bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Transactivator Function of Cottontail Rabbit Papillomavirus E2 Is Essential for Tumor Induction in Rabbits
Authors: Jeckel Sonja , Huber Evamaria , Stubenrauch Frank , Iftner Thomas 
Published: October 2002

ABSTRACT
Infection of domestic rabbits with cottontail rabbit papillomavirus (CRPV) causes local papillomas which progress to carcinomas in more than 80% of cases. This animal model system therefore allows the identification of molecular mechanisms required for the induction and progression of epithelial tumors. The viral E2 protein stimulates both viral DNA replication and transcription, and these functions can be genetically separated. We introduced the respective mutations into CRPV E2 and found, in line with published data for other papillomavirus E2 proteins, that mutation of the highly conserved amino acid 37 or 73 resulted in replication-competent but transactivation-deficient E2 proteins, whereas E2 proteins with mutations at residue 39 were replication deficient and transactivation competent. The R37A, I73L, and I73A E2 mutants, showing a loss of transactivation function, and the R37K E2 mutant, which is still transactivation competent, were introduced into the whole genome of CRPV, which was then injected into the skin of rabbits. Strikingly, the ability to induce tumors within 6 weeks was abolished by each of the E2 mutations, in contrast to the tumor induction rate (93%) obtained with wild-type CRPV DNA. Two small papillomas induced by mutant E2 I73A CRPV DNA appeared as late as 12 or 24 weeks postinjection, were significantly smaller, and showed no further extension of growth. These data suggest that functionally conserved amino acids in the transactivation domain of E2 are also required for the induction and growth of epithelial tumors in rabbits infected with CRPV.
Mandelic Acid Condensation Polymer: Novel Candidate Microbicide for Prevention of Human Immunodeficiency Virus and Herpes Simplex Virus Entry
Authors: Herold B. C. , Scordi-Bello I. , Cheshenko N. , Marcellino D. , Dzuzelewski M. , Francois F. , Morin R. , Casullo V. Mas , Anderson R. A. , Chany C. , Waller D. P. , Zaneveld L. J. D. , Klotman M. E. 
Published: October 2002

ABSTRACTPresently marketed vaginal barrier methods are cytotoxic and damaging to the vaginal epithelium and natural vaginal flora when used frequently. Novel noncytotoxic agents are needed to protect men and women from sexually transmitted diseases. One novel candidate is a mandelic acid condensation polymer, designated SAMMA. The spectrum and mechanism of antiviral activity were explored using clinical isolates and laboratory-adapted strains of human immunodeficiency virus (HIV) and herpes simplex virus (HSV). SAMMA is highly effective against all CCR5 and CXCR4 isolates of HIV in primary human macrophages and peripheral blood mononuclear cells. SAMMA also inhibits infection of cervical epithelial cells by HSV. Moreover, it exhibits little or no cytotoxicity and has an excellent selectivity index. SAMMA, although not a sulfonated or sulfated polymer, blocks the binding of HIV and HSV to cells by targeting the envelope glycoproteins gp120 and gB-2, respectively, and also inhibits HSV entry postattachment. SAMMA is an excellent, structurally novel candidate microbicide that warrants further preclinical evaluation.
Roles of Nonstructural Protein nsP2 and Alpha/Beta Interferons in Determining the Outcome of Sindbis Virus Infection
Authors: Frolova Elena I. , Fayzulin Rafik Z. , Cook Susan H. , Griffin Diane E. , Rice Charles M. , Frolov Ilya 
Published: October 2002

