bluuebunny/crossref_metadata_2025 huggingface dataset:
Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation
Authors: Liu Shan-Lu , Lerman Michael I. , Miller A. Dusty
Published: June 2003

Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway.
Mutual Interference of Adenovirus Infection and
myc
Expression
Authors: Löhr Kristina , Hartmann Oliver , Schäfer Helmut , Dobbelstein Matthias
Published: June 2003

During infection with adenovirus, massive changes in the transcription of virus genes are observed, suggesting that the expression of cellular genes may also be modulated. To characterize the levels of cellular RNA species in infected cells, cDNA arrays were screened 24 h after infection of HeLa cells with wild-type adenovirus type 5, strain dl309. Despite complete transduction of the cells, fewer than 20 cellular genes (out of 4,600 analyzed and 1,200 found detectable and expressed above background) were altered more than threefold in their corresponding RNA levels compared to mock-infected cells. In particular, the expression of the
myc
oncogene was reduced at the mRNA level. This reduction was dependent on the replication of virus DNA and partially dependent on the presence of the adenovirus gene products E1B-55 kDa and E4orf6, but not E4orf3. On the other hand, MYC protein had an increased half-life in infected cells, resulting in roughly constant steady-state protein levels. The adenovirus E1A gene product is necessary and sufficient to stabilize MYC. Overexpressed MYC inhibited adenovirus replication and the proper formation of the virus replication centers. We conclude that adenovirus infection leads to the stabilization of MYC, perhaps as a side effect of E1A activities. On the other hand,
myc
mRNA levels are negatively regulated during adenovirus infection, and this may avoid the detrimental effect of excessive MYC on adenovirus replication.
The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3
Authors: Basler Christopher F. , Mikulasova Andrea , Martinez-Sobrido Luis , Paragas Jason , Mühlberger Elke , Bray Mike , Klenk Hans-Dieter , Palese Peter , García-Sastre Adolfo
Published: June 2003

The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation.
Delivery of Adeno-Associated Virus Vectors to the Fetal Retina: Impact of Viral Capsid Proteins on Retinal Neuronal Progenitor Transduction
Authors: Surace Enrico M. , Auricchio Alberto , Reich Samuel J. , Rex Tonia , Glover Ernest , Pineles Stacey , Tang Waixing , O'Connor Erin , Lyubarsky Arkady , Savchenko Andrey , Pugh Edward N. , Maguire Albert M. , Wilson James M. , Bennett Jean
Published: June 2003

The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.
The Viral σ1 Protein and Glycoconjugates Containing α2-3-Linked Sialic Acid Are Involved in Type 1 Reovirus Adherence to M Cell Apical Surfaces
Authors: Helander Anna , Silvey Katherine J. , Mantis Nicholas J. , Hutchings Amy B. , Chandran Kartik , Lucas William T. , Nibert Max L. , Neutra Marian R.
Published: June 2003

Type 1 reoviruses invade the intestinal mucosa of mice by adhering selectively to M cells in the follicle-associated epithelium and then exploiting M cell transport activity. The purpose of this study was to identify the apical cell membrane component and viral protein that mediate the M cell adherence of these viruses. Virions and infectious subviral particles of reovirus type 1 Lang (T1L) adhered to rabbit M cells in Peyer's patch mucosal explants and to tissue sections in an overlay assay. Viral adherence was abolished by pretreatment of sections with periodate and in the presence of excess sialic acid or lectins MAL-I and MAL-II (which recognize complex oligosaccharides containing sialic acid linked α2-3 to galactose). The binding of T1L particles to polarized human intestinal (Caco-2
BBe
) cell monolayers was correlated with the presence of MAL-I and MAL-II binding sites, blocked by excess MAL-I and -II, and abolished by neuraminidase treatment. Other type 1 reovirus isolates exhibited MAL-II-sensitive binding to rabbit M cells and polarized Caco-2
BBe
cells, but type 2 or type 3 isolates including type 3 Dearing (T3D) did not. In assays using T1L-T3D reassortants and recoated viral cores containing T1L, T3D, or no σ1 protein, MAL-II-sensitive binding to rabbit M cells and polarized Caco-2
BBe
cells was consistently associated with the T1L σ1. MAL-II-recognized oligosaccharide epitopes are not restricted to M cells in vivo, but MAL-II immobilized on virus-sized microparticles bound only to the follicle-associated epithelium and M cells. The results suggest that selective binding of type 1 reoviruses to M cells in vivo involves interaction of the type 1 σ1 protein with glycoconjugates containing α2-3-linked sialic acid that are accessible to viral particles only on M cell apical surfaces.
Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Infection of Human Fibroblast Cells Occurs through Endocytosis
Authors: Akula Shaw M. , Naranatt Pramod P. , Walia Neelam-Sharma , Wang Fu-Zhang , Fegley Barbara , Chandran Bala
Published: June 2003

Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the α3β1 integrin via its glycoprotein gB, and virus binding studies suggest that α3β1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH
4
Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH
4
Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.
Subclinical Prion Disease Induced by Oral Inoculation
Authors: Thackray Alana M. , Klein Michael A. , Bujdoso Raymond
Published: June 2003

Natural transmission of prion disease is believed to occur by peripheral infection such as oral inoculation. Following this route of inoculation, both the peripheral nervous system and the lymphoreticular system may be involved in the subsequent neuroinvasion of the central nervous system by prions, which may not necessarily result in clinical signs of terminal disease. Subclinical prion disease, characterized by the presence of infectivity and PrP
Sc
in the absence of overt clinical signs, may occur. It is not known which host factors contribute to whether infection with prions culminates in a terminal or subclinical disease state. We have investigated whether the level of host PrP
c
protein expression is a factor in the development of subclinical prion disease. When RML prion inoculum was inoculated by either the i.c. or intraperitoneal route, wild-type and
tga20
mice both succumbed to terminal prion disease. In contrast, orally inoculated
tga20
mice succumbed to terminal prion disease, whereas wild-type mice showed no clinical signs. However, wild-type mice sacrificed 375 or 525 days after oral inoculation harbored significant levels of brain PrP
Sc
and infectivity. These data show that same-species transmission of prions by the oral route in animals that express normal levels of PrP
c
can result in subclinical prion disease. This indicates that the level of host PrP
c
protein expression is a contributing factor to the regulation of development of terminal prion disease. Events that increase PrP
c
expression may predispose a prion-infected animal to the more deleterious effects of prion pathology.
Bunyamwera Virus Nonstructural Protein NSs Counteracts Interferon Regulatory Factor 3-Mediated Induction of Early Cell Death
Authors: Kohl Alain , Clayton Reginald F. , Weber Friedemann , Bridgen Anne , Randall Richard E. , Elliott Richard M.
Published: June 2003

The genome of Bunyamwera virus (BUN; familyBunyaviridae, genusOrthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.
Hydrogen Bonding at a Conserved Threonine in Lentivirus Capsid Is Required for Virus Replication
Authors: Rue Sarah M. , Roos Jason W. , Amzel L. Mario , Clements Janice E. , Barber Sheila A.
Published: June 2003

The N terminus of the capsid protein (CA) undergoes a considerable conformational change when the human immunodeficiency virus (HIV) protease cleaves it free from the Pr55
Gag
polyprotein. This rearrangement is thought to facilitate the establishment of specific CA-CA interactions that are required for the formation of the mature viral core. Substitution of amino acids that are critical for this refolding of the N terminus is generally detrimental to virus replication and mature virion core morphology. Here, we identify a conserved threonine in simian immunodeficiency virus (SIV) CA, T(47)
CA
, that is requisite for viral replication. Replacement of T(47)
CA
in the infectious viral clone SIVmac239 with amino acids with different hydrogen-bonding capabilities and analysis of the effects of these substitutions at key steps in the viral life cycle demonstrate that hydrogen bonding at this position is important for virus infectivity and virion release. In the HIV-based homology model of the mature SIV CA N terminus presented in this study, T(47)
CA
forms several hydrogen bonds with a proximal aspartate, D(50)
CA
. This model, coupled with strong phenotypic similarities between viral substitution mutants of each of these two residues in all of the virological assays described herein, indicates that hydrogen bonding between T(47)
CA
and D(50)
CA
is likely required for viral replication. As hydrogen bonding between these two residues is present in HIV CA as well, this interaction presents a potential target for antiviral drug design.
The Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Manipulates the Activity of Glycogen Synthase Kinase-3β
Authors: Fujimuro Masahiro , Hayward S. Diane
Published: June 2003

