bluuebunny/crossref_metadata_2025_split huggingface dataset:
Complete In Vitro Assembly of the Reovirus Outer Capsid Produces Highly Infectious Particles Suitable for Genetic Studies of the Receptor-Binding Protein
Authors: Chandran Kartik , Zhang Xing , Olson Norman H. , Walker Stephen B. , Chappell James D. , Dermody Terence S. , Baker Timothy S. , Nibert Max L. 
Published: July 2002

ABSTRACT

Mammalian reoviruses, prototype members of the
Reoviridae
family of nonenveloped double-stranded RNA viruses, use at least three proteins—ς1, μ1, and ς3—to enter host cells. ς1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of ς1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by “recoating” genome-containing core particles that lacked ς1, μ1, and ς3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to ς1. The recoated particles bound to and infected cultured cells in a ς1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant ς1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the ς1 protein. Additional experiments showed that recoated particles containing ς1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound ς1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of ς1 with respect to its structure, assembly into particles, and roles in entry.
The U
S
3 Protein Kinase Blocks Apoptosis Induced by the
d
120 Mutant of Herpes Simplex Virus 1 at a Premitochondrial Stage
Authors: Munger Joshua , Chee Ana V. , Roizman Bernard 
Published: July 2002

ABSTRACT

Earlier studies have shown that the
d
120 mutant of herpes simplex virus 1, which lacks both copies of the α4 gene, induces caspase-3-dependent apoptosis in HEp-2 cells. Apoptosis was also induced by the α4 rescuant but was blocked by the complementation of rescuant with a DNA fragment encoding the U
S
3 protein kinase (R. Leopardi and B. Roizman, Proc. Natl. Acad. Sci. USA 93:9583–9587, 1996, and R. Leopardi, C. Van Sant, and B. Roizman, Proc. Natl. Acad. Sci. USA 94:7891–7896, 1997). To investigate its role in the apoptotic cascade, the U
S
3 open reading frame was cloned into a baculovirus (Bac-U
S
3) under the control of the human cytomegalovirus immediate-early promoter. We report the following. (i) Bac-U
S
3 blocks processing of procaspase-3 to active caspase. Procaspase-3 levels remained unaltered if superinfected with Bac-U
S
3 at 3 h after
d
120 mutant infection, but significant amounts of procaspase-3 remained in cells superinfected with Bac-Us3 at 9 h postinfection with
d
120 mutant. (ii) The U
S
3 protein kinase blocks the proapoptotic cascade upstream of mitochondrial involvement inasmuch as Bac-U
S
3 blocks release of cytochrome
c
in cells infected with the
d
120 mutant. (iii) Concurrent infection of HEp-2 cells with Bac-U
S
3 and the
d
120 mutant did not alter the pattern of accumulation or processing of ICP0, -22, or -27, and therefore U
S
3 does not appear to block apoptosis by targeting these proteins.
Cytokine Production by Vγ+-T-Cell Subsets Is an Important Factor Determining CD4+-Th-Cell Phenotype and Susceptibility of BALB/c Mice to Coxsackievirus B3-Induced Myocarditis
Authors: Huber Sally A. , Graveline Danielle , Born Willi K. , O'Brien Rebecca L. 
Published: July 2002

