bluuebunny/crossref_metadata_2025_split huggingface dataset:
Infectious Entry by Amphotropic as well as Ecotropic Murine Leukemia Viruses Occurs through an Endocytic Pathway
Authors: Katen Louis J. , Januszeski Michael M. , Anderson W. French , Hasenkrug Kim J. , Evans Leonard H. 
Published: July 2002

ABSTRACT

Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH
4
Cl and bafilomycin A. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37°C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. We also found that infectious viruses were efficiently internalized during treatment. This indicated that the arrest occurred in an intracellular compartment and that the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of this study pertains to the interpretation of experiments in which agents that block endocytic acidification inhibit infectivity. As we have found with the MuLVs, inhibition of infectivity may be secondary to the block of endocytic acidification. While this strongly suggests the involvement of an endocytic pathway, it does not necessarily indicate a requirement for an acidic compartment during the infectious process. Likewise, a lack of inhibition during transient treatment with the drugs would not preclude an endocytic pathway for viruses that are stable during the course of the treatment.
Efficient Transmission of Two Different Sheep Scrapie Isolates in Transgenic Mice Expressing the Ovine PrP Gene
Authors: Crozet Carole , Flamant Frederic , Bencsik Anna , Aubert Denise , Samarut Jacques , Baron Thierry 
Published: July 2002

ABSTRACT
We produced transgenic mice expressing the sheep prion protein to obtain a sensitive model for sheep spongiform encephalopathies (scrapie). The complete open reading frame, with alanine, arginine, and glutamine at susceptibility codons 136, 154, and 171, respectively, was inserted downstream from the neuron-specific enolase promoter. A mouse line, Tg(OvPrP4), devoid of the murine PrP gene, was obtained by crossing with PrP knockout mice. Tg(OvPrP4) mice were shown to selectively express sheep PrP in their brains, as demonstrated in mRNA and protein analysis. We showed that these mice were susceptible to infection by sheep scrapie following intracerebral inoculation with two natural sheep scrapie isolates, as demonstrated not only by the occurrence of neurological signs but also by the presence of the spongiform changes and abnormal prion protein accumulation in their brains. Mean times to death of 238 and 290 days were observed with these isolates, but the clinical course of the disease was strikingly different in the two cases. One isolate led to a very early onset of neurological signs which could last for prolonged periods before death. Independently of the incubation periods, some of the mice inoculated with this isolate showed low or undetectable levels of PrPsc, as detected by both Western blotting and immunohistochemistry. The development of experimental scrapie in these mice following inoculation of the scrapie infectious agent further confirms that neuronal expression of the PrP open reading frame alone is sufficient to mediate susceptibility to spongiform encephalopathies. More importantly, these mice provide a new and promising tool for studying the infectious agents in sheep spongiform encephalopathies.
The Late Stage of Human Immunodeficiency Virus Type 1 Assembly Is an Energy-Dependent Process
Authors: Tritel Marc , Resh Marilyn D. 
Published: July 2002

ABSTRACT
Several recent studies have indicated the involvement of host cell factors in human immunodeficiency virus type 1 (HIV-1) assembly. To ascertain whether ATP-dependent factors play a role in this process, we quantified virus-like particle (VLP) production by ATP-depleted cells. Pharmacological ATP depletion abrogated VLP production without affecting cell viability or inducing degradation of HIV-1 Gag protein. This effect occurred even when the ATP-depleting agents were added 1 h into the assembly process, and it was reversed by removal of these agents. ATP depletion did not affect Gag membrane binding or multimerization. Density gradient analysis indicated that HIV-1 assembly intermediates were stalled late in the assembly process. This conclusion was further supported by electron microscopy analysis, which revealed a preponderance of plasma membrane-associated stalk-like structures in the ATP-depleted cells. Since no HIV-1 proteins bind or hydrolyze ATP, these findings indicate that an ATP-requiring cellular factor is an obligatory participant late in the HIV-1 assembly process.
Epstein-Barr Virus Immediate-Early Protein BRLF1 Induces the Lytic Form of Viral Replication through a Mechanism Involving Phosphatidylinositol-3 Kinase Activation
Authors: Darr Catherine Dayle , Mauser Amy , Kenney Shannon 
Published: July 2002

ABSTRACT
Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.
Role of the Promyelocytic Leukemia Protein PML in the Interferon Sensitivity of Lymphocytic Choriomeningitis Virus
Authors: Djavani Mahmoud , Rodas Juan , Lukashevich Igor S. , Horejsh Douglas , Pandolfi Pier Paolo , Borden Katherine L. B. , Salvato Maria S. 
Published: July 2002