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in all KSHV-associated malignancies. LANA is essential for replication and maintenance of the viral episomes during latent infection. However, LANA also has a transcriptional regulatory role and can affect gene expression both positively and negatively. A previously performed yeast two-hybrid screen identified glycogen synthase kinase 3 (GSK-3) as a LANA-interacting protein. Interaction with both GSK-3α and GSK-3β was confirmed in transfected cells with coprecipitation assays. GSK-3β also interacted with the herpesvirus saimiri homolog ORF73. GSK-3β is an intermediate in the Wnt signaling pathway and a negative regulator of β-catenin. In transfected cells, LANA was shown to overcome GSK-3β-mediated degradation of β-catenin. Examination of primary effusion lymphoma (PEL) cells found increased levels of β-catenin relative to KSHV-negative B cells, and this translated into increased activity of a β-catenin-responsive reporter containing Tcf/Lef binding sites. In tetradecanoyl phorbol acetate-treated PEL cells, loss of LANA expression correlated temporally with loss of detectable β-catenin. LANA was found to alter the intracellular distribution of GSK-3β so that nuclear GSK-3β was more readily detectable in the presence of LANA. Mapping experiments with coimmunoprecipitation assays revealed that both N-terminal and C-terminal LANA sequences were required for efficient GSK-3β interaction. LANA mutants that were defective for GSK-3β interaction were unable to mediate GSK-3β relocalization or activate a β-catenin-responsive Tcf-luciferase reporter. This study identified manipulation of GSK-3β activity as a mechanism by which LANA may modify transcriptional activity and contribute to the phenotype of primary effusion lymphoma.
Vesicular Stomatitis Virus Glycoprotein Is a Determinant of Pathogenesis in Swine, a Natural Host
Authors: Martinez Isidoro , Rodriguez Luis L. , Jimenez Carlos , Pauszek Steven J. , Wertz Gail W.
Published: June 2003

There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-G
I
); (ii) two copies of the G gene, one from each serotype (VSIV-G
NJ
G
I
); or (iii) a single copy of the G
NJ
gene instead of the G
I
gene (VSIV-G
NJ
). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-G
I
), VSNJV (VSIV-G
NJ
), or both (VSIV-G
NJ
G
I
), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-G
NJ
and VSIV-G
NJ
G
I
were attenuated. However, in swine, a natural host for VSV, the G
NJ
glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-G
I
. These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.
Novel PKCηIs Required To Activate Replicative Functions of the Major Nonstructural Protein NS1 of Minute Virus of Mice
Authors: Lachmann Sylvie , Rommeleare Jean , Nüesch Jürg P. F.
Published: June 2003

The multifunctional protein NS1 of minute virus of mice (MVMp) is posttranslationally modified and at least in part regulated by phosphorylation. The atypical lambda isoform of protein kinase C (PKCλ) phosphorylates residues T435 and S473 in vitro and in vivo, leading directly to an activation of NS1 helicase function, but it is insufficient to activate NS1 for rolling circle replication. The present study identifies an additional cellular protein kinase phosphorylating and regulating NS1 activities. We show in vitro that the recombinant novel PKCη phosphorylates NS1 and in consequence is able to activate the viral polypeptide in concert with PKCλ for rolling circle replication. Moreover, this role of PKCη was confirmed in vivo. We thereby created stably transfected A9 mouse fibroblasts, a typical MVMp-permissive host cell line with Flag-tagged constitutively active or inactive PKCη mutants, in order to alter the activity of the NS1 regulating kinase. Indeed, tryptic phosphopeptide analyses of metabolically32P-labeled NS1 expressed in the presence of a dominant-negative mutant, PKCηDN, showed a lack of distinct NS1 phosphorylation events. This correlates with impaired synthesis of viral DNA replication intermediates, as detected by Southern blotting at the level of the whole cell population and by BrdU incorporation at the single-cell level. Remarkably, MVM infection triggers an accumulation of endogenous PKCηin the nuclear periphery, suggesting that besides being a target for PKCη, parvovirus infections may also affect the regulation of this NS1 regulating kinase. Altogether, our results underline the tight interconnection between PKC-mediated signaling and the parvoviral life cycle.
Multiple Interactions across the Surface of the gp120 Core Structure Determine the Global Neutralization Resistance Phenotype of Human Immunodeficiency Virus Type 1
Authors: Bouma Peter , Leavitt Maria , Zhang Peng Fei , Sidorov Igor A. , Dimitrov Dimiter S. , Quinnan Gerald V.
Published: June 2003

