylalanine (LXXLF) motif located in the p6
Gag
domain of the structural Gag polyprotein. Recombinant proteins spanning full-length Vpr (Vpr
1–97
) or the amino-terminal 71 amino acids (Vpr
1–71
) formed specific complexes with recombinant p6 proteins in vitro. Complex formation required an intact LXXLF motif and exhibited an intrinsic dissociation constant of ∼75 μM. Gel filtration and cross-linking analyses further revealed that Vpr
1–71
self-associated in solution. Our experiments demonstrate that Vpr can bind directly and specifically to p6 and suggest that oligomerization of both Vpr and Gag may serve to increase the avidity and longevity of Vpr-Gag complexes, thereby ensuring efficient Vpr packaging.
Transcription and RNA Replication of Tacaribe Virus Genome and Antigenome Analogs Require N and L Proteins: Z Protein Is an Inhibitor of These Processes
Authors: López Nora , Jácamo Rodrigo , Franze-Fernández Marı́a T.
Published: July 2002

Tacaribe virus (TV), the prototype of the New World group of arenaviruses, comprises a single phylogenetic lineage together with four South American pathogenic producers of hemorrhagic disease. The TV genome consists of two single-stranded RNA segments called S and L. A reconstituted transcription-replication system based on plasmid-supplied TV-like RNAs and TV proteins was established. Plasmid expression was driven by T7 RNA polymerase supplied by a recombinant vaccinia virus. Plasmids were constructed to produce TV S segment analogs containing the negative-sense copy of chloramphenicol acetyltransferase (CAT) flanked at the 5′ and 3′ termini by sequences corresponding to those of the 5′ and 3′ noncoding regions of the S genome (minigenome) or the S antigenome (miniantigenome). In cells expressing N and L proteins, input minigenome or miniantigenome produced, respectively, encapsidated miniantigenome or minigenome which in turn produced progeny minigenome or progeny miniantigenome. Both minigenome and miniantigenome in the presence of N and L mediated transcription, which was analyzed as CAT expression. Coexpression of the small RING finger Z (p11) protein was highly inhibitory to both transcription and replication mediated by the minigenome or the miniantigenome. The effect depended on synthesis of Z protein rather than on plasmid or the RNA and was not ascribed to decreased amounts of plasmid-supplied template or proteins (N or L). N and L proteins were sufficient to support full-cycle RNA replication of a plasmid-supplied S genome analog in which CAT replaced the N gene. Replication of this RNA was also inhibited by Z expression.
Biochemical Characterization of
Junonia coenia
Densovirus Nonstructural Protein NS-1
Authors: Ding Chuantian , Urabe Masashi , Bergoin Max , Kotin Robert M.
Published: July 2002

Junonia coenia
densovirus (JcDNV) is an autonomous parvovirus that infects the larvae of the common buckeye butterfly,
Junonia coenia
. Unlike vertebrate parvoviruses, the genes encoding the structural protein and nonstructural (NS) proteins of JcDNV are in opposite orientations; thus, each strand contains a sense and antisense open reading frame (ORF). The promoter at map position 93 controls expression of NS ORFs 2, 3, and 4, which encode three NS proteins, NS-1, NS-2, and NS-3. These proteins are likely to be involved in viral DNA replication, among other functions. In contrast to the nonstructural proteins of the vertebrate parvoviruses, the NS proteins of the
Densovirinae
have not been characterized. Here, we describe biochemical properties of the NS-1 protein of JcDNV. The NS-1 ORF was cloned in frame with the
Escherichia coli malE
gene, which encodes the bacterial maltose binding protein (MBP). Using electrophoretic mobility shift and DNase I protection assays, we identified the region of the JcDNV terminal sequence that is recognized specifically by the MBP-NS-1 fusion protein. The site consists of (GAC)
4
and is located on the A-A′ region of the terminal palindrome. In addition, the MBP-NS-1 fusion protein catalyzes the cleavage of single-stranded DNA (ssDNA) substrates derived from the JcDNV putative origin of replication, primarily at two sites in the motif 5′-G*TAT*TG-3′. One cleavage site is between the thymidine dinucleotide at positions 92 and 93 and the other site corresponds to thymidine at nucleotide 95; both sites are on the complementary strand of the sequence assigned GenBank accession number
A12984
. Cleavage of ssDNA is dependent on the presence of a divalent metal cofactor but does not require nucleoside triphosphate hydrolysis. Parvovirus NS proteins contain the phylogenically conserved Walker A- and B-site ATPase motifs. These sites in JcDNV NS-1 diverge from the consensus, yet despite these atypical motifs our analyses support that MBP-NS-1 has ATP-dependent helicase activity. These results indicate that JcDNV NS-1 possesses activities common to the superfamily of rolling-circle replication initiator proteins in general and the parvovirus replication proteins in particular, and they provide a basis for comparative analyses of the structure and function relationships among the parvovirus NS-1 equivalents.
Rous Sarcoma Virus Gag Protein-Oligonucleotide Interaction Suggests a Critical Role for Protein Dimer Formation in Assembly
Authors: Ma Yu May , Vogt Volker M.
Published: July 2002

