bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Macrophage Infection upon Subcutaneous Inoculation of Rhesus Macaques with Simian Immunodeficiency Virus-Loaded Dendritic Cells or T Cells but Not with Cell-Free Virus
Authors: Ignatius Ralf , Tenner-Racz Klara , Messmer Davorka , Gettie Agegnehu , Blanchard James , Luckay Amara , Russo Christine , Smith Stephen , Marx Preston A. , Steinman Ralph M. , Racz Paul , Pope Melissa
Published: September 2002

Information on the establishment of immunodeficiency virus infection through transmission of infected cells is sparse. Dendritic cells (DCs) and T cells may be central to the onset and subsequent spread of infection following mucosal exposure. To directly investigate the consequences of virus being introduced by DCs or T cells, we reinjected ex vivo simian immunodeficiency virus (SIV)-loaded autologous immature DCs and T cells subcutaneously (s.c.) into healthy macaques. s.c. injection of cell-bound virus was used to mirror what may happen if virus-loaded cells pass through an epithelium or perhaps DCs and T cells that immediately entrap cell-free virus, having just crossed an epithelial barrier. Virus load in the plasma was monitored along with combined in situ hybridization and immunohistochemistry to identify the cells replicating virus in the lymphoid tissues. Both DCs and T cells transmitted infection after being pulsed with either wild-type ornef-defective (deltanef) SIVmac239. As seen in animals infected intravenously, replication of deltanefwas attenuated compared to that of wild-type virus when introduced in either cell-bound form. Upon examination of the draining lymph nodes (LNs) during the first days of infection, virus-producing CD4+T cells predominated in control animals that received s.c. cell-free virus. In dramatic contrast, both SIV-positive macrophages and T cells were detected in the LNs of monkeys infected with cell-associated SIV. Therefore, although both cell-free and cell-associated viruses are infectious, the initial cells amplifying the virus differ. This may have important implications for the subsequent dissemination of infection and/or induction of antiretroviral immunity.
Protective Immunity to Rabbit Oral and Cutaneous Papillomaviruses by Immunization with Short Peptides of L2, the Minor Capsid Protein
Authors: Embers Monica E. , Budgeon Lynn R. , Pickel Martin , Christensen Neil D.
Published: September 2002

The papillomavirus minor capsid protein, L2, has been shown to exhibit immunogenicity, whereby a variety of B-cell epitopes, predominantly in the amino terminus of L2, have been deduced. However, immunity to L2 in vivo has not been examined extensively. Notably, a common neutralization epitope for human papillomavirus (HPV) types 6 and 16 was mapped to amino acids (aa) 108 to 120. The objectives of this study were to derive antisera from rabbits using the corresponding sequences from rabbit viruses and to assess the ability of these peptides to protect against infection. Synthetic peptides consisting of two overlapping sequences each in the region of aa 94 to 122 of the rabbit oral (ROPV) and cottontail rabbit (CRPV) papillomaviruses were used to immunize rabbits. Rabbits were then infected with both ROPV and CRPV and monitored for the development of oral and cutaneous papillomas, respectively. Serum derived from rabbits immunized with either of the two peptides was shown to (i) react to purified L2 from the cognate virus, (ii) specifically recognize L2 within virus-infected cells, and (iii) neutralize virus in vitro. Following viral challenge, cutaneous papilloma growth was completely absent in rabbits immunized with either CRPV peptide. Likewise, ROPV peptide-immunized rabbits were protected from oral papillomatosis. Challenge of CRPV peptide-immune rabbits with the viral genome resulted in efficient papilloma growth, suggesting a neutralizing antibody-mediated mechanism of protection. These results afford in vivo evidence for the immunogenicity provided by a distinct region of L2 and further support previous evidence for the ability of this region to elicit antiviral immunity.
Identification of a G
2
Arrest Domain in the E1∧E4 Protein of Human Papillomavirus Type 16
Authors: Davy Clare E. , Jackson Deborah J. , Wang Qian , Raj Kenneth , Masterson Phillip J. , Fenner Nicola F. , Southern Shirley , Cuthill Scott , Millar Jonathan B. A. , Doorbar John
Published: September 2002