Resistance to neutralization is an important characteristic of primary isolates of human immunodeficiency virus type 1 (HIV-1) that relates to the potential for successful vaccination to prevent infection and use of immunotherapeutics for treatment of established infection. In order to further elucidate mechanisms responsible for neutralization resistance, we studied the molecular mechanisms that determine the resistance of the primary virus isolate of the strain HIV-1 MN to neutralization by soluble CD4 (sCD4). As is the case for the global neutralization resistance phenotype, sCD4 resistance depended upon sequences in the amino-terminal heptad repeat region of gp41 (HR1), as well as on multiple functional interactions within the envelope complex. The functional interactions that determined the resistance included interactions between the variable loop 1 and 2 (V1/V2) region and sequences in or near the CD4 binding site (CD4bs) and with the V3 loop. Additionally, the V3 loop region was found to interact functionally with sequences in the outer domain of gp120, distant from the CD4bs and coreceptor-binding site, as well as with a residue thought to be located centrally in the coreceptor-binding site. These and previous results provide the basis for a model by which functional signals that determine the neutralization resistance, high-infectivity phenotype depend upon interactions occurring across the surface of the gp120 core structure and involving variable loop structures and gp41. This model should be useful in efforts to define epitopes that may be important for primary virus neutralization.
Structural Analysis of the Kaposi's Sarcoma-Associated Herpesvirus K1 Protein
Authors: Lee Bok-Soo , Connole Michelle , Tang Zuoquin , Harris Nancy L. , Jung Jae U.
Published: June 2003

The K1 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). In addition, the extracellular domain of K1 demonstrates regional homology with the immunoglobulin (Ig) family and contains conserved regions (C1 and C2) and variable regions (V1 and V2). To generate mouse monoclonal antibodies directed against the KSHV K1 protein, BALB/c mice were primed and given boosters with K1 protein purified from mammalian cells. Twenty-eight hybridomas were tested for reactivity with K1 protein by enzyme-linked immunosorbent assay, immunofluorescence, flow cytometry, immunohistochemistry, and immunoblotting. Deletion mutants of the K1 extracellular domain were used to map the epitope of each antibody. All antibodies were directed to the Ig, C1, and C2 regions of K1. Furthermore, antibody recognition of a short sequence (amino acids 92 to 125) of the C2 region overlapping with the Ig region of K1 efficiently induced intracellular free calcium mobilization; antibody recognition of the other regions of K1 did not. The efficient signal transduction of K1 induced by antibody stimulation required both the ITAM sequence of the cytoplasmic domain and the normal structure of the extracellular domain. Finally, immunological assays showed that K1 was expressed during the early lytic cycle of viral replication in primary effusion lymphoma cells. K1 was readily detected in multicentric Castleman's disease tissues, whereas it was not detected in Kaposi's sarcoma lesions, suggesting that K1 is preferentially expressed in lymphoid cells. Thus, these results indicate that the conserved regions, particularly the Ig and C2 regions, of the K1 extracellular domain are exposed on the outer surface and play an important role in K1 structure and signal transduction, whereas the variable regions of K1 appear to be away from the surface.
An Activity Associated with Human Chromosome 21 Permits Nuclear Colocalization of the Adenovirus E1B-55K and E4orf6 Proteins and Promotes Viral Late Gene Expression
Authors: Chastain-Moore Amy M. , Roberts Terry , Trott Deborah A. , Newbold Robert F. , Ornelles David A.
Published: June 2003

The adenovirus E1B-55K and E4orf6 proteins cooperate during virus infection while performing several tasks that contribute to a productive infection, including the selective nucleocytoplasmic transport of late viral mRNA. Previous studies have shown that the E4orf6 protein retains the E1B-55K protein in the nucleus of human and monkey cells, but not in those of rodents, suggesting that primate-specific cellular factors contribute to the E4orf6-mediated retention of the E1B-55K protein in the nucleus. In an effort to identify these proposed primate-specific cellular factors, the interaction of the E1B-55K and E4orf6 proteins was studied in a panel of stable human-rodent monochromosomal somatic cell hybrids. Analysis of this panel of cell lines has demonstrated the existence of an activity associated with human chromosome 21 that permits the E1B-55K and E4orf6 proteins to colocalize in the nucleus of a rodent cell. Additional hybrid cells bearing portions of human chromosome 21 were used to map this activity to a 10-megabase-pair segment of the chromosome, extending from 21q22.12 to a region near the q terminus. Strikingly, this region also facilitates the expression of adenovirus late genes in a rodent cell background while having little impact on the expression of early viral genes.
Human Herpesvirus 7 Open Reading Frame U12 Encodes a Functional β-Chemokine Receptor
Authors: Nakano Kazushi , Tadagaki Kenjiro , Isegawa Yuji , Aye Mya Mya , Zou Ping , Yamanishi Koichi
Published: June 2003

Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily, infects mainly CD4+T cells in vitro and infects children during infancy. After the primary infection, HHV-7 becomes latent. HHV-7 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 gene, we cloned the gene and expressed the U12 protein in cells. The U12 gene encoded a calcium-mobilizing receptor for the EBI1 ligand chemokine-macrophage inflammatory protein 3β (ELC/MIP-3β) but not for other chemokines, suggesting that the chemokine selectivity of the U12 gene product is distinct from that of the known mammalian chemokine receptors. These studies revealed that U12 activates distinct transmembrane signaling pathways that may mediate biological functions by binding with a β-chemokine, ELC/MIP-3β.
Cooperative Interactions of Simian Immunodeficiency Virus Nef, AP-2, and CD3-ζ Mediate the Selective Induction of T-Cell Receptor-CD3 Endocytosis
Authors: Swigut Tomek , Greenberg Michael , Skowronski Jacek
Published: June 2003