ABSTRACTTwo coxsackievirus B3 (CVB3) variants (H3 and H310A1) differ by a single amino acid mutation in the VP2 capsid protein. H3 induces severe myocarditis in BALB/c mice, but H310A1 is amyocarditic. Infection with H3, but not H310A1, preferentially activates Vγ4 Vδ4 cells, which are strongly positive for gamma interferon (IFN-γ), whereas Vγ1 Vδ4 cells are increased in both H3 and H310A1 virus-infected animals. Depletion of Vγ1+cells using monoclonal anti-Vγ1 antibody enhanced myocarditis and CD4+-, IFN-γ+-cell responses in both H3- and H310A1-infected mice yet decreased the CD4+-, IL-4+-cell response. Depleting Vγ4+cells suppressed myocarditis and reduced CD4+IFN-γ+cells but increased CD4+IL-4+T cells. The role of cytokine production by Vγ1+and Vγ4+T cells was investigated by adoptively transferring these cells isolated from H3-infected BALB/c Stat4 knockout (Stat4ko) (defective in IFN-γ expression) or BALB/c Stat6ko (defective in IL-4 expression) mice into H3 virus-infected wild-type BALB/c recipients. Vγ4 and Vγ1+T cells from Stat4ko mice expressed IL-4 but no or minimal IFN-γ, whereas these cell populations derived from Stat6ko mice expressed IFN-γ but no IL-4. Stat4ko Vγ1+cells (IL-4+) suppress myocarditis. Stat6ko Vγ1+cells (IFN-γ+) were not inhibitory. Stat6ko Vγ4+cells (IFN-γ+) significantly enhanced myocarditis. Stat4ko Vγ4+cells (IL-4+) neither inhibited nor enhanced disease. These results show that distinct γδ-T-cell subsets control myocarditis susceptibility and bias the CD4+-Th-cell response. The cytokines produced by the Vγ subpopulation have a significant influence on the CD4+-Th-cell phenotype.
Role of Metastability and Acidic pH in Membrane Fusion by Tick-Borne Encephalitis Virus
Authors: Stiasny Karin , Allison Steven L. , Mandl Christian W. , Heinz Franz X. 
Published: July 2002

ABSTRACT
The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus is, like the alphavirus E1 protein, a class II viral fusion protein that differs structurally and probably mechanistically from class I viral fusion proteins. The surface of the native TBE virion is covered by an icosahedrally symmetrical network of E homodimers, which mediate low-pH-induced fusion in endosomes. At the pH of fusion, the E homodimers are irreversibly converted to a homotrimeric form, which we have found by intrinsic fluorescence measurements to be more stable than the native dimers. Thus, the TBE virus E protein is analogous to the prototypical class I fusion protein, the influenza virus hemagglutinin (HA), in that it is initially synthesized in a metastable state that is energetically poised to be converted to the fusogenic state by exposure to low pH. However, in contrast to what has been observed with influenza virus HA, this transition could not be triggered by input of heat energy alone and membrane fusion could be induced only when the virus was exposed to an acidic pH. In a previous study we showed that the dimer-to-trimer transition appears to be a two-step process involving a reversible dissociation of the dimer followed by an irreversible trimerization of the dissociated monomeric subunits. Because the dimer-monomer equilibrium in the first step apparently depends on the protonation state of E, the lack of availability of monomers for the trimerization step at neutral pH could explain why low pH is essential for fusion in spite of the metastability of the native E dimer.
Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes
Authors: Balagué Cristina , Noya Francisco , Alemany Ramon , Chow Louise T. , Curiel David T. 
Published: July 2002

ABSTRACT
Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.
Antigenically Distinct Conformations of CXCR4
Authors: Baribaud Frédéric , Edwards Terri G. , Sharron Matthew , Brelot Anne , Heveker Nikolaus , Price Ken , Mortari Frank , Alizon Marc , Tsang Monica , Doms Robert W. 
Published: July 2002

ABSTRACT
The major human immunodeficiency virus type 1 (HIV-1) coreceptors are the chemokine receptors CCR5 and CXCR4. The patterns of expression of the major coreceptors and their use by HIV-1 strains largely explain viral tropism at the level of entry. However, while virus infection is dependent upon the presence of CD4 and an appropriate coreceptor, it can be influenced by a number of factors, including receptor concentration, affinity between envelope gp120 and receptors, and potentially receptor conformation. Indeed, seven-transmembrane domain receptors, such as CCR5, can exhibit conformational heterogeneity, although the significance for virus infection is uncertain. Using a panel of monoclonal antibodies (MAbs) to CXCR4, we found that CXCR4 on both primary and transformed T cells as well as on primary B cells exhibited considerable conformational heterogeneity. The conformational heterogeneity of CXCR4 explains the cell-type-dependent ability of CXCR4 antibodies to block chemotaxis to stromal cell-derived factor 1α and to inhibit HIV-1 infection. In addition, the MAb most commonly used to study CXCR4 expression, 12G5, recognizes only a subpopulation of CXCR4 molecules on all primary cell types analyzed. As a result, CXCR4 concentrations on these important cell types have been underestimated to date. Finally, while the factors responsible for altering CXCR4 conformation are not known, we found that they do not involve CXCR4 glycosylation, sulfation of the N-terminal domain of CXCR4, or pertussis toxin-sensitive G-protein coupling. The fact that this important HIV-1 coreceptor exists in multiple conformations could have implications for viral entry and for the development of receptor antagonists.
The Kaposi's Sarcoma-Associated Herpesvirus K8 Protein Interacts with CREB-Binding Protein (CBP) and Represses CBP-Mediated Transcription
Authors: Hwang Seungmin , Gwack Yousang , Byun Hyewon , Lim Chunghun , Choe Joonho 
Published: July 2002

ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame K8 encodes a basic region-leucine zipper protein of 237 amino acids that homodimerizes with its bZIP domain. KSHV K8 shows significant homology to the Epstein-Barr virus (EBV) immediate-early protein Zta, a key regulator in the reactivation and replication of EBV. In this study, we report that K8, like its homolog EBV Zta, interacts with cellular CREB-binding protein (CBP) in vivo and in vitro. This interaction requires the C/H3 domain of CBP and the basic region of K8. K8 represses CBP-mediated transcription by competing with limited amounts of cellular CBP, exemplified by the reduced expression from the AP-1 and human immunodeficiency virus long terminal repeat promoters.
In Vitro Analysis of Human Immunodeficiency Virus Type 1 Minus-Strand Strong-Stop DNA Synthesis and Genomic RNA Processing
Authors: Driscoll Mark D. , Golinelli Marie-Pierre , Hughes Stephen H. 
Published: July 2002

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), nucleocapsid protein (NC), genomic RNA, and the growing DNA strand all influence the copying of the HIV-1 RNA genome into DNA. A detailed understanding of these activities is required to understand the process of reverse transcription. HIV-1 viral DNA is initiated from a tRNA
3
Lys
primer bound to the viral genome at the primer binding site. The U3 and R regions of the RNA genome are the first sequences to be copied. The TAR hairpin, a structure found within the R region of the viral genome, is the site of increased RT pausing, RNase H activity, and RT dissociation. Template RNA was digested approximately 17 bases behind the site where polymerase paused at the base of TAR. In most template RNAs, this was the only cleavage made by the RT responsible for initiating polymerization. If the RT that initiated DNA synthesis dissociated from the base of the TAR hairpin and an RT rebound at the end of the primer, there was competition between the polymerase and RNase H activities. After the complete heteroduplex was formed, there were additional RNase H cleavages that did not involve polymerization. Levels of NC that prevented TAR DNA self-priming did not protect genomic RNA from RNase H digestion. RNase H digestion of the 100-bp heteroduplex produced a 14-base RNA from the 5′ end of the RNA that remained annealed to the 3′ end of the minus-strand strong-stop DNA only if NC was present in the reaction.
Assembly and Organization of Glycoproteins B, C, D, and H in Herpes Simplex Virus Type 1 Particles Lacking Individual Glycoproteins: No Evidence for the Formation of a Complex of These Molecules
Authors: Rodger Gaener , Boname Jessica , Bell Susanne , Minson Tony 
Published: July 2002

ABSTRACT
Glycoprotein B (gB), gC, gD, and gH:L of herpes simplex virus type 1 (HSV-1) are implicated in virus adsorption and penetration. gB, gD, and gH:L are essential for these processes, and their expression is necessary and sufficient to induce cell fusion. The current view is that these molecules act in concert as a functional complex, and cross-linking studies support this view (C. G. Handler, R. J. Eisenberg, and G. H. Cohen, J. Virol. 70:6067–6075, 1996). We examined the glycoprotein composition, with respect to gB, gC, gD, and gH, of mutant virions lacking individual glycoproteins and the sedimentation characteristics of glycoproteins extracted from these virions. The amounts of gB, gC, gD, or gH detected in virions did not alter when any one of these molecules was absent, and it therefore appears that they are incorporated into the virion independently of each other. The sedimentation characteristics of gB and gD from mutant virions were not different from those of wild-type virions. We confirmed that gB, gC, and gD could be cross-linked to each other on the virion surface but found that the absence of one glycoprotein did not alter the outcome of cross-linking reactions between the remaining molecules. The incorporation and arrangement of these glycoproteins in the virion envelope therefore appear to be independent of the individual molecular species. This is difficult to reconcile with the concept that gB, gC, gD, and gH:L are incorporated as a functional complex into the virion envelope.
Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein
Authors: Pallaoro Michele , Lahm Armin , Biasiol Gabriella , Brunetti Mirko , Nardella Caterina , Orsatti Laura , Bonelli Fabio , Orrù Stefania , Narjes Frank , Steinkühler Christian 
Published: July 2002

