bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the Rabies Virus Alpha/Beta Interferon Antagonist: Phosphoprotein P Interferes with Phosphorylation of Interferon Regulatory Factor 3
Authors: Brzózka Krzysztof , Finke Stefan , Conzelmann Karl-Klaus
Published: May 2005

Rabies virus (RV) of the
Rhabdoviridae
family grows in alpha/beta interferon (IFN)-competent cells, suggesting the existence of viral mechanisms preventing IFN gene expression. We here identify the viral phosphoprotein P as the responsible IFN antagonist. The critical involvement of P was first suggested by the observation that an RV expressing an enhanced green fluorescent protein (eGFP)-P fusion protein (SAD eGFP-P) (S. Finke, K. Brzózka, and K. K. Conzelmann, J. Virol. 78:12333-12343, 2004) was eliminated in IFN-competent HEp-2 cell cultures, in contrast to wild-type (wt) RV or an RV replicon lacking the genes for matrix protein and glycoprotein. SAD eGFP-P induced transcription of the IFN-β gene and expression of the IFN-responsive MxA and STAT-1 genes. Similarly, an RV expressing low levels of P, which was generated by moving the P gene to a promoter-distal gene position (SAD ΔPLP), lost the ability to prevent IFN induction. The analysis of RV mutants lacking expression of truncated P proteins P2, P3, or P4, which are expressed from internal AUG codons of the wt RV P open reading frame, further showed that full-length P is competent in suppressing IFN-β gene expression. In contrast to wt RV, the IFN-inducing SAD ΔPLP caused S386 phosphorylation, dimerization, and transcriptional activity of IFN regulatory factor 3 (IRF-3). Phosphorylation of IRF-3 by TANK-binding kinase-1 expressed from transfected plasmids was abolished in wt RV-infected cells or by cotransfection of P-encoding plasmids. Thus, RV P is necessary and sufficient to prevent a critical IFN response in virus-infected cells by targeting activation of IRF-3 by an upstream kinase.
Factors Required for the Uridylylation of the Foot-and-Mouth Disease Virus 3B1, 3B2, and 3B3 Peptides by the RNA-Dependent RNA Polymerase (3D
pol
) In Vitro
Authors: Nayak Arabinda , Goodfellow Ian G. , Belsham Graham J.
Published: May 2005

The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3D
pol
. To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3D
pol
in the presence of the 3CD precursor plus an internal RNA sequence termed a
c
is-acting
r
eplication
e
lement (
cre
). The FMDV
cre
has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus
cre
structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV
cre
(also known as the 3
B
-
u
ridylylation
s
ite, or
bus
) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.
Immunization of Macaques with Single-Cycle Simian Immunodeficiency Virus (SIV) Stimulates Diverse Virus-Specific Immune Responses and Reduces Viral Loads after Challenge with SIVmac239
Authors: Evans David T. , Bricker Jennifer E. , Sanford Hannah B. , Lang Sabine , Carville Angela , Richardson Barbra A. , Piatak Michael , Lifson Jeffrey D. , Mansfield Keith G. , Desrosiers Ronald C.
Published: May 2005

Genetically engineered simian immunodeficiency viruses (SIV) that is limited to a single cycle of infection was evaluated as a nonreplicating AIDS vaccine approach for rhesus macaques. Four Mamu-A*01+macaques were inoculated intravenously with three concentrated doses of single-cycle SIV (scSIV). Each dose consisted of a mixture of approximately equivalent amounts of scSIV strains expressing the SIVmac239 and SIVmac316 envelope glycoproteins with mutations innefthat prevent major histocompatibility complex (MHC) class I downregulation. Viral loads in plasma peaked between 104and 105RNA copies/ml on day 4 after the first inoculation and then steadily declined to undetectable levels over the next 4 weeks. SIV Gag-specific T-cell responses were detected in peripheral blood by MHC class I tetramer staining (peak, 0.07 to 0.2% CD8+T cells at week 2) and gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays (peak, 50 to 250 spot forming cells/106peripheral blood mononuclear cell at week 3). Following the second and third inoculations at weeks 8 and 33, respectively, viral loads in plasma peaked between 102and 104RNA copies/ml on day 2 and were cleared over a 1-week period. T-cell-proliferative responses and antibodies to SIV were also observed after the second inoculation. Six weeks after the third dose, each animal was challenged intravenously with SIVmac239. All four animals became infected. However, three of the four scSIV-immunized animals exhibited 1 to 3 log reductions in acute-phase plasma viral loads relative to two Mamu-A*01+control animals. Additionally, two of these animals were able to contain their viral loads below 2,000 RNA copies/ml as late as 35 weeks into the chronic phase of infection. Given the extraordinary difficulty in protecting against SIVmac239, these results are encouraging and support further evaluation of lentiviruses that are limited to a single cycle of infection as a preclinical AIDS vaccine approach.
[
18
F]FHPG Positron Emission Tomography for Detection of Herpes Simplex Virus (HSV) in Experimental HSV Encephalitis
Authors: Buursma A. R. , de Vries E. F. J. , Garssen J. , Kegler D. , van Waarde A. , Schirm J. , Hospers G. A. P. , Mulder N. H. , Vaalburg W. , Klein H. C.
Published: May 2005

Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial. Subtle brain infections with HSV may be causally related to neuropsychiatric disorders such as Alzheimer's dementia. We investigated the feasibility of a noninvasive positron emission tomography (PET) imaging technique using [
18
F]FHPG as a tracer for the detection of HSE. For this purpose, rats received HSV-1 (infected group) or phosphate-buffered saline (control group) by intranasal application, and dynamic PET scans were acquired. In addition, the distribution of tracer accumulation in specific brain areas was studied with phosphor storage imaging. The PET images revealed that the overall brain uptake of [
18
F]FHPG was significantly higher for the infected group than for control animals. Phosphor storage images showed an enhanced accumulation of [
18
F]FHPG in regions known to be affected after intranasal infection with HSV. High-performance liquid chromatography metabolite analysis showed phosphorylated metabolites of [
18
F]FHPG in infected brains, proving that the increased [
18
F]FHPG uptake in infected brains was due to HSV thymidine kinase-mediated trapping. Freeze lesion experiments showed that damage to the blood-brain barrier could in principle induce elevated [
18
F]FHPG uptake, but this nonspecific tracer uptake could easily be discriminated from HSE-derived uptake by differences in the tracer kinetics. Our results show that [
18
F]FHPG PET is a promising tool for the detection of HSV encephalitis.
Induction of T-Cell Immunity to Antiretroviral Drug-Resistant Human Immunodeficiency Virus Type 1
Authors: Stratov Ivan , Dale C. Jane , Chea Socheata , McCluskey James , Kent Stephen J.
Published: May 2005

Antiretroviral drug-resistant human immunodeficiency virus type 1 (HIV-1) is a major, growing, public health problem. Immune responses targeting epitopes spanning drug resistance sites could ameliorate development of drug resistance. We studied 25 individuals harboring multidrug-resistant HIV-1 for T-cell immunity to HIV-1 proteins and peptides spanning all common drug resistance mutations. CD8 T cells targeting epitopes spanning drug-induced mutations were detected but only in the 3 individuals with robust HIV-specific T-cell activity. Novel CD8 T-cell responses were detected against the common L63P and L10I protease inhibitor fitness mutations. Induction of T-cell immunity to drug-resistant variants was demonstrated in simian human immunodeficiency virus-infected macaques, where both CD8 and CD4 T-cell immune responses to reverse transcriptase and protease antiretroviral mutations were elicited using a novel peptide-based immunotherapy. T-cell responses to antiretroviral resistance mutations were strongest in the most immunocompetent animals. This study suggests feasible strategies to further evaluate the potential of limiting antiretroviral drug resistance through induction of T-cell immunity.
Identification of a Cell Population That Produces Alpha/Beta Interferon In Vitro and In Vivo in Response to Noncytopathic Bovine Viral Diarrhea Virus
Authors: Brackenbury L. S. , Carr B. V. , Stamataki Z. , Prentice H. , Lefevre E. A. , Howard C. J. , Charleston B.
Published: May 2005

In vitro infection of bovine cells of many origins with the cytopathogenic bovine viral diarrhea virus (cpBVDV) results in the induction of alpha/beta interferon (IFN-α/β), whereas noncytopathogenic BVDV (ncpBVDV) isolates have been shown not to induce IFN-α/β in vitro. Similarly, cpBVDV induces IFN-α/β in the early bovine fetus, but ncpBVDV does not. However, acute infection of naïve cattle with ncpBVDV results in IFN-α/β production. In this study, we identified and characterized a minor population of cells, present in lymph nodes that produce IFN-α in response to ncpBVDV. These cells expressed the myeloid markers CD14, CD11b, and CD172a but did not express CD4 and CD45RB. We also established that these cells produced IFN-α in the absence of detectable productive infection.
The Structure of the Poliovirus 135S Cell Entry Intermediate at 10-Angstrom Resolution Reveals the Location of an Externalized Polypeptide That Binds to Membranes
Authors: Bubeck Doryen , Filman David J. , Cheng Naiqian , Steven Alasdair C. , Hogle James M. , Belnap David M.
Published: May 2005

