bluuebunny/crossref_metadata_2025 huggingface dataset:
Activity of the Human Papillomavirus Type 16 Late Negative Regulatory Element Is Partly due to Four Weak Consensus 5′ Splice Sites That Bind a U1 snRNP-Like Complex
Authors: Cumming Sarah A. , McPhillips Maria G. , Veerapraditsin Thanaporn , Milligan Steven G. , Graham Sheila V.
Published: April 2003

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the squamous epithelia that it infects. Capsid proteins, and hence mature virions, are produced in the outermost layer of differentiated cells. As late gene transcripts are produced in the lower layers, posttranscriptional mechanisms likely prevent capsid protein production in less differentiated cells. For HPV type 16 (HPV-16), a 79-nucleotide (nt) negative regulatory element (NRE) inhibits gene expression in basal epithelial cells. To identify key NRE sequences, we carried out transient transfection in basal epithelial cells with reporter constructs containing the HPV-16 late 3′ untranslated region with deletions and mutations of the NRE. Reporter gene expression was increased over 40-fold by deletion of the entire element, 10-fold by deletion of the 5′ portion of the NRE that contains four weak consensus 5′ splice sites, and only 3-fold by deletion of the 3′ GU-rich region. Both portions of the element appear to be necessary for full repression. Inactivating mutations in the 5′ splice sites in the 5′ NRE partially alleviated repression in the context of the 79-nt NRE but caused full derepression when assayed in a construct with the 3′ NRE deleted. All four contribute to the inhibitory effect, though the second splice site is most inhibitory. Sm proteins, U1A and U1 snRNA, but not U1 70K, could be affinity purified with the wild-type NRE but not with the NRE containing mutations in the 5′ splice sites, indicating that a U1 snRNP-like complex forms upon the element.
Characterization of the Minimal DNA Binding Domain of the Human Papillomavirus E1 Helicase: Fluorescence Anisotropy Studies and Characterization of a Dimerization-Defective Mutant Protein
Authors: Titolo S. , Brault K. , Majewski J. , White P. W. , Archambault J.
Published: April 2003

The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.
A Tyrosine Motif in the Cytoplasmic Domain of Mason-Pfizer Monkey Virus Is Essential for the Incorporation of Glycoprotein into Virions
Authors: Song Chisu , Dubay Susan R. , Hunter Eric
Published: April 2003

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.
Analysis of the Mechanism by Which the Small-Molecule CCR5 Antagonists SCH-351125 and SCH-350581 Inhibit Human Immunodeficiency Virus Type 1 Entry
Authors: Tsamis Fotini , Gavrilov Svetlana , Kajumo Francis , Seibert Christoph , Kuhmann Shawn , Ketas Tom , Trkola Alexandra , Palani Anadan , Clader John W. , Tagat Jayaram R. , McCombie Stuart , Baroudy Bahige , Moore John P. , Sakmar Thomas P. , Dragic Tatjana
Published: April 2003

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.
AttenuatedSalmonella entericaSerovar Typhi andShigella flexneri2a Strains Mucosally Deliver DNA Vaccines Encoding Measles Virus Hemagglutinin, Inducing Specific Immune Responses and Protection in Cotton Rats
Authors: Pasetti Marcela F. , Barry Eileen M. , Losonsky Genevieve , Singh Mahender , Medina-Moreno Sandra M. , Polo John M. , Ulmer Jeffrey , Robinson Harriet , Sztein Marcelo B. , Levine Myron M.
Published: April 2003

