bluuebunny/crossref_metadata_2025_split huggingface dataset:
Baculovirus Caspase Inhibitors P49 and P35 Block Virus-Induced Apoptosis Downstream of Effector Caspase DrICE Activation in
Drosophila melanogaster
Cells
Authors: Lannan Erica , Vandergaast Rianna , Friesen Paul D. 
Published: June 2007

ABSTRACT

Baculoviruses induce widespread apoptosis in invertebrates. To better understand the pathways by which these DNA viruses trigger apoptosis, we have used a combination of RNA silencing and overexpression of viral and host apoptotic regulators to identify cell death components in the model system of
Drosophila melanogaster
. Here we report that the principal effector caspase DrICE is required for baculovirus-induced apoptosis of
Drosophila
DL-1 cells as demonstrated by RNA silencing. proDrICE was proteolytically cleaved and activated during infection. Activation was blocked by overexpression of the cellular inhibitor-of-apoptosis proteins DIAP1 and SfIAP but not by the baculovirus caspase inhibitor P49 or P35. Rather, the substrate inhibitors P49 and P35 prevented virus-induced apoptosis by arresting active DrICE through formation of stable inhibitory complexes. Consistent with a two-step activation mechanism, proDrICE was cleaved at the large/small subunit junction TETD
230
-G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD
28
-A by a P49/P35-sensitive protease. Confirming that P49 targeted DrICE and not the initiator caspase DRONC, depletion of DrICE by RNA silencing suppressed virus-induced cleavage of P49. Collectively, our findings indicate that whereas DIAP1 functions upstream to block DrICE activation, P49 and P35 act downstream by inhibiting active DrICE. Given that P49 has the potential to inhibit both upstream initiator caspases and downstream effector caspases, we conclude that P49 is a broad-spectrum caspase inhibitor that likely provides a selective advantage to baculoviruses in different cellular backgrounds.
Epstein-Barr Virus Latent Membrane Protein 2A Mediates Transformation through Constitutive Activation of the Ras/PI3-K/Akt Pathway
Authors: Fukuda Makoto , Longnecker Richard 
Published: June 2007

ABSTRACT
Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.
Binding-Site Interactions between Epstein-Barr Virus Fusion Proteins gp42 and gH/gL Reveal a Peptide That Inhibits both Epithelial and B-Cell Membrane Fusion
Authors: Kirschner Austin N. , Lowrey Amanda S. , Longnecker Richard , Jardetzky Theodore S. 
Published: June 2007

ABSTRACTHerpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.
Macrophages Transmit Human Immunodeficiency Virus Type 1 Products to CD4-Negative Cells: Involvement of Matrix Metalloproteinase 9
Authors: Muratori Claudia , Sistigu Antonella , Ruggiero Eliana , Falchi Mario , Bacigalupo Ilaria , Palladino Clelia , Toschi Elena , Federico Maurizio 
Published: June 2007

ABSTRACT
It was previously reported that human immunodeficiency virus type 1 (HIV-1) spreads in CD4 lymphocytes through cell-to-cell transmission. Here we report that HIV-1-infected macrophages, but not lymphocytes, transmit HIV-1 products to CD4-negative cells of either epithelial, neuronal, or endothelial origin in the absence of overt HIV-1 infection. This phenomenon was detectable as early as 1 h after the start of cocultivation and depended on cell-to-cell contact but not on the release of viral particles from donor cells. Transfer of HIV-1 products occurred upon their polarization and colocalization within zones of cell-to-cell contact similar to virological synapses. Neither HIV-1 Env nor Nef expression was required but, interestingly, we found that an HIV-1-dependent increase in matrix metalloproteinase 9 production from donor cells significantly contributed to the cell-to-cell transmission of the viral products. The macrophage-driven transfer of HIV-1 products to diverse CD4-negative cell types may have a significant role in AIDS pathogenesis.
The ORF3 Protein of Porcine Circovirus Type 2 Interacts with Porcine Ubiquitin E3 Ligase Pirh2 and Facilitates p53 Expression in Viral Infection
Authors: Liu Jue , Zhu Yu , Chen Isabelle , Lau Jennifer , He Fang , Lau Adeline , Wang Zhilong , Karuppannan Anbu K. , Kwang Jimmy 
Published: June 2007