Poliovirus provides a well-characterized system for understanding how nonenveloped viruses enter and infect cells. Upon binding its receptor, poliovirus undergoes an irreversible conformational change to the 135S cell entry intermediate. This transition involves shifts of the capsid protein β barrels, accompanied by the externalization of VP4 and the N terminus of VP1. Both polypeptides associate with membranes and are postulated to facilitate entry by forming a translocation pore for the viral RNA. We have calculated cryo-electron microscopic reconstructions of 135S particles that permit accurate placement of the β barrels, loops, and terminal extensions of the capsid proteins. The reconstructions and resulting models indicate that each N terminus of VP1 exits the capsid though an opening in the interface between VP1 and VP3 at the base of the canyon that surrounds the fivefold axis. Comparison with reconstructions of 135S particles in which the first 31 residues of VP1 were proteolytically removed revealed that the externalized N terminus is located near the tips of propeller-like features surrounding the threefold axes rather than at the fivefold axes, as had been proposed in previous models. These observations have forced a reexamination of current models for the role of the 135S particle in transmembrane pore formation and suggest testable alternatives.
Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity
Authors: Mark-Danieli Michal , Laham Nihay , Kenan-Eichler Michal , Castiel Asher , Melamed Daniel , Landau Meytal , Bouvier Nicole M. , Evans Matthew J. , Bacharach Eran
Published: May 2005

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Ψ) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.
Indoleamine 2,3-Dioxygenase Mediates Cell Type-Specific Anti-Measles Virus Activity of Gamma Interferon
Authors: Obojes Karola , Andres Oliver , Kim Kwang Sik , Däubener Walter , Schneider-Schaulies Jürgen
Published: May 2005

Gamma interferon (IFN-γ) has been shown to be increased in sera from patients with acute measles and after vaccination, to exhibit protective functions in brains of patients with subacute sclerosing panencephalitis, and to mediate a noncytolytic clearance of measles virus (MV) from rodent brains. In order to reveal a possible intracellular antiviral activity in the absence of antigen presentation and cytotoxic T cells, we investigated IFN-γ-induced effects on MV replication in various tissue culture cells. While attenuated MV strains are more sensitive to IFN-α/β than are wild-type strains, IFN-γ inhibits the replication of all MV strains in epithelial, endothelial, and astroglial cells, but not in lymphoid and neuronal cell lines. The antiviral activity induced by IFN-γ correlates with the induction of indoleamine 2,3-dioxygenase (IDO), an enzyme of the tryptophan degradation pathway known to mediate antiviral as well as antibacterial and antiparasitic effects. The IFN-γ-induced antiviral activity can be overcome by the addition of excess amounts ofl-tryptophan, which indicates a specific role of IDO in the anti-MV activity. Our data suggest that the IFN-γ-induced enzyme IDO plays an important antiviral role in MV infections of epithelial, endothelial, and astroglial cells.
Resistance of Human Immunodeficiency Virus Type 1 to the High-Mannose Binding Agents Cyanovirin N and Concanavalin A
Authors: Witvrouw Myriam , Fikkert Valery , Hantson Anke , Pannecouque Christophe , O'Keefe Barry R. , McMahon James , Stamatatos Leonidas , de Clercq Erik , Bolmstedt Anders
Published: May 2005

Due to the biological significance of the carbohydrate component of the human immunodeficiency virus type 1 (HIV-1) glycoproteins in viral pathogenesis, the glycosylation step constitutes an attractive target for anti-HIV therapy. Cyanovirin N (CV-N), which specifically targets the high-mannose (HM) glycans on gp120, has been identified as a potent HIV-1 entry inhibitor. Concanavalin A (ConA) represents another mannose-binding lectin, although it has a lower specificity for HM glycans than that of CV-N. For the present study, we selected CV-N- and ConA-resistant HIV-1 strains in the presence of CV-N and ConA, respectively. Both resistant strains exhibited a variety of mutations eliminating N-linked glycans within gp120. Strains resistant to CV-N or ConA displayed high levels of cross-resistance towards one another. The N-glycan at position 302 was eliminated in both of the lectin-resistant strains. However, the elimination of this glycan alone by site-directed mutagenesis was not sufficient to render HIV-1 resistant to CV-N or ConA, suggesting that HIV-1 needs to mutate several N-glycans to become resistant to these lectins. Both strains also demonstrated clear cross-resistance towards the carbohydrate-dependent monoclonal antibody 2G12. In contrast, the selected strains did not show a reduced susceptibility towards the nonlectin entry inhibitors AMD3100 and enfuvirtide or towards reverse transcriptase or protease inhibitors. Recombination of the mutated
gp160
genes of the strains resistant to CV-N or ConA into a wild-type background fully reproduced the (cross-)resistance profiles of the originally selected strains, pointing to the impact of the N-glycan mutations on the phenotypic resistance profiles of both selected strains.
Systematic Identification of Antiprion Drugs by High-Throughput Screening Based on Scanning for Intensely Fluorescent Targets
Authors: Bertsch Uwe , Winklhofer Konstanze F. , Hirschberger Thomas , Bieschke Jan , Weber Petra , Hartl F. Ulrich , Tavan Paul , Tatzelt Jörg , Kretzschmar Hans A. , Giese Armin
Published: May 2005