Measles remains a leading cause of child mortality in developing countries. Residual maternal measles antibodies and immunologic immaturity dampen immunogenicity of the current vaccine in young infants. Because cotton rat respiratory tract is susceptible to measles virus (MV) replication after intranasal (i.n.) challenge, this model can be used to assess the efficacy of MV vaccines. Pursuing a new measles vaccine strategy that might be effective in young infants, we used attenuatedSalmonella entericaserovar Typhi CVD 908-htrAandShigella flexneri2a CVD 1208 vaccines to deliver mucosally to cotton rats eukaryotic expression plasmid pGA3-mH and Sindbis virus-based DNA replicon pMSIN-H encoding MV hemagglutinin (H). The initial i.n. dose-response with bacterial vectors alone identified a well-tolerated dosage (1 × 109to 7 × 109CFU) and a volume (20 μl) that elicited strong antivector immune responses. Animals immunized i.n. on days 0, 28, and 76 with bacterial vectors carrying DNA plasmids encoding MV H or immunized parenterally with these naked DNA vaccine plasmids developed MV plaque reduction neutralizing antibodies and proliferative responses against MV antigens. In a subsequent experiment of identical design, cotton rats were challenged with wild-type MV 1 month after the third dose of vaccine or placebo. MV titers were significantly reduced in lung tissue of animals immunized with MV DNA vaccines delivered either via bacterial live vectors or parenterally. Since attenuated serovar Typhi andS. flexnerican deliver measles DNA vaccines mucosally in cotton rats, inducing measles immune responses (including neutralizing antibodies) and protection, boosting strategies can now be evaluated in animals primed with MV DNA vaccines.
Immunostimulant Patch Containing Heat-Labile Enterotoxin fromEscherichia coliEnhances Immune Responses to Injected Influenza Virus Vaccine through Activation of Skin Dendritic Cells
Authors: Guebre-Xabier Mimi , Hammond Scott A. , Epperson Diane E. , Yu Jianmei , Ellingsworth Larry , Glenn Gregory M.
Published: April 2003

Vaccine strategies, such as influenza virus vaccination of the elderly, are highly effective at preventing disease but provide protection for only the responding portion of the vaccinees. Adjuvants improve the magnitude and rates of responses, but their potency must be attenuated to minimize side effects. Topical delivery of strong adjuvants such as heat-labile enterotoxin fromEscherichia coli(LT) induces potent immune responses. We hypothesized that LT delivered alone in an immunostimulating (LT-IS) patch placed on the skin at the site of injection could augment the immune response to injected vaccines. This was based on the observation that topically applied LT induces migration of activated antigen-presenting cells (APCs) from the skin to the proximal draining lymph node (DLN), and that APCs loaded with antigen by injection in the same anatomical region also migrate to the same DLN. We observed that when influenza virus vaccine is injected and an LT-IS patch is placed to target the same DLN, the influenza virus antibody response is enhanced. Similarly, influenza virus-specific T cells isolated from the lungs show increased levels of gamma interferon and interleukin-4 production. An LT-IS patch placed near an injected vaccine also leads to increased levels of hemagglutination inhibition titers, enhanced mucosal immunoglobulin A responses, and enhanced antigen presentation. Although the mechanisms by which an LT-IS patch exerts its enhancing effects need further study, the enhanced immune responses, ability to safely use potent adjuvants, and simplicity of LT-IS patch application address an important unmet need and provide a new immune enhancement strategy.
Ex Vivo Profiling of CD8+-T-Cell Responses to Human Cytomegalovirus Reveals Broad and Multispecific Reactivities in Healthy Virus Carriers
Authors: Elkington Rebecca , Walker Susan , Crough Tania , Menzies Moira , Tellam Judy , Bharadwaj Mandvi , Khanna Rajiv
Published: April 2003

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8+-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8+-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8+-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8+-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.
Members of the AP-1 Family, c-Jun and c-Fos, Functionally Interact with JC Virus Early Regulatory Protein Large T Antigen
Authors: Kim Joanne , Woolridge Stefanie , Biffi Renato , Borghi Elisa , Lassak Adam , Ferrante Pasquale , Amini Shohreh , Khalili Kamel , Safak Mahmut
Published: April 2003