Human papillomavirus type 16 (HPV16) is the most common cause of cervical carcinoma. Cervical cancer develops from low-grade lesions that support the productive stages of the virus life cycle. The 16E1∧E4 protein is abundantly expressed in such lesions and can be detected in cells supporting vegetative viral genome amplification. Using an inducible mammalian expression system, we have shown that 16E1∧E4 arrests HeLa cervical epithelial cells in G
2
. 16E1∧E4 also caused a G
2
arrest in SiHa, Saos-2 and
Saccharomyces pombe
cells and, as with HeLa cells, was found in the cytoplasm. However, whereas 16E1∧E4 is found on the keratin networks in HeLa and SiHa cells, in Saos-2 and
S. pombe
cells that lack keratins, 16E1∧E4 had a punctate distribution. Mutagenesis studies revealed a proline-rich region between amino acids 17 and 45 of 16E1∧E4 to be important for arrest. This region, which we have termed the “arrest domain,” contains a putative nuclear localization signal, a cyclin-binding motif, and a single cyclin-dependent kinase (Cdk) phosphorylation site. A single point mutation in the putative Cdk phosphorylation site (T23A) abolished 16E1∧E4-mediated G
2
arrest. Arrest did not involve proteins regulating the phosphorylation state of Cdc2 and does not appear to involve the activation of the DNA damage or incomplete replication checkpoint. G
2
arrest was also mediated by the E1∧E4 protein of HPV11, a low-risk mucosal HPV type that also causes cervical lesions. The E1∧E4 protein of HPV1, which is more distantly related to that of HPV16, did not cause G
2
arrest. We conclude that, like other papillomavirus proteins, 16E1∧E4 affects cell cycle progression and that it targets a conserved component of the cell cycle machinery.
Kinetics of Expression of Rhesus Monkey Rhadinovirus (RRV) and Identification and Characterization of a Polycistronic Transcript Encoding the RRV Orf50/Rta, RRV R8, and R8.1 Genes
Authors: DeWire Scott M. , McVoy Michael A. , Damania Blossom
Published: September 2002

Rhesus monkey rhadinovirus (RRV) is a close relative of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8). RRV serves as an in vitro and an in vivo model for KSHV, and the mapping of its transcription program during lytic replication is significant since it represents de novo infection in the absence of stimulation with phorbol esters. Further, the RRV lytic system facilitates the making of recombinant viruses, and hence transcription profiling of the wild-type virus is important. Currently, the kinetics of lytic gene expression of RRV, the function of the RRV Orf50/Rta gene, and the presence of the RRV R8 and R8.1 genes are not known. This study details the transcription profile seen during RRV lytic replication and shows that RRV latency-associated nuclear antigen, viral FLIP (vFLIP), and vCyclin are transcribed during the RRV lytic phase. In addition, this study describes the identification of three new spliced products of the RRV Orf50, R8, and R8.1 genes, which are structural homologs of the KSHV Orf50, K8, and K8.1 genes, respectively. Characterization of the RRV Orf50 protein identifies it as a strong transcriptional transactivator capable of activating three early RRV promoters. Interestingly, the KSHV Orf50 transactivator can also activate these simian virus promoters, suggesting that there exists a conservation of gene function between the key transcription factors of KSHV and RRV.
A Single Mutation in the Carboxy Terminus of Reovirus Outer-Capsid Protein σ3 Confers Enhanced Kinetics of σ3 Proteolysis, Resistance to Inhibitors of Viral Disassembly, and Alterations in σ3 Structure
Authors: Wilson Gregory J. , Nason Emma L. , Hardy Charles S. , Ebert Daniel H. , Wetzel J. Denise , Venkataram Prasad B. V. , Dermody Terence S.
Published: September 2002

Mammalian reoviruses undergo acid-dependent proteolytic disassembly within endosomes, resulting in formation of infectious subvirion particles (ISVPs). ISVPs are obligate intermediates in reovirus disassembly that mediate viral penetration into the cytoplasm. The initial biochemical event in the reovirus disassembly pathway is the proteolysis of viral outer-capsid protein σ3. Mutant reoviruses selected during persistent infection of murine L929 cells (PI viruses) demonstrate enhanced kinetics of viral disassembly and resistance to inhibitors of endocytic acidification and proteolysis. To identify sequences in σ3 that modulate acid-dependent and protease-dependent steps in reovirus disassembly, the σ3 proteins of wild-type strain type 3 Dearing; PI viruses L/C, PI 2A1, and PI 3-1; and four novel mutant σ3 proteins were expressed in insect cells and used to recoat ISVPs. Treatment of recoated ISVPs (rISVPs) with either of the endocytic proteases cathepsin L or cathepsin D demonstrated that an isolated tyrosine-to-histidine mutation at amino acid 354 (Y354H) enhanced σ3 proteolysis during viral disassembly. Yields of rISVPs containing Y354H in σ3 were substantially greater than those of rISVPs lacking this mutation after growth in cells treated with either acidification inhibitor ammonium chloride or cysteine protease inhibitor E64. Image reconstructions of electron micrographs of virus particles containing wild-type or mutant σ3 proteins revealed structural alterations in σ3 that correlate with the Y354H mutation. These results indicate that a single mutation in σ3 protein alters its susceptibility to proteolysis and provide a structural framework to understand mechanisms of σ3 cleavage during reovirus disassembly.
Identification and Characterization of Three Immunodominant Structural Proteins of Fowlpox Virus
Authors: Boulanger Denise , Green Philip , Jones Brenda , Henriquet Gwenn , Hunt Lawrence G. , Laidlaw Stephen M. , Monaghan Paul , Skinner Michael A.
Published: September 2002