ABSTRACT
Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a newly identified protein, ORF3, plays a major role in virus-induced apoptosis and is involved in viral pathogenesis in vitro and in vivo. To characterize the role of the ORF3 protein in modulation of cellular function, a yeast two-hybrid system was used to screen a porcine cDNA library to find its interacting partner. We have isolated and characterized pPirh2 (for “porcine p53-induced RING-H2”), an E3 ubiquitin ligase, which specifically interacts with the ORF3 protein of PCV2. This interaction was further confirmed when the ORF3 protein coimmunoprecipitated with and colocalized to pPirh2 in PK15 cells. The ORF3 protein has been found to interact with the p53 binding domain of pPirh2 in yeast cells. Expression of the protein results in less pPirh2 expression in PCV2-infected cells. Furthermore, increases in p53 expression were observed in PCV2-infected and ORF3 (alone)-transfected cells. Phosphorylation of p53 at Ser-46, which is related to p53-induced apoptosis, was also time-dependently activated in PCV-infected and ORF3-transfected cells. Taken together, our results show that the PCV2 ORF3 protein specifically interacts with pPirh2 and inhibits its stabilization; this may lead to increasing p53 expression, resulting in apoptosis.
A Putative Leucine Zipper within the Herpes Simplex Virus Type 1 UL6 Protein Is Required for Portal Ring Formation
Authors: Nellissery Jacob K. , Szczepaniak Renata , Lamberti Carmela , Weller Sandra K. 
Published: June 2007

ABSTRACT
The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (Δ198-295), D-6 (Δ322-416), and D-LZ (Δ409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.
An RNA Pseudoknot in the 3′ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis
Authors: Beerens Nancy , Snijder Eric J. 
Published: June 2007

ABSTRACT
In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.
Antibody Recognition of Cell Surface-Associated NS1 Triggers Fc-γ Receptor-Mediated Phagocytosis and Clearance of West Nile Virus-Infected Cells
Authors: Chung Kyung Min , Thompson Bruce S. , Fremont Daved H. , Diamond Michael S. 
Published: June 2007

ABSTRACTPrevious studies have suggested that monoclonal antibodies (MAbs) to flavivirus nonstructural protein-1 (NS-1) protect against infection in mice through an Fc-γ receptor-dependent pathway. To identify a specific mechanism, we evaluated the protective activity of anti-NS1 MAbs to WNV using mice and cells with deficiencies of specific Fc-γ receptors. Our results suggest that only MAbs that recognize cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of WNV-infected cells. These findings may be relevant for generating novel therapeutic MAbs or vaccines against flaviviruses that target the NS1 protein.
Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model
Authors: Chen Zhaochun , Earl Patricia , Americo Jeffrey , Damon Inger , Smith Scott K. , Yu Fujuan , Sebrell Andrew , Emerson Suzanne , Cohen Gary , Eisenberg Roselyn J. , Gorshkova Inna , Schuck Peter , Satterfield William , Moss Bernard , Purcell Robert 
Published: June 2007

ABSTRACT

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (

K
d

of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.
Cellular Protein Modification by Poliovirus: the Two Faces of Poly(rC)-Binding Protein
Authors: Perera Rushika , Daijogo Sarah , Walter Brandon L. , Nguyen Joseph H. C. , Semler Bert L. 
Published: June 2007

ABSTRACT
During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-ΔKH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.
DNA-Dependent Oligomerization of Herpes Simplex Virus Type 1 Regulatory Protein ICP4
Authors: Kuddus Ruhul H. , DeLuca Neal A. 
Published: June 2007

ABSTRACT
The human herpes simplex virus type 1 regulatory protein ICP4 binds DNA as a dimer and forms a single protein-DNA complex (A complex) with short DNA probes. ICP4 oligomerized in a DNA-dependent manner, forming two or more protein-DNA complexes with longer DNA fragments containing a single DNA binding site. When resolved electrophoretically, one or more low-mobility DNA-protein complexes follow the fast-moving A complex. The major protein-DNA complex (B complex) formed by ICP4 with long DNA probes migrates just behind the A complex in the electric field, implying the oligomerization of ICP4 on the DNA. Binding experiments with circularly permutated DNA probes containing one ICP4 binding site revealed that about 70 bp of nonspecific DNA downstream of the cognate ICP4 binding site was required for efficient B complex formation. In addition, the C-terminal domain of ICP4 was found to be required for DNA-dependent oligomerization and B complex formation. Gel mobility shift analysis of protein-DNA complexes, combined with supershift analysis using different monoclonal antibodies, indicated that the B complex contained two ICP4 dimers. DNase I footprinting of ICP4-DNA complexes showed that one ICP4 dimer contacts the specific binding site and another ICP4 dimer contacts nonspecific DNA in the B complex. DNA-dependent oligomerization increased the affinity of ICP4 for relatively weak binding sites on large DNA molecules. The results of this study suggest how ICP4 may use multiple weak binding sites to aid in transcription activation.
Rate of Recombinational Deletion among Human Endogenous Retroviruses
Authors: Belshaw Robert , Watson Jason , Katzourakis Aris , Howe Alexis , Woolven-Allen John , Burt Austin , Tristem Michael 
Published: June 2007