The activating protein 1 (AP-1) family of regulatory proteins is characterized as immediate-early inducible transcription factors which were shown to be activated by a variety of stress-related stimuli and to be involved in numerous biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. We have recently demonstrated the involvement of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the human polyomavirus, JC virus (JCV), genome. Here, we further examined their role in JCV gene regulation and replication through their physical and functional interaction with JCV early regulatory protein large T antigen (T-Ag). Transfection and replication studies indicated that c-Jun and c-Fos can significantly diminish T-Ag-mediated JCV gene transcription and replication. Affinity chromatography and coimmunoprecipitation assays demonstrated that c-Jun and T-Ag physically interact with each other. Results from band shift assays showed that the binding efficiency of c-Jun to the AP-1 site was reduced in the presence of T-Ag. In addition, we have mapped, through the use of a series of deletion mutants, the regions of these proteins which are important for their interaction. While the c-Jun interaction domain of T-Ag is localized to the middle portion of the protein, the T-Ag interacting domain of c-Jun maps to its basic-DNA binding region. Results of transient-transfection assays with various c-Jun mutants and T-Ag expression constructs further confirm the specificity of the functional interaction between c-Jun and T-Ag. Taken together, these data demonstrate that immediate-early inducible transcription factors c-Jun and c-Fos physically and functionally interact with JCV major early regulatory protein large T-Ag and that this interaction modulates JCV transcription and replication in glial cells.
Rapid Tumor Formation of Human T-Cell Leukemia Virus Type 1-Infected Cell Lines in Novel NOD-SCID/γcnullMice: Suppression by an Inhibitor against NF-κB
Authors: Dewan M. Zahidunnabi , Terashima Kazuo , Taruishi Midori , Hasegawa Hideki , Ito Mamoru , Tanaka Yuetsu , Mori Naoki , Sata Tetsutaro , Koyanagi Yoshio , Maeda Michiyuki , Kubuki Yoko , Okayama Akihiko , Fujii Masahiro , Yamamoto Naoki
Published: April 2003

We established a novel experimental model for human T-cell leukemia virus type 1 (HTLV-1)-induced tumor using NOD-SCID/γcnull(NOG) mice. This model is very useful for investigating the mechanism of tumorigenesis and malignant cell growth of adult T-cell leukemia (ATL)/lymphoma, which still remains unclear. Nine HTLV-1-infected cell lines were inoculated subcutaneously in the postauricular region of NOG mice. As early as 2 to 3 weeks after inoculation, seven cell lines produced a visible tumor while two transformed cell lines failed to do so. Five of seven lines produced a progressively growing large tumor with leukemic infiltration of the cells in various organs that eventually killed the animals. Leukemic cell lines formed soft tumors, whereas some transformed cell lines developed into hemorrhagic hard tumors in NOG mice. One of the leukemic cell lines, ED-40515(−), was unable to produce visible tumors in NOD-SCID mice with a common γ-chain after 2 weeks. In vivo NF-κB DNA binding activity of the ED-40515(−) cell line was higher and the NF-κB components were changed compared to cells in vitro. Bay 11-7082, a specific and effective NF-κB inhibitor, prevented tumor growth at the sites of the primary region and leukemic infiltration in various organs of NOG mice. This in vivo model of ATL could provide a novel system for use in clarifying the mechanism of growth of HTLV-1-infected cells as well as for the development of new drugs against ATL.
Caspase Cleavage of the Nonstructural Protein NS1 Mediates Replication of Aleutian Mink Disease Parvovirus
Authors: Best Sonja M. , Shelton Janie F. , Pompey Justine M. , Wolfinbarger James B. , Bloom Marshall E.
Published: April 2003