Genes encoding fowlpox virus (FWPV) structural proteins have been identified mainly by sequence homology with those from vaccinia virus (VACV), but little is known about the encoded proteins. Production of monoclonal antibodies (MAbs) against Poxine and HP1-440 (Munich) clone FP9 allowed the identification of three immunodominant FWPV proteins: the 39-kDa core protein (encoded by FPV168, homologous to VACV A4L), a 30- and 35-kDa protein doublet, and an abundant 63-kDa protein. The 30- and 35-kDa proteins are nonglycosylated, antigenically related proteins present in the intracellular mature virus membrane and localizing closely with the viral factories. N-terminal sequencing identified the 35-kDa protein as encoded by FPV140 (the FWPV homolog of VACV H3L). The 63-kDa protein forms covalently linked dimers and oligomers. It remained mainly insoluble upon detergent treatment of purified virus but did not localize closely with the viral factory. N-terminal sequencing was unsuccessful, suggesting N-terminal blocking. CNBr digestion generated a peptide encoded by FPV191, predicted to encode one of two FWPV A-type inclusion (ATI) proteins. The characteristics of the 63-kDa protein were inconsistent with published observations on cowpox or VACV ATI proteins (it appears to be essential). The 63-kDa protein, however, shares characteristics with both VACV p4c virus occlusion and 14-kDa fusion proteins. Gene assignment at the poxvirus ATI locus (between VACV A24R and A28L) is complicated by sequence redundancies and variations, often due to deletions and multiple frameshift mutations. The identity of FPV191 in relation to genes at this locus is discussed.
Flock House Virus RNA Polymerase Is a Transmembrane Protein with Amino-Terminal Sequences Sufficient for Mitochondrial Localization and Membrane Insertion
Authors: Miller David J. , Ahlquist Paul
Published: September 2002

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected
Drosophila melanogaster
cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast
Saccharomyces cerevisiae
, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in
Drosophila
cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.
Memory CD4
+
T Cells Are the Earliest Detectable Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Cells in the Female Genital Mucosal Tissue during HIV-1 Transmission in an Organ Culture System
Authors: Gupta Phalguni , Collins Kelly B. , Ratner Deena , Watkins Simon , Naus Gregory J. , Landers Daniel V. , Patterson Bruce K.
Published: September 2002

The virologic and cellular factors that are involved in transmission of human immunodeficiency virus type 1 (HIV-1) across the female genital tissue are poorly understood. We have recently developed a human cervical tissue-derived organ culture model to study heterosexual transmission of HIV-1 that mimics the in vivo situation. Using this model we investigated the role of phenotypic characteristics of HIV-1 and identified the cell types that are first infected during transmission. Our data indicate that the cell-free R5 HIV-1 was more efficiently transmitted than cell-free X4 HIV-1. Cell-free and cell-associated HIV-1 had comparable transmission efficiency regardless of whether the virus was of R5 or X4 type. We have demonstrated that memory CD4
+
T cells and not Langerhans cells were the first HIV-1 RNA-positive cells detected at the epithelial-submucosal junction 6 h after virus exposure. Multicolor laser confocal microscopy demonstrated a globular distribution of HIV-1
gag-pol
mRNA in the cytoplasm, and the distribution of CD4 and the CD45RO isoform was irregular on the cellular membrane. At 96 h postinoculation, in addition to memory CD4
+
T cells, HIV-1 RNA-positive Langerhans cells and macrophages were also detected. The identification of CD4
+
T cells in the tissue at 6 h was confirmed by flow cytometric simultaneous immunophenotyping and ultrasensitive fluorescence in situ hybridization assay on immune cells isolated from disaggregated tissue. Furthermore, PMPA {9-[2-(phosphonomethoxy)propyl] adenine}, an antiretroviral compound, and UC781, a microbicide, inhibited HIV-1 transmission across the mucosa, indicating the utility of the organ culture to screen topical microbicides for their ability to block sexual transmission of HIV-1.
Activation of Terminally Differentiated Human Monocytes/Macrophages by Dengue Virus: Productive Infection, Hierarchical Production of Innate Cytokines and Chemokines, and the Synergistic Effect of Lipopolysaccharide
Authors: Chen Yun-Chi , Wang Sheng-Yuan
Published: September 2002