ABSTRACTAlphaviruses productively infect a variety of vertebrate and insect cell lines. In vertebrate cells, Sindbis virus redirects cellular processes to meet the needs of virus propagation. At the same time, cells respond to virus replication by downregulating virus growth and preventing dissemination of the infection. The balance between these two mechanisms determines the outcome of infection at the cellular and organismal levels. In this report, we demonstrate that a viral nonstructural protein, nsP2, is a significant regulator of Sindbis virus-host cell interactions. This protein not only is a component of the replicative enzyme complex required for replication and transcription of viral RNAs but also plays a role in suppressing the antiviral response in Sindbis virus-infected cells. nsP2 most likely acts by decreasing interferon (IFN) production and minimizing virus visibility. Infection of murine cells with Sindbis virus expressing a mutant nsP2 leads to higher levels of IFN secretion and the activation of 170 cellular genes that are induced by IFN and/or virus replication. Secreted IFN protects naive cells against Sindbis virus infection and also stops viral replication in productively infected cells. Mutations in nsP2 can also attenuate Sindbis virus cytopathogenicity. Such mutants can persist in mammalian cells with defects in the alpha/beta IFN (IFN-α/β) system or when IFN activity is neutralized by anti-IFN-α/β antibodies. These findings provide new insight into the alphavirus-host cell interaction and have implications for the development of improved alphavirus expression systems with better antigen-presenting potential.
Sialic Acid Functions in Enterovirus 70 Binding and Infection
Authors: Alexander David A. , Dimock Kenneth 
Published: October 2002

ABSTRACTThe interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for enterovirus 70 (EV70), a member of thePicornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.
Infectious Bursal Disease Virus Capsid Protein VP3 Interacts both with VP1, the RNA-Dependent RNA Polymerase, and with Viral Double-Stranded RNA
Authors: Tacken Mirriam G. J. , Peeters Ben P. H. , Thomas Adri A. M. , Rottier Peter J. M. , Boot Hein J. 
Published: October 2002

ABSTRACT

Infectious bursal disease virus
(IBDV) is a double-stranded RNA (dsRNA) virus of the
Birnaviridae
family. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shell capsid that is composed of VP2 and VP3. In this study we identified the domains responsible for the interaction between VP3 and VP1. Using the yeast two-hybrid system we found that VP1 binds to VP3 through an internal domain, while VP3 interacts with VP1 solely by its carboxy-terminal 10 amino acids. These results were confirmed by using a reverse-genetics system that allowed us to analyze the interaction of carboxy-terminally truncated VP3 molecules with VP1 in infected cells. Coimmunoprecipitations with VP1- and VP3-specific antibodies revealed that the interaction is extremely sensitive to truncation of VP3. The mere deletion of the C-terminal residue reduced coprecipitation almost completely and also fully abolished production of infectious virions. Surprisingly, these experiments additionally revealed that VP3 also binds to RNA. RNase treatments and reverse transcription-PCR analyses of the immunoprecipitates demonstrated that VP3 interacts with dsRNA of both viral genome segments. This interaction is not mediated by the carboxy-terminal domain of VP3 since C-terminal truncations of 1, 5, or 10 residues did not prevent formation of the VP3-dsRNA complexes. VP3 seems to be the key organizer of birnavirus structure, as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1, and the two genomic dsRNAs.
Role of the Alfalfa Mosaic Virus Methyltransferase-Like Domain in Negative-Strand RNA Synthesis
Authors: Vlot A. Corina , Menard Aymeric , Bol John F. 
Published: October 2002

ABSTRACT

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in
Nicotiana benthamiana
by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in
trans
by coexpression of wt P1.
Adenovirus RIDβ Subunit Contains a Tyrosine Residue That Is Critical for RID-Mediated Receptor Internalization and Inhibition of Fas- and TRAIL-Induced Apoptosis
Authors: Lichtenstein Drew L. , Krajcsi Peter , Esteban David J. , Tollefson Ann E. , Wold William S. M. 
Published: October 2002