The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, ΔMBDΔPR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the ΔMBDΔPR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.
Protein Kinase C-Independent Activation of the Epstein-Barr Virus Lytic Cycle
Authors: Gradoville Lyndle , Kwa David , El-Guindy Ayman , Miller George
Published: July 2002

The protein kinase C (PKC) pathway has been considered to be essential for activation of latent Epstein-Barr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene
BZLF1
, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activation in prototype cell lines that differed dramatically in their response to inducing agents. We determined whether PKC was involved in lytic cycle induction by histone deacetylase (HDAC) inhibitors. Consistent with prevailing views, B95-8 cells were activated into the lytic cycle by the phorbol ester TPA, via a PKC-dependent mechanism. B95-8 was not inducible by HDAC inhibitors such as
n
-butyrate and trichostatin A (TSA). Bisindolylmaleimide I, a selective PKC inhibitor, blocked lytic cycle activation in B95-8 cells in response to TPA. In marked contrast, in HH514-16 cells, the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors; TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in HH514-16 cells by
n
-butyrate or TSA. In an extensive exploration of the mechanism underlying these different responses we found that the variable role of the PKC pathway in the two cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, by differences in the start sites of immediate-early gene transcription, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp. While B95-8 cells contained more total PKC activity than did HH514-16 cells in an in vitro assay, another EBV-transformed marmoset lymphoblastoid cell line, FF41, in which the lytic cycle was not inducible by TPA, contained comparably high levels of PKC activity. Moreover, two marmoset lymphoblastoid cells lines in which the lytic cycle could not be triggered by TPA maintained the same profile of EBV latency proteins as B95-8 cells. Thus, the profile of EBV latency proteins did not account for susceptibility to induction by PKC agonists. PKC activation is neither obligatory nor sufficient for the switch between latency and lytic cycle gene expression of EBV in many cell backgrounds. Lytic cycle induction by HDAC inhibitors proceeds by a PKC-independent mechanism.
Asymmetric Requirement for Cholesterol in Receptor-Bearing but Not Envelope-Bearing Membranes for Fusion Mediated by Ecotropic Murine Leukemia Virus
Authors: Lu Xiongbin , Xiong Ying , Silver Jonathan
Published: July 2002

We show that fusion mediated by ecotropic murine leukemia virus envelope is dependent on cholesterol in receptor-bearing membranes. The effect is >10 times larger in insect cells than mammalian cells, probably because the former can be more extensively depleted of cholesterol. The fact that cholesterol is apparently not needed in envelope-bearing membranes suggests that it plays a role in an asymmetric step in membrane fusion and argues against a class of models in which cholesterol is important in symmetric fusion intermediates. The insect cell system has promise for clarifying the role of membrane rafts in other aspects of cell physiology.
Simian Immunodeficiency Virus Nef Protein Delays the Progression of CD4+T Cells through G1/S Phase of the Cell Cycle
Authors: Ndolo Thomas , Dhillon Navdeep K. , Nguyen Hau , Guadalupe Moraima , Mudryj Maria , Dandekar Satya
Published: July 2002