Dengue virus (DV) primarily infects blood monocytes (MO) and tissue macrophages (Mφ). We have shown in the present study that DV can productively infect primary human MO/Mφ regardless of the stage of cell differentiation. After DV infection, the in vitro-differentiated MO/Mφ secreted multiple innate cytokines and chemokines, including tumor necrosis factor alpha, alpha interferon (IFN-α), interleukin-1β (IL-1β), IL-8, IL-12, MIP-1α, and RANTES but not IL-6, IL-15, or nitric oxide. Secretion of these mediators was highlighted by distinct magnitude, onset, kinetics, duration, and induction potential. A chemokine-to-cytokine hierarchy was noted in the magnitude and induction potential of secretion, and a chemokine-to-cytokine-to-chemokine/Th1 cytokine cascade could be seen in the production kinetics. Furthermore, we found that terminally differentiated MO/Mφ cultured for more than 45 days could support productive DV infection and produce innate cytokines and chemokines, indicating that these mature cells were functionally competent in the context of a viral infection. In addition, DV replication in primary differentiated human MO/Mφ was enhanced and prolonged in the presence of lipopolysaccharide (LPS), and LPS-mediated synergistic production of IFN-α could be seen in DV-infected MO/Mφ. The secretion of innate cytokines and chemokines by differentiated MO/Mφ suggests that regional accumulation of these mediators may occur in various tissues to which DV has disseminated and may thus result in local inflammation. The LPS-mediated enhancement of virus replication and synergistic IFN-α production suggests that concurrent bacterial infection may modulate cytokine-mediated disease progression during DV infection.
Mutagenic Stabilization and/or Disruption of a CD4-Bound State Reveals Distinct Conformations of the Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein
Authors: Xiang Shi-Hua , Kwong Peter D. , Gupta Rishi , Rizzuto Carlo D. , Casper David J. , Wyatt Richard , Wang Liping , Hendrickson Wayne A. , Doyle Michael L. , Sodroski Joseph
Published: September 2002

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.
Biological Significance of a Human Enterovirus B-Specific RNA Element in the 3′ Nontranslated Region
Authors: Merkle Ingrid , van Ooij Mark J. M. , van Kuppeveld Frank J. M. , Glaudemans Dirk H. R. F. , Galama Jochem M. D. , Henke Andreas , Zell Roland , Melchers Willem J. G.
Published: September 2002

The secondary structures predicted for the enteroviral 3′ nontranslated region (3′NTR) all seem to indicate a conformation consisting of two (X and Y) hairpin structures. The higher-order RNA structure of the 3′NTR appears to exist as an intramolecular kissing interaction between the loops of these two hairpin structures. The enterovirus B-like subgroup possesses an additional stem-loop structure, domain Z, which is not present in the poliovirus-like enteroviruses. It has been suggested that the Z domain originated from a burst of short sequence repetitions (E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol, Nucleic Acids Res. 20:1739-1745, 1992). However, no functional features have yet been ascribed to this enterovirus B-like-specific RNA element in the 3′NTR. In this study, we tested the functional characteristics and biological significance of domain Z. A mutant of the cardiovirulent coxsackievirus group B3 strain Nancy which completely lacked the Z domain and which therefore acquired enterovirus C-like secondary structures exhibited a wild-type growth phenotype, as determined by single-cycle growth analysis with BGM cells. This result proves that the Z domain is virtually dispensable for viral growth in tissue cultures. Partial distortion of the Z domain structure resulted in a disabled virus with reduced growth kinetics, probably due to alternative conformations of the overall structure of the domain. Infection of mice showed that the recombinant coxsackievirus group B3 mutant which completely lacked the Z domain was less virulent. Pancreatic tissues from mice infected with wild-type virus and recombinant virus were equally affected. However, the heart tissue from mice infected with the recombinant virus showed only slight signs of myocarditis. These results suggest that the enterovirus B-like-specific Z domain plays a role in coxsackievirus-induced pathogenesis.
Large Hepatitis Delta Antigen Is Not a Suppressor of Hepatitis Delta Virus RNA Synthesis once RNA Replication Is Established
Authors: Macnaughton Thomas B. , Lai Michael M. C.
Published: September 2002

Moderation of hepatitis delta virus (HDV) replication is a likely prerequisite in the establishment of chronic infections and is thought to be mediated by the intracellular accumulation of large hepatitis delta antigen (L-HDAg). The regulatory role of this protein was suggested from several studies showing that cotransfection of plasmid cDNAs expressing both L-HDAg and HDV RNA results in a potent inhibition of HDV RNA replication. However, since this approach differs significantly from natural HDV infections, where HDV RNA replication is initiated from an RNA template, and L-HDAg appears only late in the replication cycle, it remains unclear whether L-HDAg can modulate HDV RNA replication in the natural HDV replication cycle. In this study, we investigated the effect of L-HDAg, produced as a result of the natural HDV RNA editing event, on HDV RNA replication. The results showed that following cDNA-free HDV RNA transfection, a steady-state level of RNA was established at 3 to 4 days posttransfection. The same level of HDV RNA was reached when a mutant HDV genome unable to make L-HDAg was used, suggesting that L-HDAg did not play a role. The rates of HDV RNA synthesis, as measured by metabolic labeling experiments, were identical at 4 and 8 days posttransfection and in the wild type and the L-HDAg-deficient mutant. We further examined the effect of overexpression of L-HDAg at various stages of the HDV replication cycle, showing that HDV RNA synthesis was resistant to L-HDAg when it was overexpressed 3 days after HDV RNA replication had initiated. Finally, we showed that, contrary to conventional thinking, L-HDAg alone, at a certain molar ratio with HDV RNA, can initiate HDV RNA replication. Thus, L-HDAg does not inherently inhibit HDV RNA synthesis. Taken together, these results indicated that L-HDAg affects neither the rate of HDV RNA synthesis nor the final steady-state level of HDV RNA and that L-HDAg is unlikely to act as an inhibitor of HDV RNA replication in the natural HDV replication cycle.
Strategy for Nonenveloped Virus Entry: a Hydrophobic Conformer of the Reovirus Membrane Penetration Protein μ1 Mediates Membrane Disruption
Authors: Chandran Kartik , Farsetta Diane L. , Nibert Max L.
Published: September 2002