Virus-induced apoptosis of infected cells can limit both the time and the cellular machinery available for virus replication. Hence, many viruses have evolved strategies to specifically inhibit apoptosis. However, Aleutian mink disease parvovirus (ADV) is the first example of a DNA virus that not only induces apoptosis but also utilizes caspase activity to facilitate virus replication. To determine the function of caspase activity during ADV replication, virus-infected cell lysates or purified ADV proteins were incubated with various purified caspases. Caspases cleaved the major nonstructural protein of ADV (NS1) at two caspase recognition sequences, whereas ADV structural proteins could not be cleaved. Importantly, the NS1 products could be identified in ADV-infected cells but were not present in infected cells pretreated with caspase inhibitors. By mutating putative caspase cleavage sites (D to E), we mapped the two cleavage sites to amino acid residues NS1:227 (INTD↓S) and NS1:285 (DQTD↓S). Replication of ADV containing either of these mutations was reduced 10
3
- to 10
4
-fold compared to that of wild-type virus, and a construct containing both mutations was replication defective. Immunofluorescent studies revealed that cleavage was required for nuclear localization of NS1. The requirement for caspase activity during permissive replication suggests that limitation of caspase activation and apoptosis in vivo may be a novel approach to restricting virus replication.
Inhibition of Human Immunodeficiency Virus Type 1 Env-Mediated Fusion by DC-SIGN
Authors: Nobile Cinzia , Moris Arnaud , Porrot Françoise , Sol-Foulon Nathalie , Schwartz Olivier
Published: April 2003

DC-SIGN, a lectin expressed on dendritic cell and macrophage subsets, binds to human immunodeficiency virus Env glycoproteins, allowing capture of viral particles. Captured virions either infect target cells or are efficiently transmitted to lymphocytes. Cellular mechanisms underlying the effects of DC-SIGN remain poorly understood. Here we have analyzed the effects of DC-SIGN on viral entry and on syncytium formation induced by Env glycoproteins. The lectin enhanced susceptibility to viral infection and dramatically increased virion internalization. Captured virions accumulated in the vesicular pathway, and their access to the cytosol was altered. Strikingly, the presence of DC-SIGN on target cells inhibited their ability to form syncytia with Env-expressing cells. However, increasing CD4 surface levels on target cells alleviated this inhibitory effect of DC-SIGN. Moreover, the potency of the viral fusion inhibitor T-20 was not affected in DC-SIGN-expressing cells. Altogether, our results indicate that DC-SIGN exerts subtle and complex effects during early steps of HIV type 1 replication. DC-SIGN facilitates capture and accumulation of viral particles in a vesicular compartment and inhibits viral fusion. Competition between CD4 and DC-SIGN for Env binding likely affects virus access to the cytosol and syncytium formation.
Replication Studies Using Genotype 1a Subgenomic Hepatitis C Virus Replicons
Authors: Gu Baohua , Gates Adam T. , Isken Olaf , Behrens Sven-Erik , Sarisky Robert T.
Published: April 2003

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-α), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-α may be attributed directly to the type 1a polymerase.
Manganese-Dependent Polioviruses Caused by Mutations within the Viral Polymerase
Authors: Crotty Shane , Gohara David , Gilligan Devin K. , Karelsky Sveta , Cameron Craig E. , Andino Raul
Published: April 2003

Viral RNA-dependent RNA polymerases exhibit great sequence diversity. Only six core amino acids are conserved across all polymerases of positive-strand RNA viruses of eukaryotes. While exploring the function of one of these completely conserved residues, asparagine 297 in the prototypic poliovirus polymerase 3D
pol
, we identified three viable mutants with noncanonical amino acids at this conserved position. Although asparagine 297 could be replaced by glycine or alanine in these mutants, the viruses exhibited Mn
2+
-dependent RNA replication and viral growth. All known RNA polymerases and replicative polymerases of bacterial, eukaryotic, and viral organisms are thought to be magnesium dependent in vivo, and therefore these mutant polioviruses may represent the first viruses with a requirement for an alternative polymerase cation. These results demonstrate the extreme functional flexibility of viral RNA-dependent RNA polymerases. Furthermore, the finding that strictly conserved residues in the nucleotide binding pocket of the polymerase can be altered in a manner that supports virus production suggests that drugs targeting this region of the enzyme will still be susceptible to the problem of drug-resistant escape mutants.
Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins
Authors: Dimitrova Maria , Imbert Isabelle , Kieny Marie Paule , Schuster Catherine
Published: April 2003

Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione
S
-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.
Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B
Authors: Lundin Marika , Monné Magnus , Widell Anders , von Heijne Gunnar , Persson Mats A. A.
Published: April 2003

Hepatitis C virus
(HCV) belongs to the
Hepacivirus
genus in the
Flaviviridae
family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the
Flaviviridae
family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in
Flaviviridae
.
Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harboring Subgenomic Replicons
Authors: Gosert Rainer , Egger Denise , Lohmann Volker , Bartenschlager Ralf , Blum Hubert E. , Bienz Kurt , Moradpour Darius
Published: April 2003

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5′-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.
Inhibition of Protease-Resistant Prion Protein Accumulation In Vitro by Curcumin
Authors: Caughey Byron , Raymond Lynne D. , Raymond Gregory J. , Maxson Laura , Silveira Jay , Baron Gerald S.
Published: April 2003

Inhibition of the accumulation of protease-resistant prion protein (PrP-res) is a prime strategy in the development of potential transmissible spongiform encephalopathy (TSE) therapeutics. Here we show that curcumin (diferoylmethane), a major component of the spice turmeric, potently inhibits PrP-res accumulation in scrapie agent-infected neuroblastoma cells (50% inhibitory concentration, ∼10 nM) and partially inhibits the cell-free conversion of PrP to PrP-res. In vivo studies showed that dietary administration of curcumin had no significant effect on the onset of scrapie in hamsters. Nonetheless, other studies have shown that curcumin is nontoxic and can penetrate the brain, properties that give curcumin advantages over inhibitors previously identified as potential prophylactic and/or therapeutic anti-TSE compounds.
Role of N Glycosylation of Hepatitis B Virus Envelope Proteins in Morphogenesis and Infectivity of Hepatitis Delta Virus
Authors: Sureau Camille , Fournier-Wirth Chantal , Maurel Patrick
Published: April 2003

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.
Psychopharmacological treatments in persons with dual diagnosis of psychiatric disorders and developmental disabilities
Authors: Antochi R , Stavrakaki C , Emery P C
Published: April 2003

People with developmental disabilities are at considerable risk for the development of comorbid psychiatric conditions. Psychopharmacological treatments may have a crucial role in a multidisciplinary and multimodal approach to the management of psychopathology in this population. Psychiatric illnesses that are particularly amenable include mood disorders, anxiety disorders, schizophrenia, and attention deficit hyperactivity disorders (ADHDs) and antidepressants, mood stabilisers, anxiolytics, antipsychotics, and stimulants should be considered, respectively. ADHD may also respond to α2-agonists. Psychotropic agents such as β-antagonists can target aggressive, self injurious, and stereotypical behaviours and opioid antagonists may be helpful in treating self injurious behaviour and stereotypy. Selective serotonin reuptake inhibitors, newer anticonvulsants, and atypical neuroleptics are preferred when treating psychiatric disorders among people with developmental disabilities. This paper will review the major studies of pharmacological treatment of mental illness in individuals with developmental disabilities.
Essentiality of nickel and homeostatic mechanisms for its regulation in terrestrial organisms
Authors: Phipps T , Tank S L , Wirtz J , Brewer L , Coyner A , Ortego L S , Fairbrother A
Published: April 2003