ABSTRACTThe adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDα and RIDβ subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these “death” receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDβ that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDβ were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDβ C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxφ). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDβ but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.
A Limited Number of Transducible Hepatocytes Restricts a Wide-Range Linear Vector Dose Response in Recombinant Adeno-Associated Virus-Mediated Liver Transduction
Authors: Nakai Hiroyuki , Thomas Clare E. , Storm Theresa A. , Fuess Sally , Powell Sharon , Wright J. Fraser , Kay Mark A. 
Published: October 2002

ABSTRACT

Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or
Escherichia coli lacZ
-expressing AAV serotype 2-based vectors at doses ranging between 4.0 × 10
8
and 1.1 × 10
13
vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 × 10
9
to 3.0 × 10
11
vg/mouse. Vector doses above 3.0 × 10
11
vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 × 10
12
vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 × 10
12
vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.
Regulation of Bovine Papillomavirus Replicative Helicase E1 by the Ubiquitin-Proteasome Pathway
Authors: Malcles Marie-Helene , Cueille Nathalie , Mechali Francisca , Coux Olivier , Bonne-Andrea Catherine 
Published: October 2002

ABSTRACT

Papillomaviruses maintain their genomes in a relatively constant copy number as stable extrachromosomal plasmids in the nuclei of dividing host cells. The viral initiator of replication, E1, is not detected in papillomavirus-infected cells. Here, we present evidence that E1 encoded by bovine papillomavirus type 1 is an unstable protein that is degraded through the ubiquitin-proteasome pathway. In a cell-free system derived from
Xenopus
egg extracts, E1 degradation is regulated by both cyclin E/Cdk2 binding and E1 replication activity. Free E1 is readily ubiquitinated and degraded by the proteasome, while it becomes resistant to this degradation pathway when bound to cyclin E/Cdk2 complexes before the start of DNA synthesis. This stabilization is reversed in a process involving E1-dependent replication activity. In transiently transfected cells, E1 is also polyubiquitinated and accumulates when proteasome activity is inhibited. Thus, the establishment and maintenance of a stable number of papillomavirus genomes in latently infected cells are in part a function of regulated ubiquitin-mediated degradation of E1.
The p127 Subunit (DDB1) of the UV-DNA Damage Repair Binding Protein Is Essential for the Targeted Degradation of STAT1 by the V Protein of the Paramyxovirus Simian Virus 5
Authors: Andrejeva J. , Poole E. , Young D. F. , Goodbourn S. , Randall R. E. 
Published: October 2002

ABSTRACTThe V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.
Use of Immunostimulatory Sequence-Containing Oligonucleotides as Topical Therapy for Genital Herpes Simplex Virus Type 2 Infection
Authors: Pyles Richard B. , Higgins Debbie , Chalk Claudia , Zalar Anthony , Eiden Joseph , Brown Carrie , Van Nest Gary , Stanberry Lawrence R. 
Published: October 2002

ABSTRACTSynthetic oligonucleotides containing CpG motifs in specific sequence contexts have been shown to induce potent immune responses. We have evaluated mucosal administration of two immunostimulatory sequence (ISS)-containing phosphorothioate-stabilized oligonucleotides for antiherpetic efficacy in animal models. The ISS oligonucleotides, suspended in phosphate-buffered saline, were tested in mouse and guinea pig vaginal models of herpes simplex virus type 2 (HSV-2) infection. For comparison, groups of untreated, non-ISS oligonucleotide-treated, and acyclovir-treated animals also were monitored. The results indicated that vaginal epithelial application of ISS (up to 6 h after viral inoculation) with mice lethally challenged with HSV-2 delayed disease onset and reduced the number of animals that developed signs of disease (P= 0.003). ISS application significantly increased survival rates over those of controls (P= 0.0014). The ISS also impacted an established infection in the guinea pig model of HSV-2 disease. A single administration of ISS (21 days after viral inoculation) significantly reduced the frequency and severity of HSV-2 lesions compared to results with non-ISS oligonucleotide-treated and untreated guinea pigs (P< 0.01). HSV-2 is shed from the vaginal cavity of the guinea pig in the absence of lesions, similar to the case with humans. As an additional indication of ISS efficacy, the magnitude of viral shedding also was significantly reduced in ISS-treated animals (P< 0.001). These effects appeared to be immunologically mediated, since ISS had no direct effect on HSV-2 replication in vitro using standard plaque assays. These data suggest that ISS may be useful in the treatment and control of genital herpes in humans.
Effective Postexposure Treatment of Retrovirus-Induced Disease with Immunostimulatory DNA Containing CpG Motifs
Authors: Olbrich Anke R. M. , Schimmer Simone , Heeg Klaus , Schepers Koen , Schumacher Ton N. M. , Dittmer Ulf 
Published: October 2002