ABSTRACT

Lymphocytic choriomeningitis virus (LCMV) induces type I interferon (alpha and beta interferon [IFN-α and IFN-β]) upon infection and yet is sensitive to the addition of type II interferon (gamma interferon [IFN-γ]) to the culture media. This sensitivity is biologically important because it correlates inversely with the ability of certain LCMV strains to persist in mice (D. Moskophidis, M. Battegay, M. A. Bruendler, E. Laine, I. Gresser, and R. M. Zinkernagel, J. Virol. 68:1951-1955, 1994). The cellular oncoprotein PML is induced by both IFN-
α
/
β
and IFN-
γ
, and PML binds the LCMV Z protein and becomes redistributed within cells from nucleus to cytoplasm upon LCMV infection. In the present study, increased PML expression results in diminished LCMV replication, implicating PML in the IFN sensitivity of LCMV. Virus production in PML −/− murine embryonic fibroblasts (MEF) exceeds virus production in PML +/+ MEF, and this difference is exacerbated by IFN treatment that upregulates PML expression. IFN-γ also diminishes LCMV production in PML −/− cells; therefore, viral IFN sensitivity is not entirely due to PML. Both viral mRNA production and viral protein production decrease as PML expression increases. Here we propose that PML reduces LCMV transcription through its interaction with the Z protein.
Population Genetic Analysis of the Protease Locus of Human Immunodeficiency Virus Type 1 Quasispecies Undergoing Drug Selection, Using a Denaturing Gradient-Heteroduplex Tracking Assay
Authors: Doukhan Laurence , Delwart Eric 
Published: July 2002

ABSTRACT
Monitoring the evolution of human immunodeficiency virus type 1 (HIV-1) drug resistance requires measuring the frequency of closely related genetic variants making up the complex viral quasispecies found in vivo. In order to resolve both major and minor (≥2%) protease gene variants differing by one or more nucleotide substitutions, we analyzed PCR products derived from plasma viral quasispecies by using a combination of denaturing gradient gel electrophoresis and DNA heteroduplex tracking assays. Correct population sampling of the high level of genetic diversity present within viral quasispecies could be documented by parallel analysis of duplicate, independently generated PCR products. The composition of genetically complex protease gene quasispecies remained constant over short periods of time in the absence of treatment and while plasma viremia fell >100-fold following the initiation of protease inhibitor ritonavir monotherapy. Within a month of initiating therapy, a strong reduction in the genetic diversity of plasma viral populations at the selected protease locus was associated with rising plasma viremia and the emergence of drug resistance. The high levels of protease genetic diversity seen before treatment reemerged only months later. In one patient, reduction in genetic diversity at the protease gene was observed concomitantly with an increase in diversity at the envelope gene (E. L. Delwart, P. Heng, A. Neumann, and M. Markowitz, J. Virol. 72:2416-2421, 1998), indicating that opposite population genetic changes can take place in different HIV-1 loci. The rapid emergence of drug-resistant HIV-1 was therefore associated with a strong, although only transient, reduction in genetic diversity at the selected locus. The denaturing gradient-heteroduplex tracking assay is a simple method for the separation and quantitation of very closely related, low-frequency, genetic variants within complex viral populations.
E Box-Dependent Activation of Telomerase by Human Papillomavirus Type 16 E6 Does Not Require Induction of c-
myc
Authors: Gewin Lindy , Galloway Denise A. 
Published: July 2002

ABSTRACT

Human papillomavirus type 16 (HPV-16) E6 activates telomerase specifically in epithelial cells. The oncogene c-
myc
has also been shown to activate telomerase in several cell types. Here we show that while both HPV-16 E6 and c-
myc
require intact E boxes to transactivate the hTERT promoter, E6 does not induce hTERT transcription simply by inducing expression of c-
myc
. Moreover, hTERT transactivation by HPV-16 E6 correlates with its ability to bind the cellular E6-associated protein (E6AP), suggesting that E6 and E6AP may target a regulator of hTERT expression.
Binding of Human Immunodeficiency Virus Type 1 gp120 to CXCR4 Induces Mitochondrial Transmembrane Depolarization and Cytochromec-Mediated Apoptosis Independently of Fas Signaling
Authors: Roggero Rodolphe , Robert-Hebmann Véronique , Harrington Steve , Roland Joachim , Vergne Laurence , Jaleco Sara , Devaux Christian , Biard-Piechaczyk Martine 
Published: July 2002

ABSTRACTApoptosis of CD4+T lymphocytes, induced by contact between human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120) and its receptors, could contribute to the cell depletion observed in HIV-infected individuals. CXCR4 appears to play an important role in gp120-induced cell death, but the mechanisms involved in this apoptotic process remain poorly understood. To get insight into the signal transduction pathways connecting CXCR4 to apoptosis following gp120 binding, we used different cell lines expressing wild-type CXCR4 and a truncated form of CD4 that binds gp120 but lacks the ability to transduce signals. The present study demonstrates that (i) the interaction of cell-associated gp120 with CXCR4-expressing target cells triggers a rapid dissipation of the mitochondrial transmembrane potential resulting in the cytosolic release of cytochromecfrom the mitochondria to cytosol, concurrent with activation of caspase-9 and -3; (ii) this apoptotic process is independent of Fas signaling; and (iii) cooperation with a CD4 signal is not required. In addition, following coculture with cells expressing gp120, a Fas-independent apoptosis involving mitochondria and caspase activation is also observed in primary umbilical cord blood CD4+T lymphocytes expressing high levels of CXCR4. Thus, this gp120-mediated apoptotic pathway may contribute to CD4+T-cell depletion in AIDS.
Effect of Soluble CD4 on Exposure of Epitopes on Primary, Intact, Native Human Immunodeficiency Virus Type 1 Virions of Different Genetic Clades
Authors: Mbah Henry A. , Burda Sherri , Gorny Miroslaw K. , Williams Constance , Revesz Kathy , Zolla-Pazner Susan , Nyambi Phillipe N. 
Published: July 2002