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrPSc), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrPC/PrPScinteraction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 μM. Among these, six compounds displayed an inhibitory effect on PrPScpropagation in scrapie-infected N2a cells. Four of these candidate drugs share anN′-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrPCand PrPScis a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.
Interaction between the Human Cytomegalovirus UL82 Gene Product (pp71) and hDaxx Regulates Immediate-Early Gene Expression and Viral Replication
Authors: Cantrell Stacy R. , Bresnahan Wade A.
Published: May 2005

The human cytomegalovirus UL82-encoded pp71 protein is required for efficient virus replication and immediate-early gene expression when cells are infected at a low multiplicity. Functions attributed to pp71 include the ability to enhance the infectivity of viral DNA, bind to and target hypophosphorylated Rb family member proteins for degradation, drive quiescent cells into the cell cycle, and bind to the cellular protein hDaxx. Using UL82 mutant viruses, we demonstrate that the LXCXD motif within pp71 is not necessary for efficient virus replication in fibroblasts, suggesting that pp71's ability to degrade hypophosphorylated Rb family members and induce quiescent cells into the cell cycle is not responsible for the growth defect associated with a UL82 deletion mutant. However, UL82 mutants that cannot bind to hDaxx are unable to induce immediate-early gene expression and are severely attenuated for viral replication. These results indicate that the interaction between the human cytomegalovirus UL82 gene product (pp71) and hDaxx regulates immediate-early gene expression and viral replication.
Allosteric Effects of Ligands and Mutations on Poliovirus RNA-Dependent RNA Polymerase
Authors: Boerner Joanna E. , Lyle John M. , Daijogo Sarah , Semler Bert L. , Schultz Stephen C. , Kirkegaard Karla , Richards Oliver C.
Published: May 2005

Protein priming of viral RNA synthesis plays an essential role in the replication of picornavirus RNA. Both poliovirus and coxsackievirus encode a small polypeptide, VPg, which serves as a primer for addition of the first nucleotide during synthesis of both positive and negative strands. This study examined the effects on the VPg uridylylation reaction of the RNA template sequence, the origin of VPg (coxsackievirus or poliovirus), the origin of 3D polymerase (coxsackievirus or poliovirus), the presence and origin of interacting protein 3CD, and the introduction of mutations at specific regions in the poliovirus 3D polymerase. Substantial effects associated with VPg origin were traced to differences in VPg-polymerase interactions. The effects of 3CD proteins and mutations at polymerase-polymerase intermolecular Interface I were most consistent with allosteric effects on the catalytic 3D polymerase molecule. In conclusion, the efficiency and specificity of VPg uridylylation by picornavirus polymerases is greatly influenced by allosteric effects of ligand binding that are likely to be relevant during the viral replicative cycle.
Plant Virus-Derived Small Interfering RNAs Originate Predominantly from Highly Structured Single-Stranded Viral RNAs
Authors: Molnár Attila , Csorba Tibor , Lakatos Lóránt , Várallyay Éva , Lacomme Christophe , Burgyán József
Published: May 2005

RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected withCymbidium ringspot tombusvirus(CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show thatTobacco mosaic virus(TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation ofPDSsiRNAs than the corresponding antisensePDSsequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.
Stringent Requirement for the C Protein of Wild-Type Measles Virus for Growth both In Vitro and in Macaques
Authors: Takeuchi Kaoru , Takeda Makoto , Miyajima Naoko , Ami Yasushi , Nagata Noriyo , Suzaki Yuriko , Shahnewaz Jamila , Kadota Shin-ichi , Nagata Kyosuke
Published: May 2005