Human and simian immunodeficiency virus (HIV and SIV, respectively) infections are characterized by gradual depletion of CD4+T cells. The underlying mechanisms of CD4+T-cell depletion and HIV and SIV persistence are not fully determined. The Nef protein is expressed early in infection and is necessary for pathogenesis. Nef can cause T-cell activation and downmodulates cell surface signaling molecules. However, the effect of Nef on the cell cycle has not been well characterized. To determine the role of Nef in the cell cycle, we investigated whether the SIV Nef protein can modulate cell proliferation and apoptosis in CD4+Jurkat T cells. We developed a CD4+Jurkat T-cell line that stably expresses SIV Nef under the control of an inducible promoter. Alterations in cell proliferation were determined by flow cytometry using stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester and bromodeoxyuridine incorporation. Apoptotic cell death was measured by annexin V and propidium iodide staining. Our results demonstrated that SIV Nef inhibited Fas-induced apoptosis in these cells and that the mechanism involved upregulation of the Bcl-2 protein. SIV Nef suppressed CD4+T-cell proliferation by inhibiting the progression of cells into S phase of the cell cycle. Suppression involved an upregulation of cyclin-dependent kinase inhibitors p21 and p27 and the downregulation of cyclin D1and cyclin A. In summary, inhibition of apoptosis by Nef can lead to persistence of infected cells and can support viral replication. In addition, a Nef-mediated delay in cell cycle progression may contribute to CD4+T-cell anergy/depletion seen in HIV and SIV disease.
Brillouin-Wigner Perturbation Theory as a Possible more Effective Alternative to Many-Body Rayleigh-Schrodinger Perturbation Theory and Coupled Cluster Theory
Authors: Čársky Petr , Hrouda Vojtěch , Sychrovský Vladimír , Hubač Ivan , Babinec Peter , Mach Pavol , Urban Ján , Mášik Jozef
Published: July 2002

Brillouin-Wigner perturbation theory is revisited using the Lippmann-Schwinger equation and applied to the H2O molecule. The results obtained are examined from the view-point of the development of practical computational methods.
Chromatin structure of eukaryotic promoters: a changing perspective
Authors: Georgel Philippe T
Published: July 2002

Over the past few years, many studies have attempted to determine the role of nucleosomes as both positive and negative transcription regulators. The emphasis has mostly centered on chromatin remodeling activities and histone modifications, leaving the question of the influence of the higher-order structure out of the spotlight. Recent technical developments allowing direct measurements of size and mechanical properties of in vivo assembled chromatin may shed light on this poorly understood area. This article presents a brief summary of the current knowledge on transcription-dependent chromatin dynamics and how a rather simple agarose electrophoresis method may change the current view on structural changes linked to transcriptional activation of chromatin.Key words: chromatin, higher-order structure, quantitative agarose gel electrophoresis.
Glyconeogenic pathway in isolated skeletal muscles of rats
Authors: Xavier Analúcia Rampazzo , Salles Roselino José Eduardo de , Resano Neusa Maria Zanon , Garófalo Maria Antonieta Rissato , Migliorini Renato Helios , Kettelhut Isis do Carmo
Published: July 2002

Although the conversion of lactate to glycogen (glyconeogenesis) in muscle was demonstrated a long time ago, the biochemical reactions responsible for this process are still a controversial matter. In the present study, advantage was taken from the specific inhibition induced by phenylalanine on muscle pyruvate kinase (PK) to investigate the role of reverse PK activity in muscle glyconeogenesis. Addition of phenylalanine to the incubation medium of a preparation of isolated, intact skeletal muscles that maintain metabolic activity for several hours reduced by 50% the rate of incorporation of [14C]lactate or [14C]bicarbonate into muscle glycogen. Muscle extracts presented high levels of maximal activity of PK in the reverse direction, which was completely blocked in the presence of phenylalanine. In contrast, mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), did not affect the incorporation of14C from either lactate or bicarbonate into muscle glycogen. Maximal PEPCK activity was much lower in muscle extracts than in gluconeogenic or glyceroneogenic tissues and was suppressed in the presence of mercaptopicolinic acid. The data suggest that a reversal of the metabolic flux through the reaction catalyzed by PK contributes to the accumulation of lactate-derived glycogen that occurs in skeletal muscle under certain physiological conditions.Key words: lactate, glyconeogenesis, skeletal muscle, reverse pyruvate kinase reaction, phenylalanine.
GENERIC ∊-REMOVAL AND INPUT ∊-NORMALIZATION ALGORITHMS FOR WEIGHTED TRANSDUCERS
Authors: MOHRI MEHRYAR
Published: July 2002