The Nef proteins of human immunodeficiency virus and simian immunodeficiency virus (SIV) bind the AP-1 and AP-2 clathrin adaptors to downmodulate the expression of CD4 and CD28 by recruiting them to sites of AP-2 clathrin-dependent endocytosis. Additionally, SIV Nef directly binds the CD3-ζ subunit of the CD3 complex and downmodulates the T-cell receptor (TCR)-CD3 complex. We report here that SIV mac239 Nef induces the endocytosis of TCR-CD3 in Jurkat T cells. SIV Nef also induces the endocytosis of a chimeric CD8-CD3-ζ protein containing only the CD3-ζ cytoplasmic domain (8-ζ), in the absence of other CD3 subunits. Thus, the interaction of SIV Nef with CD3-ζ likely mediates the induction of TCR-CD3 endocytosis. In cells expressing SIV Nef and 8-ζ, both proteins colocalize with AP-2, indicating that Nef induces 8-ζ internalization via this pathway. Surprisingly, deletion of constitutively strong AP-2 binding determinants (CAIDs) in SIV Nef had little effect on its ability to induce TCR-CD3, or 8-ζ endocytosis, even though these determinants are required for the induction of CD4 and CD28 endocytosis via this pathway. Fluorescent microscopic analyses revealed that while neither the mutant SIV Nef protein nor 8-ζ colocalized with AP-2 when expressed independently, both proteins colocalized with AP-2 when coexpressed. In vitro binding studies using recombinant SIV Nef proteins lacking CAIDs and recombinant CD3-ζ cytoplasmic domain demonstrated that SIV Nef and CD3-ζ cooperate to bind AP-2 via a novel interaction. The fact that Nef uses distinct AP-2 interaction surfaces to recruit specific membrane receptors demonstrates how Nef independently selects distinct types of target receptors and recruits them to AP-2 for endocytosis.
Biphasic DNA Synthesis in Spumaviruses
Authors: Delelis Olivier , Saïb Ali , Sonigo Pierre
Published: June 2003

Spumaviruses are complex retroviruses whose replication cycle resembles that of hepadnaviruses, especially by a late-occurring reverse transcription step. The possible existence of an early reverse transcription as observed in other retroviruses was not documented. Using real-time quantitative PCR, we addressed directly the kinetics of DNA synthesis during spumavirus infection. An early phase of viral DNA synthesis developed until 3 h postinfection, followed by a second phase, culminating 10 h postinfection. Both phases were abolished by the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine. Similar to other retroviruses, circular forms of viral DNA harboring two long terminal repeats were mainly found in the nucleus of infected cells. Interestingly, a fraction of these circular forms were detected in the cytoplasm and in extracellular virions, a feature shared with hepadnaviruses. Combined with packaging of both viral DNA and RNA genomes in virions, early and late reverse transcription might allow spumavirus to maximize its genome replication.
Characterization of Entry Mechanisms of Human Herpesvirus 8 by Using an Rta-Dependent Reporter Cell Line
Authors: Inoue Naoki , Winter Jörn , Lal Renu B. , Offermann Margaret K. , Koyano Shin
Published: June 2003

To analyze the mechanisms of entry of human herpesvirus 8 (HHV-8), we established a reporter cell line T1H6 that contains the
lac
Z gene under the control of the polyadenylated nuclear RNA promoter, known to be strongly activated by a viral transactivator, Rta. We found that infection with cell-free virus, as well as cocultivation with HHV-8-positive primary effusion lymphoma cell lines, activated the
lac
Z gene of T1H6 in a sensitive and dose-dependent manner. Addition of Polybrene and centrifugation enhanced, but polysulfonate compounds inhibited, the HHV-8 infectivity. RGD-motif-containing polypeptides and integrins did not decrease the infectivity, suggesting the presence of an additional cellular receptor other than the reported one. The entry was dependent on pH acidification but not on the clathrin pathway. Although conditioned media obtained from human immunodeficiency virus (HIV)-infected cells did not have any effect on the early steps of HHV-8 infection, intracellular expression of a proviral HIV type 1, but not of Tat alone, increased the HHV-8-dependent reporter activation slightly, suggesting a potential of HIV-mediated enhancement of an early step of HHV-8 infection.
Identification and Simian Immunodeficiency Virus Infection of CD1d-Restricted Macaque Natural Killer T Cells
Authors: Motsinger Alison , Azimzadeh Agnes , Stanic Aleksandar K. , Johnson R. Paul , Van Kaer Luc , Joyce Sebastian , Unutmaz Derya
Published: June 2003

