bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction of Id1 and Id3 by Latent Membrane Protein 1 of Epstein-Barr Virus and Regulation of p27/Kip and Cyclin-Dependent Kinase 2 in Rodent Fibroblast Transformation
Authors: Everly David N. , Mainou Bernardo A. , Raab-Traub Nancy 
Published: November 2004

ABSTRACTLatent membrane protein 1 (LMP1), the Epstein-Barr virus oncoprotein, activates NF-κB, phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and c-Jun N-terminal kinase signaling. To determine global transcriptional changes induced by LMP1 in epithelial cells, genomic analysis of C33A cells stably expressing LMP1 was performed. Relatively few genes were induced by LMP1. Expression of two members of the Id (inhibitor of differentiation) family of proteins, Id1 and Id3, was induced in the presence of LMP1 and confirmed by mRNA and protein in C33A and Rat-1 cells. In Rat-1 foci transformed by LMP1, Id1 protein was also increased. Id proteins are known negative regulators of E-box proteins that positively regulate p16 and potentially other cyclin-dependent kinase inhibitors (cdki's). In LMP1-expressing Rat-1 cells, cdki p27 was specifically downregulated. Decreased p27 was correlated with increased levels of Cdk2 and increased levels of phosphorylated retinoblastoma protein. This study describes new properties of LMP1 that likely contribute to transformation and oncogenesis.
Coxsackievirus B3 Infection Induces
cyr61
Activation via JNK To Mediate Cell Death
Authors: Kim Sun-Mi , Park Jung-Hyun , Chung Sun-Ku , Kim Joo-Young , Hwang Ha-Young , Chung Kwang-Chul , Jo Inho , Park Sang-Ick , Nam Jae-Hwan 
Published: November 2004

ABSTRACT

Coxsackievirus B3 (CVB3), an enterovirus in the
Picornavirus
family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (
cyr61
) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells.
cyr61
is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the
cyr61
promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed
cyr61
and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of
cyr61
with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for
cyr61
in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of
cyr61
in virus-induced cell death and provide new insights into the cellular factors involved.
A Conserved Histidine in the ij Loop of the Semliki Forest Virus E1 Protein Plays an Important Role in Membrane Fusion
Authors: Chanel-Vos Chantal , Kielian Margaret 
Published: November 2004

ABSTRACT
The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low pH-triggered membrane fusion reaction mediated by the E1 protein. E1 is a class II fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable homotrimer during the fusion reaction. Intriguingly, the fusion loop is closely associated with a loop connecting the i and j β-strands. This ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to proteolysis in the protease-resistant E1 homotrimer. The SFV ij loop contains a histidine residue at position 230. Sequence comparisons revealed that an analogous histidine is completely conserved in all alphavirus and flavivirus fusion proteins. An E1 H230A mutant was constructed using the SFV infectious clone. Although cells infected with H230A RNA produced virus particles, these virions were completely noninfectious and were blocked in both cell-cell fusion and lipid mixing assays. The H230A virions efficiently bound to cell surface receptors and responded to low pH by undergoing acid-dependent conformational changes including dissociation of the E1/E2 dimer, exposure of the fusion loop, association with target liposomes, exposure of acid-conformation-specific epitopes, and formation of the stable E1 homotrimer. Studies with a soluble fragment of E1 showed that the mutant protein was defective in lipid-dependent conformational changes. Our results indicate that the E1 ij loop and the conserved H230 residue play a critical role in alphavirus-membrane fusion and suggest the presence of a previously undescribed late intermediate in the fusion reaction.
Furin-Mediated Cleavage of the Feline Foamy Virus Env Leader Protein
Authors: Geiselhart Verena , Bastone Patrizia , Kempf Tore , Schnölzer Martina , Löchelt Martin 
Published: November 2004

ABSTRACT

The molecular biology of spuma or foamy retroviruses is different from that of the other members of the
Retroviridae
. Among the distinguishing features, the N-terminal domain of the foamy virus Env glycoprotein, the 16-kDa Env leader protein Elp, is a component of released, infectious virions and is required for particle budding. The transmembrane protein Elp specifically interacts with N-terminal Gag sequences during morphogenesis. In this study, we investigate the mechanism of Elp release from the Env precursor protein. By a combination of genetic, biochemical, and biophysical methods, we show that the feline foamy virus (FFV) Elp is released by a cellular furin-like protease, most likely furin itself, generating an Elp protein consisting of 127 amino acid residues. The cleavage site fully conforms to the rules for an optimal furin site. Proteolytic processing at the furin cleavage site is required for full infectivity of FFV. However, utilization of other furin proteases and/or cleavage at a suboptimal signal peptidase cleavage site can partially rescue virus viability. In addition, we show that FFV Elp carries an N-linked oligosaccharide that is not conserved among the known foamy viruses.
C-Terminal nsP1a Protein of Human Astrovirus Colocalizes with the Endoplasmic Reticulum and Viral RNA
Authors: Guix Susana , Caballero Santiago , Bosch Albert , Pintó Rosa M. 
Published: November 2004