We present a new generic ∊-removal algorithm for weighted automata and transducers defined over a semiring. The algorithm can be used with any semiring covered by our framework and works with any queue discipline adopted. It can be used in particular in the case of unweighted automata and transducers and weighted automata and transducers defined over the tropical semiring. It is based on a general shortest-distance algorithm that we briefly describe. We give a full description of the algorithm including its pseudocode and its running time complexity, discuss the more efficient case of acyclic automata, an on-the-fly implementation of the algorithm and an approximation algorithm in the case of the semirings not covered by our framework. We illustrate the use of the algorithm with several semirings. We also describe an input ∊-normalization algorithm for weighted transducers based on the general shortest-distance algorithm. The algorithm, which works with all semirings covered by our framework, admits an on-the-fly implementation.
NORM INEQUALITIES EQUIVALENT TO LÖWNER-HEINZ THEOREM
Authors: FURUTA TAKAYUKI
Published: July 2002

We give several norm inequalities equivalent to the famous Löwner-Heinz inequality.
ULTRAVIOLET LASER EMISSION FROM OPTICALLY PUMPED ZnO
Authors: MITRA ANIRBAN , THAREJA R. K.
Published: July 2002

We report UV laser emission from an optically pumped zinc oxide powder. The laser emission spectra depend on the angle of observation which is random. The lasing is attributed to coherent feedback due to recurrent light scattering in the powder.
Linearized chord diagrams and an upper bound for vassiliev invariants
Authors: Bollobás Béla , Riordan Oliver
Published: July 2002

Recently, Stoimenow [J. Knot Th. Ram. 7 (1998), 93–114] gave an upper bound on the dimension dn of the space of order n Vassiliev knot invariants, by considering chord diagrams of a certain type. We present a simpler argument which gives a better bound on the number of these chord diagrams, and hence on dn.
FRACTAL ANALYSIS OF US SETTLEMENT PATTERNS
Authors: SAMBROOK ROGER C. , VOSS RICHARD F.
Published: July 2002

Estimates of fractal mass dimension D are used to characterize the spatial distribution or clustering behavior of human settlements in Pennsylvania, Kansas and Utah. Uniform random and rectangular lattice models are then compared to actual settlement patterns using mass dimension as a quantitative measure of model validity. Data was taken from a United States Geographical Survey geo-coded census database. The distribution of pairwise distances between settlements is used to estimate fractal dimension D in the scaling range from 3 to 300 km. Novel analysis methods are shown to reduce small and large scale distortions and increase the range of scaling behaviors for estimating mass dimension. Results show that the settlement patterns have mass dimensions which vary systematically with the visual impression of clustering visible in maps of these regions: Kansas D = 1.9, Pennsylvania D = 1.7, Utah D = 1.4, Uniform Random and Rectangular Lattice D = 2. The results demonstrate the utility of mass dimension when used to compare patterns in areas of different size and with differing numbers of settlements. Such comparisons are often impossible using traditional statistical methods. We also demonstrate how neither model captures the clustering behavior of actual settlement patterns, and propose that a measure of mass dimension might prove useful in validating future models of settlement behavior.
QUICK NUMERICAL ASSESSMENT OF SKIN BURN INJURY WITH SPREADSHEET IN PC
Authors: NG E. Y.-K. , CHUA L. T.
Published: July 2002

This paper presents a blood-perfused skin model as implemented in a spreadsheet software application for accessing burn injuries resulting from exposure of skin surface to flowing hot fluids or constant heat sources. Finite-difference analysis of heat transfer in skin burns provides an accurate prediction of tissue time–temperature relationships throughout the duration of thermal insult. The Henriques theory of skin burns is used for determining the spatial and temporal extent of tissue damage. The epidermis, dermis, and subcutaneous fat were modeled as uniform elements with distinct thermal properties. Method for implementing finite-difference solution in spreadsheet software has been described. A comparison of the injury threshold computed by this model against Henriques's result has been done and the results are in good agreement.
Semantics-based concurrency control
Authors: Badrinath B. R. , Ramamritham Krithi
Published: July 2002

The concurrency of transactions executing on atomic data types can be enhanced through the use of semantic information about operations defined on these types. Hitherto, commutativity of operations has been exploited to provide enchanced concurrency while avoiding cascading aborts. We have identified a property known as
recoverability
which can be used to decrease the delay involved in processing noncommuting operations while still avoiding cascading aborts. When an invoked operation is
recoverable
with respect to an uncommitted operation, the invoked operation can be executed by forcing a commit dependency between the invoked operation and the uncommitted operation; the transaction invoking the operation will not have to wait for the uncommitted operation to abort or commit. Further, this commit dependency only affects the order in which the operations should commit, if both commit; if either operation aborts, the other can still commit thus avoiding cascading aborts. To ensure the serializability of transactions, we force the recoverability relationship between transactions to be acyclic. Simulation studies, based on the model presented by Agrawal et al. [1], indicate that using recoverability, the turnaround time of transactions can be reduced. Further, our studies show enchancement in concurrency even when
resource constraints
are taken into consideration. The magnitude of enchancement is dependent on the resource contention; the lower the resource contention, the higher the improvement.
On completeness of historical relational query languages
Authors: Clifford James , Croker Albert , Tuzhilin Alexander
Published: July 2002