Nickel (Ni) is a naturally occurring element with many industrial uses, including in stainless steel, electroplating, pigments, and ceramics. Consequently, Ni may enter the environment from anthropogenic sources, resulting in locally elevated concentrations in soils. However, Ni is a minor essential element, and, therefore, biota have established systems that maintain Ni homeostasis. This paper discusses the role of Ni as an essential element and reviews storage, uptake, and transport systems used to maintain homeostasis within terrestrial biota. The bioaccumulation and distribution of metals in these organisms are also addressed. In all cases, information on Ni essentiality is very limited compared to other essential metals. However, the available data indicate that Ni behaves in a similar manner to other metals. Therefore, inferences specific to Ni may be made from an understanding of metal homeostasis in general. Nevertheless, it is evident that tissue and organ Ni concentrations and requirements vary considerably within and between species, and metal accumulation in various tissues within a single organism differs as well. High rates of Ni deposition around smelters indicate that Ni in acidic soils may reach concentrations that are toxic to plants and soil decomposers. However, with the exception of hyperaccumulator plants, Ni does not biomagnify in the terrestrial food web, suggesting that toxicity to higher trophic levels is unlikely.Key words: nickel, essentiality, homeostasis, bioaccumulation, uptake, transport.
Phylogeny of the rust fungi: an approach using nuclear large subunit ribosomal DNA sequences
Authors: Maier Wolfgang , Begerow Dominik , Weiß Michael , Oberwinkler Franz
Published: April 2003

Sequence data from nuclear large subunit ribosomal DNA was used to infer phylogenetic relationships of selected genera of the Uredinales. We investigated 52 rust fungi representing nine families and three outgroup species. Neighbor joining analysis and a Bayesian method of phylogenetic inference using Monte Carlo Markov chains confirm the rust fungi as a natural group and indicate that Puccinia, Uromyces, Endophyllum, and Cumminsiella have a common origin. The autoecious Rosaceae-rusts Phragmidium, Kuehneola, Triphragmium, and Trachyspora are a monophyletic group. The gasteroid genus Ochropsora is closely related to Tranzschelia. While the Pucciniastreae sensu Dietel (1938) is recognized as a monophyletic group in neighbor joining analysis, the Pucciniaceae s.l. (Dietel 1928) is supported by Bayesian analysis. The following genera appear to be monophyletic: Chrysomyxa, Coleosporium, Cronartium, Gymnosporangium, Melampsora, Phragmidium, and Tranzschelia, whereas the genera Puccinia, Pucciniastrum, Thekopsora, and Uromyces are not.Key words: molecular phylogeny, systematics, nuclear large subunit rDNA, Basidiomycota, Urediniomycetes, Uredinales.
The South AfricanHordeum capenseis more closely related to some AmericanHordeumspecies than to the EuropeanHordeum secalinum: a perspective based on the 5S DNA units (Triticeae: Poaceae)
Authors: Baum Bernard R , Johnson Douglas A
Published: April 2003

Several authors have proposed that the European Hordeum secalinum and the morphologically similar South African Hordeum capense are conspecific. In this paper we provide evidence that the two species differ in their 5S DNA unit class composition. We also report on the diversity of 5S DNA units in Hordeum muticum, a South American species. When the 5S rDNA unit class composition for these three species is compared with the unit class composition for all Hordeum species thus far investigated, it appears that H. capense is more closely related to the American Hordeum species containing the long Y2 unit class, than to H. secalinum, which lacks the long Y2 unit class but contains the long X2 unit class found in H. marinum. This analysis suggests H. capense may have originated from a stock common to the South American species, such as H. muticum.Key words: 5S DNA unit class, Hordeum capense, Hordeum secalinum, Hordeum muticum, continental drift.
Ecology and spatial distributions of surface-sediment diatoms from 77 lakes in the subarctic Canadian treeline region
Authors: Rühland Kathleen M , Smol John P , Pienitz Reinhard
Published: April 2003