ABSTRACTComputational and biological approaches were undertaken to characterize the role of the human astrovirus nonstructural protein nsP1a/4, located at the C-terminal fragment of nsP1a. Computer analysis reveals sequence similarities to other nonstructural viral proteins involved in RNA replication and/or transcription and allows the identification of a glutamine- and proline-rich region, the prediction of many phosphorylation and O-glycosylation sites, and the occurrence of a KKXX-like endoplasmic reticulum retention signal. Immunoprecipitation analysis with an antibody against a synthetic peptide of the nsP1a/4 sequence detected polyprotein precursors of 160, 75, and 38 to 40 kDa as well as five smaller proteins in the range of 21 to 27 kDa. Immunofluorescence labeling showed that the nsP1a/4 protein is accumulated at the perinuclear region, in association with the endoplasmic reticulum and the viral RNA. These results suggest the involvement of nsP1a/4 protein in the RNA replication process in endoplasmic reticulum-derived intracellular membranes.
Oligomerization of Hantavirus N Protein: C-Terminal α-Helices Interact To Form a Shared Hydrophobic Space
Authors: Kaukinen Pasi , Kumar Vibhor , Tulimäki Kirsi , Engelhardt Peter , Vaheri Antti , Plyusnin Alexander 
Published: November 2004

ABSTRACT
The structure of the nucleocapsid protein of bunyaviruses has not been defined. Earlier we have shown that Tula hantavirus N protein oligomerization is dependent on the C-terminal domains. Of them, the helix-loop-helix motif was found to be an essential structure. Computer modeling predicted that oligomerization occurs via helix protrusions, and the shared hydrophobic space formed by amino acids residues 380-IILLF-384 in the first helix and 413-LI-414 in the second helix is responsible for stabilizing the interaction. The model was validated by two approaches. First, analysis of the oligomerization capacity of the N protein mutants performed with the mammalian two-hybrid system showed that both preservation of the helix structure and formation of the shared hydrophobic space are crucial for the interaction. Second, oligomerization was shown to be a prerequisite for the granular pattern of transiently expressed N protein in transfected cells. N protein trimerization was supported by three-dimensional reconstruction of the N protein by electron microscopy after negative staining. Finally, we discuss how N protein trimerization could occur.
Identification of Residues in the Dengue Virus Type 2 NS2B Cofactor That Are Critical for NS3 Protease Activation
Authors: Niyomrattanakit Pornwaratt , Winoyanuwattikun Pakorn , Chanprapaph Santad , Angsuthanasombat Chanan , Panyim Sakol , Katzenmeier Gerd 
Published: November 2004

ABSTRACT

Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Φx
3
Φ motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a
k
cat
/
K

m

value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced
k
cat
values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.
Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State
Authors: Russell Charles J. , Jardetzky Theodore S. , Lamb Robert A. 
Published: November 2004

ABSTRACT

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol.
66
:
2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation.
Human Papillomavirus Type 16 E6 Promotes Retinoblastoma Protein Phosphorylation and Cell Cycle Progression
Authors: Malanchi Ilaria , Accardi Rosita , Diehl Frank , Smet Anouk , Androphy Elliot , Hoheisel Jörg , Tommasino Massimo 
Published: November 2004

ABSTRACTWe show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G1/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16INK4a, CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16INK4a. The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21WAF1/CIP1were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21WAF1/CIP1gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21WAF1/CIP1levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G1arrest imposed by oncogenicras. Immunofluorescence staining of cells coexpressingrasand E6 from either HPV16 or HPV1 revealed that antiproliferative (p16INK4a) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.
Replicon System for Lassa Virus
Authors: Hass Meike , Gölnitz Uta , Müller Stefanie , Becker-Ziaja Beate , Günther Stephan 
Published: November 2004

ABSTRACT

Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (
Renilla
luciferase) in antisense orientation, flanked by the 5′ and 3′ untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of
Renilla
luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5′ end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.
Recombinant Infectious Bronchitis Coronavirus Beaudette with the Spike Protein Gene of the Pathogenic M41 Strain Remains Attenuated but Induces Protective Immunity
Authors: Hodgson Teri , Casais Rosa , Dove Brian , Britton Paul , Cavanagh Dave 
Published: November 2004