The P gene of measles virus (MV) encodes the P protein and three accessory proteins (C, V, and R). However, the role of these accessory proteins in the natural course of MV infection remains unclear. For this study, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C−), by using a reverse genetics system (M. Takeda, K. Takeuchi, N. Miyajima, F. Kobune, Y. Ami, N. Nagata, Y. Suzaki, Y. Nagai, and M. Tashiro, J. Virol.
74:
6643-6647). When 293 cells expressing the MV receptor SLAM (293/hSLAM) were infected with wtMV(C−) or parental wild-type MV (wtMV), the growth of wtMV(C−) was restricted, particularly during late stages. Enhanced green fluorescent protein-expressing wtMV(C−) consistently induced late-stage cell rounding and cell death in the presence of a fusion-inhibiting peptide, suggesting that the C protein can prevent cell death and is required for long-term MV infection. Neutralizing antibodies against alpha/beta interferon did not restore the growth restriction of wtMV(C−) in 293/hSLAM cells. When cynomolgus monkeys were infected with wtMV(C−) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C−) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV but dramatically reduced expression in the same tissues in a cynomolgus monkey infected with wtMV(C−). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.
Modified Vaccinia Virus Ankara Protects Macaques against Respiratory Challenge with Monkeypox Virus
Authors: Stittelaar Koert J. , van Amerongen Geert , Kondova Ivanela , Kuiken Thijs , van Lavieren Rob F. , Pistoor Frank H. M. , Niesters Hubert G. M. , van Doornum Gerard , van der Zeijst Ben A. M. , Mateo Luis , Chaplin Paul J. , Osterhaus Albert D. M. E.
Published: May 2005

The use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naïve and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and immunocompromised animals, and its efficacy against smallpox is currently being addressed. Here we directly compare the efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus and is therefore an important candidate for the protection of humans against smallpox.
T Cells with a CD4+CD25+Regulatory Phenotype Suppress In Vitro Proliferation of Virus-Specific CD8+T Cells during Chronic Hepatitis C Virus Infection
Authors: Boettler Tobias , Spangenberg Hans Christian , Neumann-Haefelin Christoph , Panther Elisabeth , Urbani Simonetta , Ferrari Carlo , Blum Hubert E. , von Weizsäcker Fritz , Thimme Robert
Published: May 2005

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8+T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4+CD25+regulatory phenotype in suppressing virus-specific CD8+T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8+T cells were inhibited by CD4+CD25+T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8+T cells but also to influenza virus-specific CD8+T cells. Importantly, CD4+CD25+T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8+T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4+CD25+cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4+CD25+T cells that are able to suppress CD8+T-cell responses to different viral antigens. Our results further suggest that CD4+CD25+T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.
Involvement of the C-Terminal Disulfide-Bonded Loop of Murine Leukemia Virus SU Protein in a Postbinding Step Critical for Viral Entry
Authors: Burkhart Michael D. , D'Agostino Paul , Kayman Samuel C. , Pinter Abraham
Published: May 2005

A role for the C-terminal domain (CTD) of murine leukemia virus (MuLV) Env protein in viral fusion was indicated by the potent inhibition of MuLV-induced fusion, but not receptor binding, by two rat monoclonal antibodies (MAbs) specific for epitopes in the CTD. Although these two MAbs, 35/56 and 83A25, have very different patterns of reactivity with viral isolates, determinants of both epitopes were mapped to the last C-terminal disulfide-bonded loop of SU (loop 10), and residues in this loop responsible for the different specificities of these MAbs were identified. Both MAbs reacted with a minor fraction of a truncated SU fragment terminating four residues after loop 10, indicating that while the deleted C-terminal residues were not part of these epitopes, they promoted their formation. Neither MAb recognized the loop 10 region expressed in isolated form, suggesting that these epitopes were not completely localized within loop 10 but required additional sequences located N terminal to the loop. Direct support for a role for loop 10 in fusion was provided by the demonstration that Env mutants containing an extra serine or threonine residue between the second and third positions of the loop were highly attenuated for infectivity and defective in fusion assays, despite wild-type levels of expression, processing, and receptor binding. Other mutations at positions 1 to 3 of loop 10 inhibited processing of the gPr80 precursor protein or led to increased shedding of SU, suggesting that loop 10 also affects Env folding and the stability of the interaction between SU and TM.
Inhibition of STAT 1 Phosphorylation by Human Parainfluenza Virus Type 3 C Protein
Authors: Malur Achut G. , Chattopadhyay Santanu , Maitra Ratan K. , Banerjee Amiya K.
Published: May 2005