Diatom ecology and species compositional patterns across current arctic treeline can provide important paleoecological information associated with climatic and environmental change. In this paper we examine the relationships between measured environmental variables and modern diatom assemblage composition from 77 lakes across the treeline ecozones of the Central Canadian Arctic. The weighted-average optima for selected environmental variables were calculated for 74 of the most common diatoms, and photographic plates of these taxa are included. Our results indicated that both forest-tundra and arctic tundra lakes differed significantly in diatom assemblage composition from boreal forest lakes. In general, planktonic diatom taxa (e.g., Cyclotella species) were more common in forested lakes, which may be due to ecological conditions related to climate. Small, benthic, alkaliphilic Fragilaria taxa reached their highest abundances in forested lakes, likely because of the more alkaline nature of these lakes. Arctic tundra lakes were characterized by higher abundances of circumneutral to acidophilic taxa. Heavily silicified Aulacoseira taxa (e.g., Aulacoseira lirata, Aulacoseira perglabra) were more common in deeper tundra lakes, likely because of the less alkaline nature of these lakes and greater wind-induced turbulence in this zone. These trends provide important information on the variability of aquatic ecosystems across this climatically sensitive vegetational gradient.Key words: arctic treeline, Canada, diatoms, paleolimnology, weighted-average optima, climate change.
Analysis of morphogenesis in hyphomycetes: new characters derived from considering some conidiophores and conidia as condensed hyphal systems
Authors: Kendrick Bryce
Published: April 2003

Hyphomycetes (ascomycetous and basidiomycetous anamorphs) comprise numerous genera and species that have been difficult to place in fungal phylogeny. In this commentary, autobiographical notes preface a new analysis of reproductive structures in many hyphomycetes. This adopts the premise that many of the more complex examples of conidiophores and conidia represent condensed hyphal systems. Many new and potentially useful taxonomic characters are described, which are particularly applicable to computer-based synoptic keys or databases, but should also be considered by those describing new taxa or redescribing existing ones.Key words: moulds, taxonomy, conidia, conidiophores, morphogenesis, branching.
Three new species of Ophiostoma and notes on Cornuvesica falcata
Authors: Hausner G , Eyjólfsdóttir G G , Reid J
Published: April 2003

Sampling of beetles, beetle galleries, and stained tree tissues by ourselves and others to obtain isolates of ophiostomatoid fungal species yielded three Ceratocystiopsis-like entities. Using partial rDNA sequences, these were previously identified as being different from both each other and all other described species of Ceratocystiopsis and Ophiostoma. As Ceratocystiopsis Upadhyay et Kendrick has been reduced to synonymy with Ophiostoma Syd. et P. Syd., and sufficient dried material is now available, these are described herein as Ophiostoma carpenteri sp. nov., Ophiostoma rollhansenianum sp. nov., and Ophiostoma manitobense sp. nov. We found O. carpenteri to be closely related to Ophiostoma retusum (R.W. Davidson et T.E. Hinds) Hausner et al., and that both species may actually be fungal symbionts and could represent a discrete genus. Although morphologically O. rollhansenianum appears similar to Ophiostoma minutum Siemaszko, a clearly variable species, and O. manitobense to Ophiostoma minus (Hedgc.) Syd. et P. Syd., earlier rDNA data indicate that O. rollhansenianum and O. manitobense are actually more closely related to Ophiostoma coliferum (Marmolejo et Butin) Hausner et al., and Ophiostoma ranaculosum (J.R. Bridges et T.J. Perry) Hausner et al. We also comment on some morphological features that have previously been overlooked or misreported in Cornuvesica falcata (E.F. Wright et Cain) C.D. Viljoen et al., such as the ascospores actually being hyaline and the presence of two distinct Chalara anamorphs.Key words: Ophiostoma, molecular and morphological criteria, Ceratocystiopsis, Cornuvesica.
Recruitment and life-history traits of sparse plant species in subalpine grasslands
Authors: Austrheim Gunnar , Eriksson Ove
Published: April 2003