ABSTRACT

We have replaced the ectodomain of the spike (S) protein of the Beaudette strain (Beau-R; apathogenic for
Gallus domesticus
chickens) of avian infectious bronchitis coronavirus (IBV) with that from the pathogenic M41 strain to produce recombinant IBV BeauR-M41(S). We have previously shown that this changed the tropism of the virus in vitro (R. Casais, B. Dove, D. Cavanagh, and P. Britton, J. Virol. 77:9084-9089, 2003). Herein we have assessed the pathogenicity and immunogenicity of BeauR-M41(S). There were no consistent differences in pathogenicity between the recombinant BeauR-M41(S) and its apathogenic parent Beau-R (based on snicking, nasal discharge, wheezing, watery eyes, rales, and ciliostasis in trachea), and both replicated poorly in trachea and nose compared to M41; the S protein from the pathogenic M41 had not altered the apathogenic nature of Beau-R. Both Beau-R and BeauR-M41(S) induced protection against challenge with M41 as assessed by absence of recovery of challenge virus and nasal exudate. With regard to snicking and ciliostasis, BeauR-M41(S) induced greater protection (seven out of nine chicks [77%]; assessed by ciliostasis) than Beau-R (one out of nine; 11%) but less than M41 (100%). The greater protection induced by BeauR-M41(S) against M41 may be related to the ectodomain of the spike protein of Beau-R differing from that of M41 by 4.1%; a small number of epitopes on the S protein may play a disproportionate role in the induction of immunity. The results are promising for the prospects of S-gene exchange for IBV vaccine development.
Variable Methylation of the Epstein-Barr Virus Wp EBNA Gene Promoter in B-Lymphoblastoid Cell Lines
Authors: Elliott Jennifer , Goodhew E. Brook , Krug Laurie T. , Shakhnovsky Natalie , Yoo Lina , Speck Samuel H. 
Published: November 2004

ABSTRACT

During the initial stages of Epstein-Barr virus (EBV) infection of peripheral resting B cells, transcription of the six genes encoding the EBV latency-associated nuclear antigens (EBNAs) is driven from Wp, a promoter that is present in multiple copies within the EBV major internal repeat. As infection progresses, transcription from Wp is downregulated following upregulation of EBNA gene transcription driven from a promoter, Cp, located ca. 3 kb upstream of the first copy of Wp. Recently published data have provided evidence that, concomitant with the switch in EBNA gene promoter usage, Wp becomes heavily methylated (R. J. Tierney et al., J. Virol.
74
:10468-10479, 2000). Based on this observation, it has been argued that methylation of Wp plays a pivotal role in suppressing Wp activity in EBV-immortalized B-lymphoblastoid cell lines (LCLs). Here we present data compiled from analyses of Wp methylation in eight randomly selected low-passage-number B-LCLs. These data demonstrate that there is considerable variability in Wp methylation, both between different cell lines and within clonal LCLs. Overall, less methylation of Wp was noted in established, low-passage-number LCLs than was previously observed in bulk cultures of infected B cells at days 18 and 21 postinfection. Importantly, the majority of LCLs examined harbored both unmethylated and methylated copies of Wp. In addition, all low-passage-number LCLs examined contained both Cp- and Wp-initiated EBNA transcripts, arguing for the presence of some transcriptionally active copies of Wp. Taken together, these data argue that other factors, perhaps in conjunction with Wp methylation, play a role in suppressing Wp activity in LCLs.
Perception of Nigerian athletes of the use of mouth guards to prevent the stresses of sports injury
Authors: Adegbesan O A , Onyeaso C O 
Published: November 2004

Objective:The perception of Nigerian athletes of the use of mouth guards to prevent the stresses of sports injuries was examined using psychological, sociological, and physical stress variables.Methods:The descriptive survey research design was used in this study. Participants (n = 333) were selected using the purposive random sampling technique and data were collected using the 4 point Likert type instrument. The coefficient α was used to determine the reliability of the instrument with r = 0.79 for psychological, r = 0.66 for physical, and r = 0.74 for sociological stress.Results:The result reveals that female athletes perceived the use of mouth guards as being more important to prevent the stresses of sports injuries than male athletes, while athletes who had used mouth guards for longer periods also perceived them as being more important to prevent the stresses of sports injuries compared to those who had used them less. The result also revealed a significant difference (p<0.05) among the various sport groups as independent variables on the psychological and physical stress variables. Scheffé post hoc analysis was used to identify the group where significant difference was found.Conclusions:The study concluded that since the impact of injury is perceived to affect the cognitive function of athletes, athletes should be educated on the use of mouth guards in order to reduce the incidence of oral and dental injuries to the barest minimum.
Patellar tendinosis as an adaptive process: a new hypothesis: Figure 1
Authors: Hamilton B , Purdam C 
Published: November 2004

Background:Patellar tendinosis (PT), or “jumper’s knee” is a common condition in athletes participating in jumping sports, and is characterised by proximal patellar tendon pain and focal tenderness to palpation. Hypoechoic lesions observed in the proximal patellar tendon associated with the tendinosis are typically described as being a result of degenerative change or “failed healing”. We propose a new model for the development of the hypoechoic lesion observed in PT, in which the aetiology is an adaptive response to differential forces within the tendon.Methods:We assessed the clinical, histopathological, and biomechanical literature surrounding the patellar tendon and integrated this with research into the response of tendons to differential forces.Results and conclusions:We propose that the hypoechoic lesion commonly described in PT is the result of adaptation or partial adaptation of the proximal patellar tendon to a compressive load. We postulate that the biomechanics of the patellar–patellar tendon interface creates this compressive environment. Secondary failure of the surrounding tensile adapted tendon tissue may result in tissue overload and failure, with resultant stimulation of nociceptors. We believe that this “adaptive model” of patellar tendinosis is consistent with the clinical and histological findings.