Natural killer T (NKT) cells express a highly conserved T-cell receptor (TCR) and recognize glycolipids in the context of CD1d molecules. We recently demonstrated that CD4+NKT cells are highly susceptible to human immunodeficiency virus type 1 (HIV-1) infection and are selectively depleted in HIV-infected individuals. Here, we identified macaque NKT cells using CD1d tetramers and human Vα24 antibodies. Similar to human NKT cells, α-galactosylceramide (α-GalCer)-pulsed dendritic cells activate and expand macaque NKT cells. Upon restimulation with α-GalCer-pulsed CD1d+cells, macaque NKT cells secreted high levels of cytokines, a characteristic of these T cells. Remarkably, the majority of resting and activated macaque NKT cells expressed CD8, and a smaller portion expressed CD4. Macaque NKT cells also expressed the HIV-1/simian immunodeficiency virus (SIV) coreceptor CCR5, and the CD4+subset was susceptible to SIV infection. Identification of macaque NKT cells has major implications for delineating the role of these cells in nonhuman primate disease models of HIV as well as other pathological conditions, such as allograft rejection and autoimmunity.
Novel Kaposi's Sarcoma-Associated Herpesvirus Homolog in Baboons
Authors: Whitby Denise , Stossel Andrea , Gamache Christine , Papin James , Bosch Marnix , Smith Anne , Kedes Dean H. , White Gary , Kennedy Ronald , Dittmer Dirk P.
Published: June 2003

Kaposi's sarcoma (KS) and lymphoproliferative diseases induced by KS-associated herpesvirus (KSHV/human herpesvirus 8) cause substantial morbidity and mortality in human immunodeficiency virus-infected individuals. To understand KSHV biology it is useful to investigate closely related rhadinoviruses naturally occurring in nonhuman primates. Here we report evidence for a novel KSHV homolog in captive baboon species (Papio anubisand other). Using degenerate PCR we identified a novel rhadinovirus, PapRV2, that has substantial sequence identity to two essential KSHV genes, the viral polymerase and thymidylate synthase. A subset of animals exhibited detectable PapRV2 viral load in peripheral blood mononuclear cells. Extensive serological analysis of nearly 200 animals in the colony demonstrated that the majority carried cross-reacting antibodies that recognize KSHV or macaque rhadinovirus antigens. Seroreactivity increased with age, similar to the age-specific prevalence of KSHV in the human population. This establishes baboons as a novel resource to investigate rhadinovirus biology, which can be developed into an animal model system for KSHV-associated human diseases, vaccine development, and therapy evaluation.
Differential Hyperacetylation of Histones H3 and H4 upon Promoter-Specific Recruitment of EBNA2 in Epstein-Barr Virus Chromatin
Authors: Alazard Nathalie , Gruffat Henri , Hiriart Edwige , Sergeant Alain , Manet Evelyne
Published: June 2003

Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transcriptional activator involved in the immortalization of B lymphocytes by the virus. EBNA2 is targeted to the promoters of its responsive genes, via interaction with cellular DNA-binding proteins. Using chromatin immunoprecipitation assays, we show for the first time the conditional recruitment of EBNA2 on two specific viral promoters in vivo and demonstrate a correlation between this recruitment and a local change in the acetylation of histones H3 and H4, which is promoter dependent.
Genetic and temporal patterns of multiple parasitism by brown-headed cowbirds (Molothrus ater) on song sparrows (Melospiza melodia)
Authors: McLaren C M , Woolfenden B E , Gibbs H L , Sealy S G
Published: June 2003

Identifying the factors used by an avian brood parasite to select host nests is important in understanding the determinates of individual reproductive success, yet such factors are poorly known for most parasitic species. Insights into these factors may come from understanding the conditions under which female parasites lay more than one egg per host nest (multiple parasitism). Using genetic techniques we examined patterns of multiple parasitism on a preferred host, the song sparrow (Melospiza melodia), to determine some basic patterns of multiple parasitism. Multiple parasitism involved equal frequencies of the same female parasitizing the same nest again and two or more females parasitizing the same nest. The frequency of multiple parasitism increased as the season progressed. We also documented a high frequency of parasitism that was not synchronized with host laying. These laying patterns may be the result of cowbirds "making the best of a bad situation" or of suboptimal host choice by inexperienced, nonselective females.
Intraspecific variation in testis size of small mammals: implications for muscle mass
Authors: Schulte-Hostedde Albrecht I , Millar John S , Hickling Graham J
Published: June 2003