The mechanisms employed by nonenveloped animal viruses to penetrate the membranes of their host cells remain enigmatic. Membrane penetration by the nonenveloped mammalian reoviruses is believed to deliver a partially uncoated, but still large (∼70-nm), particle with active transcriptases for viral mRNA synthesis directly into the cytoplasm. This process is likely initiated by a particle form that resembles infectious subvirion particles (ISVPs), disassembly intermediates produced from virions by proteolytic uncoating. Consistent with that idea, ISVPs, but not virions, can induce disruption of membranes in vitro. Both activities ascribed to ISVP-like particles, membrane disruption in vitro and membrane penetration within cells, are linked to N-myristoylated outer-capsid protein μ1, present in 600 copies at the surfaces of ISVPs. To understand how μ1 fulfills its role as the reovirus penetration protein, we monitored changes in ISVPs during the permeabilization of red blood cells induced by these particles. Hemolysis was preceded by a major structural transition in ISVPs, characterized by conformational change in μ1 and elution of fibrous attachment protein σ1. The altered conformer of μ1 was required for hemolysis and was markedly hydrophobic. The structural transition in ISVPs was further accompanied by derepression of genome-dependent mRNA synthesis by the particle-associated transcriptases. We propose a model for reovirus entry in which (i) primed and triggered conformational changes, analogous to those in enveloped-virus fusion proteins, generate a hydrophobic μ1 conformer capable of inserting into and disrupting cell membranes and (ii) activation of the viral particles for membrane interaction and mRNA synthesis are concurrent events. Reoviruses provide an opportune system for defining the molecular details of membrane penetration by a large nonenveloped animal virus.
Inhibitory Activity of Synthetic Peptide Antibiotics on Feline Immunodeficiency Virus Infectivity In Vitro
Authors: Ma Jia , Kennedy-Stoskopf Suzanne , Jaynes Jesse M. , Thurmond Linda M. , Tompkins Wayne A.
Published: September 2002

Natural peptide antibiotics are part of host innate immunity against a wide range of microbes, including some viruses. Synthetic peptides modeled after natural peptide antibiotics interfere with microbial membranes and are termed peptidyl membrane-interactive molecules (peptidyl-MIM [Demegen Inc, Pittsburgh, Pa.]). Sixteen peptidyl-MIM candidates were tested for activity against feline immunodeficiency virus (FIV) on infected CrFK cells. Three of them (D4E1, DC1, and D1D6) showed potent anti-FIV activity in chronically infected CrFK cells as measured by decreased reverse transcriptase (RT) activity, having 50% inhibitory concentrations of 0.46, 0.75, and 0.94 μM, respectively, which were approximately 10 times lower than their direct cytotoxic concentrations. Treatment of chronically infected CrFK cells with 2 μM D4E1 for 3 days completely reversed virus-induced cytopathic effect. Immunofluorescence revealed reduced p26 staining in these cells. Treatment of chronically infected CrFK cells with 2 μM D4E1 suppressed virus production (∼50%) for up to 7 days, The virions from the D4E1-treated culture had impaired infectivity, as measured by the 50% tissue culture infectious dose and nested PCR analysis of proviral DNA. However, these noninfectious virions were able to bind and internalize, suggesting a defect at some postentry step. After chronically infected CrFK cells were treated with D4E1 for 24 h, increased cell-associated mature p26 Gag and decreased extracellular virus-associated p26 Gag were observed by Western blot analysis, suggesting that virus assembly and/or release may be blocked by D4E1 treatment, whereas virus binding, penetration, RNA synthesis, and protein synthesis appear to be unaffected. Synthetic peptide antibiotics may be useful tools in the search for antiviral drugs having a wide therapeutic window for host cells.
Intact Microtubules Support Adenovirus and Herpes Simplex Virus Infections
Authors: Mabit Hélène , Nakano Michel Y. , Prank Ute , Saam Bianca , Döhner Katinka , Sodeik Beate , Greber Urs F.
Published: September 2002