ABSTRACTTherapeutic strategies for the treatment of acute retroviral infections have relied mainly on antiviral drugs. In this study we used the Friend virus model system to demonstrate that enhancement of the immune system can also have dramatic therapeutic effects. Since resistance to Friend virus-induced leukemia in mice is associated with T helper cell type 1 (Th1) immune responses, we enhanced these responses in susceptible mice by treatment with synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN). Treatments begun at 4 days postinfection increased recovery from 6% in the control group to 74% in the CpG-treated group. CpG-mediated recovery was associated with a significant reduction of viral loads in the blood and spleens of treated mice compared to those of control animals. The treatment promoted Th1-type cytokine production by splenocytes of Friend virus-infected mice and augmented Friend virus-specific cytotoxic T-cell responses, but no influence on the virus-specific neutralizing antibody response was observed. Friend virus-specific CD8+T cells were critical for effective treatment with CpG-ODN, since in vivo depletion of these cells from treated mice prevented their recovery. Our results demonstrate that CpG-ODN therapy can significantly enhance virus-specific cellular immune responses and prevent retrovirus-induced disease. These findings may have implications for antiviral therapy in general.
Tropism of Varicella-Zoster Virus for Human Tonsillar CD4+T Lymphocytes That Express Activation, Memory, and Skin Homing Markers
Authors: Ku Chia-Chi , Padilla Jorge A. , Grose Charles , Butcher Eugene C. , Arvin Ann M. 
Published: October 2002

ABSTRACTVaricella-zoster virus (VZV) is an alphaherpesvirus with the characteristic neurotropism of this group, but VZV also infects T cells productively and downregulates major histocompatibility complex (MHC) class I expression on infected T cells, as shown in the SCID-hu mouse model. T-cell tropism is likely to be critical for the cell-associated viremia associated with primary VZV infection. In these experiments, we found that VZV infects human tonsillar CD4+T cells in culture, with 15 to 25% being positive for VZV proteins as detected by polyclonal anti-VZV immunoglobulin G (IgG) staining and monitored by flow cytometry analysis. RNA transcripts for VZV gE, a late gene product, were detected in T-cell populations that expressed VZV surface proteins, but not in the VZV-negative cell fraction. Exposure to phorbol myristate acetate resulted in an increase in VZV-positive T cells, indicating that viral DNA was present within these cells and that VZV gene expression could be induced by T-cell activation. By immune scanning electron microscopy, VZV virions were detected in abundance on the surfaces of infected tonsillar T cells. The predominant CD4+T-lymphocyte subpopulations that became infected were activated CD69+T cells with the CD45RA−memory phenotype. Subsets of CD4+T cells that expressed skin homing markers, cutaneous leukocyte antigen, and chemokine receptor 4 were also infected with VZV. By chemotaxis assay, VZV-infected T cells migrated to SDF-1, demonstrating that skin migratory function was intact despite VZV infection. The susceptibility of tonsil T cells to VZV suggests that these cells may be important targets during the initial VZV infection of upper respiratory tract sites. Viral transfer to migrating T cells in the tonsils may facilitate cell-associated viremia, and preferential infection of CD4 T cells that express skin homing markers may enhance VZV transport to cutaneous sites of replication.