Intraspecific variation in testis size is usually interpreted in the context of sperm competition, yet an unconsidered consequence of increased testis size may be an increase in the production of testosterone, which can affect the growth of muscle mass. After muscle mass is corrected for body size, male small mammals have more muscle mass than females, which suggests that it may be a sexually selected trait. An enhanced musculature may have fitness consequences with respect to male mate-searching activities and malemale competition for access to females. We tested the prediction that males with large testes have more muscle mass (measured as lean dry mass) by examining testis size and body composition in three species of small mammals (bushy-tailed wood rat (Neotoma cinerea), deer mouse (Peromyscus maniculatus), and red-backed vole (Clethrionomys gapperi)) from the Kananaskis Valley, Alberta. In all three species, males with relatively large testes had relatively more lean dry mass than males with relatively small testes. This suggests that a secondary consequence of relatively large testes may be a relative increase in muscle mass. Further research should investigate alternative effects of intraspecific variation in testis size on individual fitness within wild populations to gain further insight into sexual selection.
Influence of food abundance on individual behaviour strategy and growth rate in juvenile brown trout (Salmo trutta)
Authors: Brännäs Eva , Jonsson Sara , Lundqvist Hans
Published: June 2003

We studied the benefit of being territorial as an effect of food abundance by measuring the proportions of individuals that displayed a territorial, floating (individuals occasionally displayed territorial behaviour), or nonterritorial (shoaling) behaviour strategy, and individual growth rates. Also, swimming activity was monitored as an indicator of emigration. Replicate groups of 12 juvenile brown trout (Salmo trutta) were released into an artificial stream channel, fed according to one of four food regimes for 10 days, and allocated to one of three behaviour categories. There was no significant relationship between food abundance and the number of territorial individuals. Instead, the proportions of individuals that displayed the alternative behaviour strategies, i.e., floating and nonterritorial, changed with food abundance. At the lowest food level, more individuals displayed nonterritorial than floating behaviour, but these two strategies were equally represented at the highest food level. The difference in growth rates with respect to behaviour category was highest at intermediate food levels. At the highest and lowest food levels, nonterritorial fish and floaters grew nearly as fast or as poorly, respectively, as the territorial individuals. Swimming activity between the two sections of the stream tank was greatest at the lowest food level, and there was a general tendency (not significant) for the floaters to exhibit the greatest activity.
New observations on urine contents in water-deprived Negev Desert rodents
Authors: Korine Carmi , Vatnick Itzick , Tets Ian G. van , Pinshow Berry
Published: June 2003

In past studies, several rodent species of the murid subfamilies Gerbillinae and Cricetomyinae from the Namib Desert, when deprived of water, excreted allantoin precipitate in their urine. Shifting nitrogen excretion from urea to allantoin allows them to save much water. This phenomenon has not been reported in other rodents, and whether it is a trait that is common among desert rodents, but undocumented, or is unique to these Namib Desert species, is not known. We found no allantoin precipitate in the urine of any of five additional species of water-deprived murid rodents of two subfamilies from the Negev Desert. There was no discernible precipitate in the urine of Gerbillus andersoni allenbyi (Gerbillinae), Gerbillus pyramidum (Gerbillinae), or Acomys cahirinus (Murinae). Sodium oxalate was found in both the precipitate and the liquid urine of Psammomys obesus (Gerbillinae), and the as yet unidentified precipitate in the urine of Gerbillus dasyurus (Gerbillinae) was not allantoin. This preliminary study suggests that not all gerbilline rodents have the capacity to switch from urea to allantoin excretion. The Namib Desert gerbilline and cricetomyine rodents may be examples of closely related mammalian taxa that have evolved a new metabolic pathway to produce a nitrogenous product that results in sizable water savings, i.e., by switching from urea to allantoin.
MOLECULAR DYNAMIC STUDY OF A SINGLE DISLOCATION IN A TWO-DIMENSIONAL LENNARD–JONES SYSTEM
Authors: ROBLES MIGUEL , MUSTONEN VILLE , KASKI KIMMO
Published: June 2003