The P mRNA of the viruses belonging to the subfamily
Paramyxovirinae
possesses a unique property of giving rise to several accessory proteins by a process that involves the utilization of overlapping open reading frames (the C proteins) and by an “RNA-editing” mechanism (the V proteins). Although these proteins are considered accessory, numerous studies have highlighted the importance of these proteins in virus transcription and interferon signaling, including our previous observation on the role of human parainfluenza virus type 3 (HPIV 3) C protein in the transcription of viral genome (Malur et al., Virus Res.
99:
199-204, 2004). In this report, we have addressed its role in interferon signaling by generating a stable cell line, L-C6, by using the lentiviral expression system which expresses HPIV 3 C protein. The L-C6 cells were efficient in abrogating both alpha and gamma interferon-induced antiviral states and demonstrated a drastic reduction in the formation of gamma-activated factor complexes in the cell extracts. Western blot analysis subsequently revealed a defect in the phosphorylation of STAT 1 in these cells. Taken together, our results indicate that HPIV 3 C protein is capable of counteracting the interferon signaling pathway by specifically inhibiting the activation of STAT 1.
Complementation of Human Papillomavirus Type 16 E6 and E7 by Jagged1-Specific Notch1-Phosphatidylinositol 3-Kinase Signaling Involves Pleiotropic Oncogenic Functions Independent of CBF1;Su(H);Lag-1 Activation
Authors: Veeraraghavalu Karthikeyan , Subbaiah Vanitha K. , Srivastava Sweta , Chakrabarti Oishee , Syal Ruchi , Krishna Sudhir
Published: May 2005

We have analyzed the induction and role of phosphatidylinositol 3-kinase (PI3K) by Notch signaling in human papillomavirus (HPV)-derived cancers. Jagged1, in contrast to Delta1, is preferentially upregulated in human cervical tumors. Jagged1 and not Delta1 expression sustained in vivo tumors by HPV16 oncogenes in HaCaT cells. Further, Jagged1 expression correlates with the rapid induction of PI3K-mediated epithelial-mesenchymal transition in both HaCaT cells and a human cervical tumor-derived cell line, suggestive of Delta1;Serrate/Jagged;Lag2 ligand-specific roles. Microarray analysis and dominant-negatives reveal that Notch-PI3K oncogenic functions can be independent of CBF1;Su(H);Lag-1 activation and instead relies on Deltex1, an alternative Notch effector.
Epstein-Barr Virus BZLF1 Protein Binds to Mitotic Chromosomes
Authors: Adamson Amy L.
Published: May 2005

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and is associated with several types of cancers, including nasopharyngeal carcinoma and Burkitt's lymphoma. An EBV protein that plays an integral role during lytic replication is the immediate-early protein BZLF1. Our laboratory has found that BZLF1 (Z) localizes to host chromosomes during mitosis. Two Z-interacting proteins are also found localized to mitotic chromosomes in the presence of Z. The association between Z and mitotic chromosomes may lead to the sequestering of Z-interacting proteins within the cell and potentially cause an alteration of chromosome compaction or chromatin structure.
Characterization of Nonstructural Protein Membrane Anchor Deletion Mutants Expressed in the Context of the Hepatitis C Virus Polyprotein
Authors: Gosert Rainer , Jendrsczok Wiebke , Berke Jan Martin , Brass Volker , Blum Hubert E. , Moradpour Darius
Published: May 2005

Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.
Role of N-Linked Glycosylation of the Hendra Virus Fusion Protein
Authors: Carter James Richard , Pager Cara Theresia , Fowler Stephen Derrick , Dutch Rebecca Ellis
Published: May 2005

The Hendra virus fusion (F) protein contains five potential sites for N-linked glycosylation in the ectodomain. Examination of F protein mutants with single asparagine-to-alanine mutations indicated that two sites in the F
2
subunit (N67 and N99) and two sites in the F
1
subunit (N414 and N464) normally undergo N-linked glycosylation. While N-linked modification at N414 is critical for protein folding and transport, F proteins lacking carbohydrates at N67, N99, or N464 remained fusogenically active. As N464 lies within heptad repeat B, these results contrast with those seen for several paramyxovirus F proteins.
G-Protein Signaling Triggered by R5 Human Immunodeficiency Virus Type 1 Increases Virus Replication Efficiency in Primary T Lymphocytes
Authors: Lin Yea-Lih , Mettling Clément , Portalès Pierre , Réant Brigitte , Clot Jacques , Corbeau Pierre
Published: May 2005