Capsids and the enclosed DNA of adenoviruses, including the species C viruses adenovirus type 2 (Ad2) and Ad5, and herpesviruses, such as herpes simplex virus type 1 (HSV-1), are targeted to the nuclei of epithelial, endothelial, fibroblastic, and neuronal cells. Cytoplasmic transport of fluorophore-tagged Ad2 and immunologically detected HSV-1 capsids required intact microtubules and the microtubule-dependent minus-end-directed motor complex dynein-dynactin. A recent study with epithelial cells suggested that Ad5 was transported to the nucleus and expressed its genes independently of a microtubule network. To clarify the mechanisms by which Ad2 and, as an independent control, HSV-1 were targeted to the nucleus, we treated epithelial cells with nocodazole (NOC) to depolymerize microtubules and measured viral gene expression at different times and multiplicities of infections. Our results indicate that in NOC-treated cells, viral transgene expression was significantly reduced at up to 48 h postinfection (p.i.). A quantitative analysis of subcellular capsid localization indicated that NOC blocked the nuclear targeting of Ad2 and also HSV-1 by more than 90% at up to 7 h p.i. About 10% of the incoming Texas Red-coupled Ad2 (Ad2-TR) was enriched at the nucleus in microtubule-depleted cells at 5 h p.i. This result is consistent with earlier observations that Ad2-TR capsids move randomly in NOC-treated cells at less than 0.1 μm/s and over distances of less than 5 μm, characteristic of Brownian motion. We conclude that fluorophore-tagged Ad2 and HSV-1 particles are infectious and that microtubules play a prominent role in efficient nuclear targeting during entry and gene expression of species C Ads and HSV-1.
Large-Scale, pH-Dependent, Quaternary Structure Changes in an RNA Virus Capsid Are Reversible in the Absence of Subunit Autoproteolysis
Authors: Taylor Derek J. , Krishna Neel K. , Canady Mary A. , Schneemann Anette , Johnson John E.
Published: September 2002

The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus,
Nudaurelia capensis
ω
virus
(NωV), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 Å in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-Å-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, NωV provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in NωV.
Decreased Levels of Recent Thymic Emigrants in Peripheral Blood of Simian Immunodeficiency Virus-Infected Macaques Correlate with Alterations within the Thymus
Authors: Sodora Donald L. , Milush Jeffrey M. , Ware Felecia , Wozniakowski Aneta , Montgomery Lisa , McClure Harold M. , Lackner Andrew A. , Marthas Marta , Hirsch Vanessa , Johnson R. Paul , Douek Daniel C. , Koup Richard A.
Published: September 2002

The thymus is responsible for de novo production of CD4+and CD8+T cells and therefore is essential for T-cell renewal. The goal of this study was to assess the impact of simian immunodeficiency virus (SIV) infection on the production of T cells by the thymus. Levels of recent thymic emigrants within the peripheral blood were assessed through quantification of macaque T-cell receptor excision circles (TREC). Comparison of SIV-infected macaques (n= 15) to uninfected macaques (n= 23) revealed stable or increased TREC levels at 20 to 34 weeks postinfection. Further assessment of SIV-infected macaques (n= 4) determined that TREC levels decreased between 24 and 48 weeks postinfection. Through the assessment of longitudinal time points in three additional SIVmac239-infected macaques, the SIV infection was divided into two distinct phases. During phase 1 (16 to 30 weeks), TREC levels remained stable or increased within both the CD4 and CD8 T-cell populations. During phase 2 (after 16 to 30 weeks), TREC levels declined in both T-cell populations. As has been described for human immunodeficiency virus (HIV)-infected patients, this decline in TREC levels did at times correlate with an increased level of T-cell proliferation (Ki67+cells). However, not all TREC decreases could be attributed to increased T-cell proliferation. Further evidence for thymic dysfunction was observed directly in a SIVmac239-infected macaque that succumbed to simian AIDS at 65 weeks postinfection. The thymus of this macaque contained an increased number of memory/effector CD8+T cells and an increased level of apoptotic cells. In summary, reduced levels of TREC can be observed beginning at 16 to 30 weeks post-SIV infection and correlate with changes indicative of dysfunction within the thymic tissue. SIV infection of macaques will be a useful model system to elucidate the mechanisms responsible for the thymic dysfunction observed in HIV-infected patients.
The African Swine Fever Virus Protein j4R Binds to the Alpha Chain of Nascent Polypeptide-Associated Complex
Authors: Goatley Lynnette C. , Twigg Steve R. F. , Miskin James E. , Monaghan Paul , St-Arnaud René , Smith Geoffrey L. , Dixon Linda K.
Published: September 2002

The African swine fever virus (ASFV) j4R protein is expressed late during the virus replication cycle and is present in both the nucleus and the cytoplasm of infected cells. By using the yeast two-hybrid system, direct binding, and coprecipitation from cells, we showed that the j4R protein binds to the alpha chain of nascent polypeptide-associated complex (αNAC). Confocal microscopy indicated that a proportion of j4R and αNAC interact in areas close to the plasma membrane, as well as through the cytoplasm in cells. In vitro binding studies suggested that binding of j4R to αNAC did not interfere with the binding of α- and βNAC subunits (the BTF3 transcription factor).
COMPONENT-ISOTOPY OF SEIFERT COMPLEXES
Authors: KADOKAMI TERUHISA
Published: September 2002

In this paper, we treat C-complexes for links defined by D. Cooper, and show the Component-isotopy Lemma for arbitrary links, which is a generalization of Cooper's result for 2-component links. By applying the technique in the present paper, we show a similar result for knots, which is called Surface-homotopy Lemma.
NUMERICAL METHODS FOR FUZZY INITIAL VALUE PROBLEMS
Authors: HÜLLERMEIER EYKE
Published: September 2002