Recruitment is critical for the maintenance of plant populations and community diversity, but sexual regeneration is considered to be infrequent in climatically harsh habitats such as subalpine grasslands. This study examines the importance of regeneration through seed for 16 sparse herb species, and we asked whether their populations are limited by safe sites or the availability of seeds. Seedling recruitment and winter survival were recorded after sowing in an experimental split-plot design in (i) pasture grazed by livestock, and (ii) exclosed grassland cultivated for annual mowing. In addition we examined the effect of disturbance and local seedbank recruitment. All species were able to recruit and survive the first winter in at least some of the experimental plots, although none were initially present. Recruitment mainly occurred in disturbed plots, and disturbed pasture plots had a significantly higher recruitment than disturbed exclosures for all species except Silene dioica. We further examined whether specific plant traits were related to variation in recruitment. Lower recruitment in the disturbed exclosure was associated with higher specific leaf area, leaf dry weight, and seed number. In contrast, seeds sown in disturbed pasture recruited more independently of species traits. The exception was a negative correlation between recruitment and leaf dry weight, and an unexpected negative correlation with seed weight. We suggest that recruitment differences among habitats mainly reflect lower humidity in the exclosure. Consequently, small stature plants with small specific leaf area should have the highest recruiting probability when exposed to drought. The almost exclusive recruitment on disturbed plots indicates a strong competitive effect on the target species in vegetated plots, and suggests that safe sites for regeneration through seed are rare in subalpine grasslands.Key words: colonization, herbs, plant abundance and distribution, disturbance, pastures, cultivated exclosures, sowing experiment.
Changes in the responses to light quality during ontogeny in Chenopodium album
Authors: Causin Humberto Fabio , Wulff Renata D
Published: April 2003

Morphological shade-avoidance responses have been hypothesized to be a form of adaptive plasticity to improve competition for light; however, little is known about their intraspecific variability and their effect on reproductive fitness. To compare plant responses either at a common age or at a common phenological stage, two experiments were conducted with early- and late-flowering Chenopodium album plants exposed to different red (660 nm) to far red (730 nm) ratios. In the first experiment, plant height and number of leaves were recorded at several times during the vegetative stage, and at the onset of flowering, each plant was harvested and other growth traits were measured. In the second experiment, three destructive harvests were performed across the whole plant cycle. Plant growth and development markedly differed between early- and late-flowering plants in all of the conditions tested. Light treatments significantly affected stem length, total leaf number, total leaf area, and relative allocation to leaf biomass. In all families, the response of stem elongation to light treatments decreased later in the development, while changes in the other plastic responses were mostly due to variations in plant growth. No significant treatment effect was found on relative biomass allocation to reproductive structures. However, individual seed mass significantly differed between certain groups, indicating that light quality can affect reproductive fitness through changes in traits other than fruit or seed set.Key words: Chenopodium album, fitness, intraspecific variability, phenotypic plasticity, red to far red ratio, shade-avoidance responses.
Postglacial spread of hemlock (Tsuga) and vegetation history in Mount Revelstoke National Park, British Columbia, Canada
Authors: Rosenberg Sandra M , Walker Ian R , Mathewes Rolf W
Published: April 2003

To investigate postglacial environmental changes in southeastern British Columbia, subfossil pollen was analyzed from Eagle Lake, Mount Revelstoke National Park. Palynological evidence suggests that during the early Holocene, Eagle Lake was surrounded by an open forest. The pollen assemblage indicates that climatic conditions were warmer and drier than at present with high alder pollen values. Spruce and fir were common in local forests throughout the Holocene. Western hemlock pollen first occurs at approximately 3500 14C years BP. The migration path of western hemlock into this region is currently unclear. Data from palynological records appear insufficient to resolve the migration pattern, although several routes are possible. The available data support northward migration into southeastern British Columbia. The arrival of this conifer may be in response to the late Holocene increase in precipitation, as inferred from many lower elevation and coastal sites. Mountain hemlock pollen does not appear until about 2100 14C years BP. Mountain hemlock's migration route into southeastern British Columbia is also unresolved. In the late Holocene, the presence of mountain hemlock, and increases of Ericales and Valeriana sitchensis pollen, suggest a shift to the more open, cool, and moist conditions characteristic of the modern-day subalpine parkland. These climatic inferences correspond closely to those derived from other palynological and geological evidence throughout southern British Columbia.Key words: pollen, vegetation, Tsuga, hemlock, British Columbia, Holocene, climate.