The binding of R5 envelope to CCR5 during human immunodeficiency virus type 1 (HIV-1) entry provokes cell activation, which has so far been considered to have no effect on virus replication, since signaling-defective CCR5 molecules have been shown to function normally as HIV-1 coreceptors on transformed cells or mitogen-stimulated T lymphocytes. As the background state of activation of these cells might have biased the results, we performed experiments using the same approach but with nonactivated primary T lymphocytes. We now report that the single R126N mutation in the DRY motif, involved in G-protein coupling, results in a signaling-defective CCR5 coreceptor with a drastically impaired capacity to support HIV-1 infection.
Keratinolytic potential ofBacillus licheniformisRG1: structural and biochemical mechanism of feather degradation
Authors: Ramnani Priya , Singh Rajni , Gupta Rani
Published: May 2005

Keratinolytic Bacillus licheniformis RG1 was used to study the mechanism of keratinolysis. Scanning electron microscopy studies revealed that bacterial cells grew closely adhered to the barbules of feathers, completely degrading them within 24 h. Biochemical studies indicated that the Bacillus strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (425 U) was synergistically enhanced by the addition of intracellular disulfide reductases (1712 U). However, these enzymes alone (keratinase and disulfide reductase), without live bacterial cells, failed to degrade the feather. Complete feather degradation was obtained only when living bacterial cells were present, emphasizing that bacterial adhesion plays a key role during the degradation process. The bacterial cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfite detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium.Key words: Bacillus licheniformis, disulfide reductase, keratinase, sulfitolysis.
Tricalcium phosphate solubilizing abilities ofTrichodermaspp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinumL.)
Authors: Rudresh D L , Shivaprakash M K , Prasad R D
Published: May 2005

Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 µg·mL–1), Trichoderma virens (PDBCTVs 12) (9.0 µg·mL–1), and Trichoderma virens (PDBCTVs 13) (8.83 µg·mL–1) solubilized 70% of that solubilzed by the reference strain Bacillus megaterium (12.43 µg·mL–1). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.Key words: Cicer arietinum, nutrient uptake, phosphate solubilization, plant yield, Trichoderma spp.
Functional characterization ofKlHEM13, a hypoxic gene ofKluyveromyces lactis
Authors: Blanco Moisés , Becerra Manuel , González-Siso M Isabel , Cerdán M Esperanza
Published: May 2005

The KlHEM13 gene of Kluyveromyces lactis encoding the coproporphyrinogen oxidase (EC 1.3.3.3), an oxygen-requiring enzyme that catalyzes the sixth step of heme biosynthesis, was cloned and functionally characterized. The coding and upstream regions of KlHEM13 were analyzed and the putative cis regulatory elements were discussed in relation to the mechanisms of regulation of this hypoxic gene in K. lactis.Key words: coproporphyrinogen oxidase (CPO), HEM13, hypoxic genes, Kluyveromyces lactis.
Lactobacillus reuteri β-galactosidase activity and low milk acidification ability
Authors: Hidalgo-Morales Madeleine , Robles-Olvera Victor , García Hugo S
Published: May 2005

β-galactosidase activity was studied as a possible cause of the low milk acidification ability observed in Lactobacillus reuteri NRRL 14171. Enzymatic activity was determined in MRS broth supplemented with either glucose or lactose and milk at the middle and final stage of the exponential phase, as well as at the stationary phase. Results were compared with β-galactosidase activity in Lactobacillus casei NRRL-B1922, a strain that shows the milk acidification ability. The effects of the types of carbon and nitrogen sources were established by comparison of growth parameters (higher maximum cell concentration and specific growth rate) in broth culture and skim milk supplemented with 2% glucose or 1% casein peptone. In milk, L. reuteri showed higher β-galactosidase activity in all growth phases compared with L. casei. Greater cell concentration maxima, specific growth rates, and acidification abilities were observed in L. reuteri when it was cultured in milk supplemented with 1% casein peptone compared with non-supplemented milk cultures. Results suggest that the poor milk acidification ability observed in L. reuteri may be more related to a weak proteolytic system than to deficient β-galactosidase activity.Key words: Lactobacillus reuteri, growth parameters, β-galactosidase, acidification.
Direct cloning of genes encoding novel xylanases from the human gut
Authors: Hayashi Hidenori , Abe Takashi , Sakamoto Mitsuo , Ohara Hiroki , Ikemura Toshimichi , Sakka Kazuo , Benno Yoshimi
Published: May 2005