In this paper, fuzzy initial value problems for modelling aspects of uncertainty in dynamical systems are introduced and interpreted from a probabilistic point of view. Due to the uncertainty incorporated in the model, the behavior of dynamical systems modelled in this way will generally not be unique. Rather, we obtain a large set of trajectories which are more or less compatible with the description of the system. We propose so-called fuzzy reachable sets for characterizing the (fuzzy) set of solutions to a fuzzy initial value problem. Loosely spoken, a fuzzy reachable set is defined as the (fuzzy) set of possible system states at a certain point of time, with given constraints concerning the initial system state and the system evolution. The main-part of the paper is devoted to the development of the numerical methods for the approximation of such sets. Algorithms for precise as well as outer approximations are presented. It is shown that fuzzy reachable sets can be approximated to any degree of accuracy under certain assumptions. Our method is illustrated by means of an example from the field of economics.
DESIGN OF ADAPTIVE FUZZY SLIDING MODE CONTROL FOR NONLINEAR SYSTEMS
Authors: CHEN JEN-YANG
Published: September 2002

In this paper, a fuzzy sliding mode controller (FSMC), which is synthesized by a collection of linguistic control rules whose membership functions of THEN-part is adapted, is proposed. Both the membership functions of IF-part and THEN-part are arranged symmetrically and distributed equally in the individual universe of discourse. In particular, the membership functions of the THEN-part can be adapted via one parameter adaptation to meet the required system specification. The proposed direct adaptive FSMC can be synthesized through the following stages. First, the control rules are constructed according to the concepts of SMC, and the fuzzy sets whose membership functions are symmetrically covered in state space. Then, the derived adaptive law is used to adjust the membership functions of the THEN-part. The FSMC is employed to approximate the equivalent control of SMC without knowing the mathematical model of the controlled system. Third, a hitting control is developed to guarantee the stability of the control system. Finally, we apply this FSMC to control a nonlinear inverted pendulum system for confirming the validity of the proposed approach.
SEARCHING FOR MEANING ON DEFUZZIFICATION
Authors: RECASENS J. , BOIXADER D. , JACAS J.
Published: September 2002

Defuzzification is an essential problem in fuzzy systems that it is always solved in a heuristic way. The aim of this work is to give a semantic interpretation to this process with the help of indistinguishability operators.
DECISION MAKING UNDER UNCERTAINTY WITH ORDINAL INFORMATION
Authors: YAGER RONALD R.
Published: September 2002

We consider the problem of decision making under uncertainty where the payoffs are measured on an ordinal scale. First the case in which no information is available with respect to the uncertainty is studied. Here decision functions are obtained which reflect the attitude of the decision maker. Next the case where a possibility distribution exists with respect to uncertain variable is considered. Finally we look at the case where both possibilistic and probabilistic uncertainty co-exist.
DIAGNOSIS THROUGH BILATERAL MEMBERSHIP FUNCTIONS AND PATTERN RECOGNITION
Authors: BOUTLEUX EMMANUEL , DUBUISSON BERNARD
Published: September 2002

On real processes many functional states are usually observed. however some of them may represent difference significant levels (or rates) of the same functional mode. They do not represent by themselves a functional mode, they are only sub-modes of the same functional mode (e.g. one of the functional mode of my car is tank empty; some functional states observed may be 10% empty tank, 50% empty tank, 96% empty tank). Thus the interest is not only to diagnose the functional mode (e.g. tank empty, sleeping driver) but also to highlight the gravity level of this functional mode (e.g. the reaction in the middle of desert, confronted to a 4% empty tank is not the same as confronted to a 98% empty tank). The aim of this paper is to present a diagnosis of the current functional mode of a process and its gravity level. Usually in pattern recognition area, a membership function is a monotonic decreasing function of a Euclidean distance between two objects. Those objects represent two states of the process and a distance here is a dissimilarity measure between those states. Such a distance is defined in all the fuzzy subset associated with this membership function. So that function is monotonic decreasing in all subset directions. In this paper directional membership functions are proposed. In this case the distance is defined only by reference to a path describing the evolution from one functional state to another one. Then the obtained membership function is oriented according to this path and do not decrease identically within all directions in the subset. Such membership functions are then suitable in order to diagnose the state associated with data evoluting between known functional modes. An application to the French telephone network illustrates this method.
OPTIMIZATION ISSUES FOR FUZZY MEASURES
Authors: MIRANDA PEDRO , GRABISCH MICHEL
Published: September 2002

In this paper, we address the problem of identification of fuzzy measures through different representations, namely the Möbius, the Shapley and the Banzhaf interaction representations. In the first part of the paper, we recall the main results concerning these representations, and give a simple algorithm to compute them. Then we determine the bounds of the Möbius and the interaction representations for fuzzy measures. Lastly, the identification of fuzzy measures by minimizing a quadratic error criterion is addressed. We give expressions of the quadratic program for all the considered representations, and study the uniqueness of the solution.
k–ORDER ADDITIVE FUZZY MEASURES
Authors: MESIAR RADKO
Published: September 2002