ABSTRACT

The fate of most human endogenous retroviruses (HERVs) has been to undergo recombinational deletion. This process involves homologous recombination between the flanking long terminal repeats (LTRs) of a full-length element, leaving a relic structure in the genome termed a solo LTR. We examined loci in one family, HERV-K(HML2), and found that the deletion rate decreased markedly with age: the rate among recently integrated loci was almost 200-fold higher than that among loci whose insertion predated the divergence of humans and chimpanzees (8 × 10
−5
and 4 × 10
−7
recombinational deletion events per locus per generation, respectively). One hypothesis for this finding is that increasing mutational divergence between the flanking LTRs reduces the probability of homologous recombination and thus the rate of solo LTR formation. Consistent with this idea, we were able to replicate the observed rates by a simulation in which the probability of recombinational deletion was reduced 10-fold by a single mutation and 100-fold by any additional mutations. We also discuss the evidence for other factors that may influence the relationship between locus age and the rate of deletion, for example, host recombination rates and selection, and highlight the consequences of recombinational deletion for dating recent HERV integrations.
Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System
Authors: Zhang Zhen , Rowe Jenny , Wang Weijia , Sommer Marvin , Arvin Ann , Moffat Jennifer , Zhu Hua 
Published: June 2007

ABSTRACT

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV
BAC
) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV
Luc
). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV
Luc
genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.
Inhibition of T-Cell Receptor Signal Transduction and Viral Expression by the Linker for Activation of T Cells-Interacting p12
I
Protein of Human T-Cell Leukemia/Lymphoma Virus Type 1
Authors: Fukumoto Risaku , Dundr Miroslav , Nicot Christophe , Adams Anthony , Valeri Valerio W. , Samelson Lawrence E. , Franchini Genoveffa 
Published: June 2007

ABSTRACT

The p12
I
protein of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is a small oncoprotein that increases calcium release following protein kinase C activation by phorbol myristate acetate, and importantly, this effect is linker for activation of T cells (LAT) independent. Here, we demonstrate that p12
I
inhibits the phosphorylation of LAT, Vav, and phospholipase C-γ1 and decreases NFAT (nuclear factor of activated T cells) activation upon engagement of the T-cell receptor (TCR) with anti-CD3 antibody. Furthermore, we demonstrate that p12
I
localizes to membrane lipid rafts and, upon engagement of the TCR, relocalizes to the interface between T cells and antigen-presenting cells, defined as the immunological synapse. A p12
I
knockout molecular clone of HTLV-1 expresses more virus upon antigen stimulation than the isogenic wild type, suggesting that, by decreasing T-cell responsiveness, p12
I
curtails viral expression. Thus, p12
I
has contrasting effects on TCR signaling: it down-regulates TCR in a LAT-dependent manner on one hand, and on the other, it increases calcium release in a LAT-independent manner. The negative regulation of T-cell activation by p12
I
may have evolved to minimize immune recognition of infected CD4
+
T cells, to impair the function of infected cytotoxic CD8
+
T cells, and to favor viral persistence in the infected host.
Power in Voxel-based Lesion-Symptom Mapping
Authors: Kimberg Daniel Y. , Coslett H. Branch , Schwartz Myrna F. 
Published: June 2007

AbstractLesion analysis in brain-injured populations complements what can be learned from functional neuroimaging. Voxel-based approaches to mapping lesion-behavior correlations in brain-injured populations are increasingly popular, and have the potential to leverage image analysis methods drawn from functional magnetic resonance imaging. However, power is a major concern for these studies, and is likely to vary regionally due to the distribution of lesion locations. Here, we outline general considerations for voxel-based methods, characterize the use of a nonparametric permutation test adapted from functional neuroimaging, and present methods for regional power analysis in lesion studies.