In this work the motion of a single dislocation in a two-dimensional triangular lattice is studied by using classical Molecular Dynamics method with the Lennard–Jones inter-atomic potential. The dislocation motion is investigated with an interactive simulation program developed to track automatically the movement of lattice defects. Constant strain and constant strain-rate deformations were applied to the system. From constant strain simulations a curve of shear stress versus dislocation velocity is obtained, showing a nonlinear power law relation. An equation of motion for the dislocation is proposed and found to be applicable when the movement of dislocation follows a quasi-static process. Numerical simulations at different strain rates show an elastic-to-plastic transition that modifies the dynamics of the dislocation motion.
STRUCTURAL AND ELECTRONIC PROPERTIES OF MAGNESIUM DIBORIDE
Authors: ERKOÇ ŞAKIR
Published: June 2003

Structural, electronic, and vibrational properties of magnesium diboride have been investigated by performing ab initio calculation within DFT level considering full MP2 correlation correction through B3LYP exchange-correlation functional. The structural, electronic, and vibrational properties of MgB 2 triatom have been calculated in its ground state.
SOLITONS ON NONCOMMUTATIVE TORUS AS ELLIPTIC CALOGERO–GAUDIN MODELS, BRANES AND LAUGHLIN WAVE FUNCTIONS
Authors: HOU BO-YU , PENG DAN-TAO , SHI KANG-JIE , YUE RUI-HONG
Published: June 2003

For the noncommutative torus [Formula: see text], in the case of the noncommutative parameter [Formula: see text], we construct the basis of Hilbert space ℋn in terms of θ functions of the positions zi of n solitons. The wrapping around the torus generates the algebra [Formula: see text], which is the Zn × Zn Heisenberg group on θ functions. We find the generators g of a local elliptic su (n), which transform covariantly by the global gauge transformation of [Formula: see text]. By acting on ℋn we establish the isomorphism of [Formula: see text] and g. We embed this g into the L-matrix of the elliptic Gaudin and Calogero–Moser models to give the dynamics. The moment map of this twisted cotangent [Formula: see text] bundle is matched to the D-equation with the Fayet–Illiopoulos source term, so the dynamics of the noncommutative solitons become that of the brane. The geometric configuration (k, u) of the spectral curve det |L(u) - k| = 0 describes the brane configuration, with the dynamical variables zi of the noncommutative solitons as the moduli T⊗ n/Sn. Furthermore, in the noncommutative Chern–Simons theory for the quantum Hall effect, the constrain equation with quasiparticle source is identified also with the moment map equation of the noncommutative [Formula: see text] cotangent bundle with marked points. The eigenfunction of the Gaudin differential L-operators as the Laughlin wave function is solved by Bethe ansatz.
STATISTICAL MODELING OF DIFFUSION COEFFICIENT IN THE MASS HETEROGENEOUS STRUCTURES
Authors: MAŠKOVIĆ Lj. , VUJOVIĆ R. , TOŠIĆ B. S. , MOHORA E.
Published: June 2003

The statistical modeling was used in the analysis of structures containing heterogeneous masses. The statistical probability of a system of coupled oscillators was found taking into account the mass dependence of energy levels. It is essentially a new approach. Both the low as well as high temperature expressions for diffusion coefficient were found. Comparison with experimental data gave satisfactory agreement.
A SIMPLE DYNAMIC PREISACH HYSTERESIS MODEL FOR FeSi MATERIALS
Authors: LU M. , LEONARD P. J. , MARKETOS P. , MEYDAN T. , MOSES A. J.
Published: June 2003

Dynamic hysteresis property is a common phenomenon in FeSi materials under time-varied applied field. This paper presented a dynamic hysteresis model based on Preisach scheme. The rectangular-shaped elementary hysteresis operator with two states in classical Preisach model is replaced by a non-rectangular shaped one with multiple states. The output of each state is calculated by a cosine function. The proposed dynamic hysteresis model is experimently tested by comparing the simulated hysteresis loops to experimental ones. The model can be used to describe the dynamic hysteresis in FeSi material for magnetizing frequencies from quasi-static to several hundred Hertz.
ON EFFICIENT METHODS FOR DETECTING HOPF BIFURCATION WITH APPLICATIONS TO POWER SYSTEM INSTABILITY PREDICTION
Authors: HUSSEIN A. , CHEN K.
Published: June 2003

Efficient measurement of the performance index (the distance of a loading parameter from the voltage collapse or instability point) is one of the key problems in power system operations and planning such an index indicates the severity of a power system with regard to voltage collapse or instability. While much work has been done on the former problem, the latter problem poses a more severe challenge both to the engineering and mathematical communities.

This paper first presents a review of the main methods for detecting Hopf bifurcation in the general field of bifurcation theory and then attempts to generalize the previously studied fold detection and index methods to the Hopf case. The proposed Hopf test functions should be of wide interest while the proposed test function based index method gives an indication of the distance of the current operating point from the Hopf instability. Promising numerical results have been obtained using some standard dynamical test systems.