Numerous proposals for extending the relational data model to incorporate the temporal dimension of data have appeared in the past several years. These proposals have differed considerably in the way that the temporal dimension has been incorporated both into the
structure
of the extended relations of these temporal models and into the extended relational
algebra
or
calculus
that they define. Because of these differences, it has been difficult to compare the proposed models and to make judgments as to which of them might in some sense be equivalent or even
better
. In this paper we define
temporally grouped
and
temporally ungrouped
historical data models and propose two notions of
historical relational completeness
, analogous to Codd's notion of relational completeness, one for each type of model. We show that the temporally ungrouped models are less expressive than the grouped models, but demonstrate a technique for extending the ungrouped models with a grouping mechanism to capture the additional semantic power of temporal grouping. For the ungrouped models, we define three different languages, a logic with explicit reference to time, a temporal logic, and a temporal algebra, and motivate our choice for the first of these as the basis for completeness for these models. For the grouped models, we define a many-sorted logic with variables over ordinary values, historical values, and times. Finally, we demonstrate the equivalence of this grouped calculus and the ungrouped calculus extended with a grouping mechanism. We believe the classification of historical data models into grouped and ungrouped models provides a useful framework for the comparison of models in the literature, and furthermore, the exposition of equivalent languages for each type provides reasonable standards for common, and minimal, notions of historical relational completeness.
Informedia Digital Video Library
Authors: Christel M. , Kanade T. , Mauldin M. , Reddy R. , Sirbu M. , Stevens S. , Wactlar H.
Published: July 2002

The Informedia Digital Video Library Project is developing new technologies for creating full-content search and retrieval digital video libraries. Working in collaboration with WQED Pittsburgh, the project is creating a testbed that will enable K-12 students to access, explore, and retrieve science and mathematics materials from the digital video library. The library will initially contain 1,000 hours of video from the archives of project partners: WQED, Fairfax Co. VA Schools' Electronic BBC-produced video courses. (Industrial partners include Digital Equipment Corp., Bell Atlantic, Intel Corp., and Microsoft, Inc.) This library will be installed at Winchester Thurston School, an independent K-12 school in Pittsburgh.
Minimal degrees for polynomial reducibilities
Authors: Homer Steven
Published: July 2002

The existence of minimal degrees is investigated for several polynomial reducibilities. It is shown that no set has minimal degree with respect to polynomial many-one or Turing reducibility. This extends a result of Ladner in which only recursive sets are considered. A polynomial reducibility ≤

h

T

is defined. This reducibility is a strengthening of polynomial Turing reducibility, and its properties relate to the P = ? NP question. For this new reducibility, a set of minimal degree is constructed under the assumption that P = NP. However, the set constructed is nonrecursive, and it is shown that no recursive set is of minimal ≤

h

T

degree.
Toward formalizing structured analysis
Authors: Baresi Luciano , Pezzè Mauro
Published: July 2002

Real-time extensions to structured analysis (SA/RT) are popular in industrial practice. Despite the large industrial experience and the attempts to formalize the various “dialects,” SA/RT notations are still imprecise and ambiguous. This article tries to identify the semantic problems of the requirements definition notation defined by Hatley and Pirbhai, one of the popular SA/RT “dialects,” and discusses possible solutions. As opposed to other articles that give their own interpretation, this article does not propose a specific semantics for the notation. This article identifies imprecisions, i.e., missing or partial information about features of the notation; it discusses ambiguities, i.e., elements of the definition that allow at least two different (“reasonable”) interpretations of features of the notation; and it lists extensions, i.e., features not belonging to the notation, but required by many industrial users and often supported by CASE tools. This article contributes by clarifying whether specific interpretations can be given unique semantics or retain ambiguities of the original definition. The article allows for the evaluation of formal definitions by indicating alternatives and consequences of the specific choices.