The aim of this study was to identify a novel 1,4-β-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43 552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 °C and stable up to 50 °C at pH 6.5 and over the pH range 4.0–11.0 at 25 °C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.Key words: xylanase, human gut, fecal microbiota, phylogenetic analysis, self-organizing map.
Characterization of a second gene encoding γ-glutamyl transpeptidase from Schizosaccharomyces pombe
Authors: Park Hey-Jung , Moon Jeong-Su , Kim Hong-Gyum , Kim Il-Han , Kim Kanghwa , Park Eun-Hee , Lim Chang-Jin
Published: May 2005

The first gene encoding γ-glutamyl transpeptidase (GGTI) of the fission yeast has previously been characterized, and its expression was found to be regulated by various oxidative stress-inducing agents. In this work, a second gene, encoding GGTII, was cloned and characterized from the fission yeast Schizosaccharomyces pombe. The structural gene encoding GGTII was amplified from the genomic DNA of the fission yeast and ligated into the shuttle vector pRS316 to generate the recombinant plasmid pPHJ02. The determined sequence contains 3040 bp and is able to encode the putative 611 amino acid sequence of GGTII, which resembles the counterparts of Saccharomyces cerevisiae, Homo sapiens, Rattus norvegicus, and Escherichia coli. The DNA sequence also contains 940-bp upstream and 289-bp downstream regions of the GGTII gene. The Schizosaccharomyces pombe cells harboring plasmid pPHJ02 showed about 4-fold higher GGT activity in the exponential phase than the cells harboring the vector only, indicating that the cloned GGTII gene is functional. The S. pombe cells containing the cloned GGTII gene were found to contain higher levels of both intracellular glutathione (GSH) content and GSH uptake. The S. pombe cells harboring plasmid pPHJ02 showed increased survival on solid media containing hydrogen peroxide, diethylmaleate, aluminum chloride, cadmium chloride, or mercuric chloride. The GGTII mRNA level was significantly elevated by treatment with GSH-depleting diethylmaleate. These results imply that the S. pombe GGTII gene produces functional GGTII protein and is involved in the response to oxidative stresses in S. pombe cells.Key words: fission yeast, genomic DNA, γ-glutamyl transpeptidase, regulation, Schizosaccharomyces pombe, stress response.
A GENERALIZATION OF SEMISIMPLE ARTINIAN RINGS
Authors: HIRANO YASUYUKI , TSUTSUI HISAYA
Published: May 2005

We investigate a ring R with the property that for every right R-module M and every ideal I of R the annihilator of I in M is a direct summand of M, and determine conditions under which such a ring is semisimple Artinian.
SOME ASPECTS OF MODULAR LATTICES WITH DUAL KRULL DIMENSION
Authors: TEPLY MARK L. , RIM SEOG HOON
Published: May 2005

For an ordinal α, a modular lattice L with 0 and 1 is α-atomic if L has dual Krull dimension α but each interval [0,x] with x < 1 has dual Krull dimension <α. The properties of α-atomic lattices are presented and applied to module theory. The endomorphism ring of certain types of α-atomic modules is a local domain and hence there is a Krull–Schmidt type theorem for those α-atomic modules.
INVARIANTS OF MIXED REPRESENTATIONS OF QUIVERS I
Authors: ZUBKOV A. N.
Published: May 2005

We define new concept of mixed representations of quivers which naturally arose from the adjoint action of orthogonal or symplectic groups on several matrices. We find generating invariants of mixed representations of quivers and prepare necessary facts to describe defining relations between them.
INVARIANTS OF MIXED REPRESENTATIONS OF QUIVERS II: DEFINING RELATIONS AND APPLICATIONS
Authors: ZUBKOV A. N.
Published: May 2005

We describe defining relations between invariants of mixed representations of quivers introduced in the first part. Some applications for invariants of the adjoint action of orthogonal or symplectic groups on several matrices are given.
ON THE COXETER COMPLEX AND ALVIS–CURTIS DUALITY FOR PRINCIPAL ℓ-BLOCKS OF GLn(q)
Authors: LINCKELMANN MARKUS , SCHROLL SIBYLLE
Published: May 2005

M. Cabanes and J. Rickard showed in [3] that the Alvis–Curtis character duality of a finite group of Lie type is induced in non defining characteristic ℓ by a derived equivalence given by tensoring with a bounded complex X, and they further conjecture that this derived equivalence should actually be a homotopy equivalence. Following a suggestion of R. Kessar, we show here for the special case of principal blocks of general linear groups with abelian Sylow-ℓ-subgroups that this is true, by an explicit verification relating the complex X to the Coxeter complex of the corresponding Weyl group.