ABSTRACT
We have used a virus-binding assay to examine conformational changes that occur when soluble CD4 (sCD4) binds to the surface of intact, native, primary human immunodeficiency virus type 1 virions. The isolates examined belong to seven genetic clades (A to H) and are representative of syncytium-inducing and non-syncytium-inducing phenotypes. Conformational changes in epitopes in the C2, V2, V3, C5, and CD4 binding domain (CD4bd) of gp120 and the cluster I and II regions of gp41 of these viruses were examined using human monoclonal antibodies that are directed at these regions. The studies revealed that sCD4 binding causes a marked increase in exposure of epitopes in the V3 loop, irrespective of the clade or the phenotype of the virus. Sporadic increases in exposure were observed in some epitopes in the V2 region, while no changes were observed in the C2, C5, or CD4bd of gp120 or the cluster I and II regions of gp41.
Differential Immunogenicity of Epstein-Barr Virus Latent-Cycle Proteins for Human CD4+T-Helper 1 Responses
Authors: Leen Ann , Meij Pauline , Redchenko Irina , Middeldorp Jaap , Bloemena Elisabeth , Rickinson Alan , Blake Neil 
Published: July 2002

ABSTRACTHuman CD4+T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8+memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4+epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4+T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4+T-helper 1 cells (EBNA1, EBNA3C ≫ LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8+-T-cell responses (EBNA3C > EBNA1 > LMP2 ≫ LMP1). Furthermore, the range of CD4+memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8+-T-cell frequencies.
Inducible Expression of Inflammatory Chemokines in Respiratory Syncytial Virus-Infected Mice: Role of MIP-1α in Lung Pathology
Authors: Haeberle Helene A. , Kuziel William A. , Dieterich Hans-Juergen , Casola Antonella , Gatalica Zoran , Garofalo Roberto P. 
Published: July 2002

ABSTRACTLower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1β, MIP-1α, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (107PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1α, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1α gene (−/− mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of −/− mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.
Involvement of Toll-Like Receptor 4 in Innate Immunity to Respiratory Syncytial Virus
Authors: Haynes Lia M. , Moore Deborah D. , Kurt-Jones Evelyn A. , Finberg Robert W. , Anderson Larry J. , Tripp Ralph A. 
Published: July 2002

ABSTRACTThe mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14+cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.
Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein
Authors: Deml Ludwig , Bojak Alexandra , Steck Stephanie , Graf Marcus , Wild Jens , Schirmbeck Reinhold , Wolf Hans , Wagner Ralf 
Published: July 2002

ABSTRACTWe have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt)gaggene flanked by essentialcis-acting sites such as the 5′-untranslated region and 3′-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with thesyngagplasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt)gagvector system. In contrast, intradermal immunization of both wtgagandsyngagvector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated withinsyngagby codon optimization do not contribute significantly to the high immunogenicity of thesyngagplasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of thesyngagDNA vaccine.
Common Origin of Four Diverse Families of Large Eukaryotic DNA Viruses
Authors: Iyer Lakshminarayan M. , Aravind L. , Koonin Eugene V. 
Published: July 2002

ABSTRACTComparative analysis of the protein sequences encoded in the genomes of three families of large DNA viruses that replicate, completely or partly, in the cytoplasm of eukaryotic cells (poxviruses, asfarviruses, and iridoviruses) and phycodnaviruses that replicate in the nucleus reveals 9 genes that are shared by all of these viruses and 22 more genes that are present in at least three of the four compared viral families. Although orthologous proteins from different viral families typically show weak sequence similarity, because of which some of them have not been identified previously, at least five of the conserved genes appear to be synapomorphies (shared derived characters) that unite these four viral families, to the exclusion of all other known viruses and cellular life forms. Cladistic analysis with the genes shared by at least two viral families as evolutionary characters supports the monophyly of poxviruses, asfarviruses, iridoviruses, and phycodnaviruses. The results of genome comparison allow a tentative reconstruction of the ancestral viral genome and suggest that the common ancestor of all of these viral families was a nucleocytoplasmic virus with an icosahedral capsid, which encoded complex systems for DNA replication and transcription, a redox protein involved in disulfide bond formation in virion membrane proteins, and probably inhibitors of apoptosis. The conservation of the disulfide-oxidoreductase, a major capsid protein, and two virion membrane proteins indicates that the odd-shaped virions of poxviruses have evolved from the more common icosahedral virion seen in asfarviruses, iridoviruses, and phycodnaviruses.