A generalization of k-order additive discrete fuzzy measures recently introduced by Grabisch is shown. k-order additive fuzzy measures on general spaces are introduced. Connection of the proposed generalization with the general Möbius transform of Shafer is shown. General evaluation formula for the Choquet integral is given. Further generalizations concerning the type of applied arithmetics are proposed.
AGGREGATION OF k-ORDER MAXITIVE FUZZY MEASURES
Authors: KOLESÁROVÁ ANNA
Published: September 2002

The k-order maxitive fuzzy measures on general spaces are discussed. The sum of ki-order maxitive measures is shown to be a ∑ ki-order maxitive fuzzy measure. The sum of two maxitive measures (possibility measures) is discussed and completely analyzed.
LOGICAL INTERPRETATION OF DISCRETE CHOQUET INTEGRAL DEFINED BY GENERAL MEASURE
Authors: RADOJEVIĆ DRAGAN
Published: September 2002

Aggregation of object or action attribute values in the presence of interaction among attributes, is of great practical importance in many fields. A discrete fuzzy Choquet integral is a very promising tool for the interaction modeling, but it allows the modeling of positive interaction and redundancy and their combinations. In this paper, the modeling of logically more complex interaction by Choquet integral is performed using a general discrete measure.
EVIDENCE MEASURES INDUCED BY KRIPKE'S ACCESSIBILITY RELATIONS
Authors: TSIPORKOVA ELENA , BOEVA VESELKA , DE BAETS BERNARD
Published: September 2002

Modal logic interpretations of plausibility and belief measures are developed based on the observation that the accessibility relation in a model of modal logic, regarded as a multivalued mapping, induces a plausibility measure and a belief measure on the set of possible worlds.
ON THE POSSIBILISTIC DECISION MODEL: FROM DECISION UNDER UNCERTAINTY TO CASE-BASED DECISION
Authors: DUBOIS DIDIER , GODO LLUÍS , PRADE HENRI , ZAPICO ADRIANA
Published: September 2002

This paper improves a previously proposed axiomatic setting for qualitative decision under uncertainty in the von Neumann and Morgenstern' style, where only ordinal linear scales are required for assessing uncertainty and utility. Two qualitative criteria are axiomatized in a finite setting: a pessimistic one and an optimistic one, respectively obeying an uncertainty aversion axiom and an uncertainty-attraction axiom. These criteria generalize the well-known maximin and maximax criteria, making them more realistic. They are suited to one-shot decisions and they are not based on the notion of mean value, but take the form of medians. Elements for a qualitative case-based decision methodology are also proposed, with pessimistic and optimistic evaluations formally similar to the expressions which cope with uncertainty, up to modifying factors which cope with the lack of normalization of similarity evaluations. Finally two extensions of the model are analysed: (i) the case of generalized possibilistic mixtures, using a t-norm instead of min, and (ii) the case of evaluating either preferences or uncertainty on Cartesian products of ordinal scales.
ON THE SUM OF OBSERVABLES IN PRODUCT MV ALGEBRAS
Authors: RIEČAN BELOSLAV
Published: September 2002

As an alternative to quantum logics MV algebras have been suggested2,20. In a quite general class of MV algebras there have been constructed sum of any two observables10,14,20. Recently the notion of product MV algebra has been introduced4,5,12,17. In any weakly σ-distributive product MV algebra the joint observable of any observables can be constructed and on the base also the sum of any observables. In this paper we show that the two concepts of constructing of the sum are equivalent.
INTELLIGENT AGENTS FOR MOBILE AND EMBEDDED DEVICES
Authors: FININ TIM , JOSHI ANUPAM , KAGAL LALANA , RATSIMOR OLGA , AVANCHA SASIKANTH , KOROLEV VLAD , CHEN HARRY , PERICH FILIP , SCOTT COST R.
Published: September 2002

The pervasive computing environments of the near future will involve the interactions, coordination and cooperation of numerous, casually accessible, and often invisible computing devices. These devices, whether carried on our person or embedded in our homes, businesses and classrooms, will connect via wireless and wired links to one another and to the global networking infrastructure. The result will be a networking milieu with a new level of openness. The localized and dynamic nature of their interactions raises many new issues that draw on and challenge the disciplines of agents, distributed systems, and security. This paper describes recent work by the UMBC Ebiquity research group which addresses some of these issues.
UBIQUITOUS INFORMATION AGENTS
Authors: BERGENTI FEDERICO , POGGI AGOSTINO
Published: September 2002

Ubiquity is one of the most important keyword of the next generation software systems. We want our systems to follow users wherever they go, and we want our systems to extend their reach to the telephony world and to its base of several hundred million users.

The ever-increasing diffusion of handy appliances, e.g. palmtops and cellular phones, is familiarizing people with the use of computers for their everyday job and, more impressively, for their everyday life. This suggests that the market of software systems for mobile applications will increase dramatically in the near future.