ABSTRACTThe NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed inEscherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observetranscleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.
cdc2 Cyclin-Dependent Kinase Binds and Phosphorylates Herpes Simplex Virus 1 U
L
42 DNA Synthesis Processivity Factor
Authors: Advani Sunil J. , Weichselbaum Ralph R. , Roizman Bernard 
Published: July 2002

ABSTRACT

Earlier studies have shown that cdc2 kinase is activated during herpes simplex virus 1 infection and that its activity is enhanced late in infection even though the levels of cyclin A and B are decreased below levels of detection. Furthermore, activation of cdc2 requires the presence of infected cell protein no. 22 and the U
L
13 protein kinase, the same gene products required for optimal expression of a subset of late genes exemplified by U
S
11, U
L
38, and U
L
41. The possibility that the activation of cdc2 and expression of this subset may be connected emerged from the observation that dominant negative cdc2 specifically blocked the expression of U
S
11 protein in cells infected and expressing dominant negative cdc2. Here we report that in the course of searching for a putative cognate partner for cdc2 that may have replaced cyclins A and B, we noted that the DNA polymerase processivity factor encoded by the U
L
42 gene contains a degenerate cyclin box and has been reported to be structurally related to proliferating cell nuclear antigen, which also binds cdk2. Consistent with this finding, we report that (i) U
L
42 is able to physically interact with cdc2 at both the amino-terminal and carboxyl-terminal domains, (ii) the carboxyl-terminal domain of U
L
42 can be phosphorylated by cdc2, (iii) immunoprecipitates obtained with anti U
L
42 antibody contained a roscovitine-sensitive kinase activity, (iv) kinase activity associated with U
L
42 could be immunodepleted by antibody to cdc2, and (v) U
L
42 transfected into cells associates with a nocodazole-enhanced kinase. We conclude that U
L
42 can associate with cdc2 and that the kinase activity has the characteristic traits of cdc2 kinase.
Emergence and Kinetics of Simian Immunodeficiency Virus-Specific CD8+T Cells in the Intestines of Macaques during Primary Infection
Authors: Veazey Ronald S. , Gauduin Marie-Claire , Mansfield Keith G. , Tham Irene C. , Altman John D. , Lifson Jeffrey D. , Lackner Andrew A. , Johnson R. Paul 
Published: July 2002

ABSTRACTIn this report, three Mamu-A*01+rhesus macaques were examined to compare the emergence of simian immunodeficiency virus (SIV)-specific CD8+T cells in the intestines and blood in early SIV infection using a major histocompatibility complex class I tetramer complexed with the Gag181–189peptide. Fourteen days after intravenous inoculation with SIVmac251, large numbers of SIV Gag181–189-specific CD8+T cells were detected in the intestinal mucosa (3.1 to 11.5% of CD3+CD8+lymphocytes) as well as in the blood (3.1 to 13.4%) of all three macaques. By 21 days postinoculation, levels of tetramer-binding cells had dropped in both the intestines and blood. At day 63, however, levels of SIV Gag181–189-specific CD8+T cells in the intestines had rebounded in all three macaques to levels that were higher (8.6 to 18.7%) than those at day 21. In contrast, percentages of tetramer-binding cells in the peripheral blood remained comparatively stable (2.5 to 4.5%) at this time point. In summary, SIV Gag181–189-specific CD8+T cells appeared in both the intestinal mucosa and peripheral blood at a comparable rate and magnitude in primary SIV infection. Given that the intestine is a major site of early viral replication as well as the site where most of the total body lymphocyte pool resides, these data indicate that it is also an early and important site of development of antiviral immune responses.
Cell Surface Heparan Sulfate Is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8.1
Authors: Birkmann Alexander , Mahr Kerstin , Ensser Armin , Yağuboğlu Svenja , Titgemeyer Fritz , Fleckenstein Bernhard , Neipel Frank 
Published: July 2002

ABSTRACTAn immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposi's sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1ΔTMFc). K8.1ΔTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1ΔTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.