bluuebunny/crossref_metadata_2025 huggingface dataset:
Description of pain provocation tests used for the diagnosis of sports‐related chronic groin pain: relationship of tests to defined clinical (pain and tenderness) and MRI (pubic bone marrow oedema) criteria
Authors: Verrall Geoffrey M. , Slavotinek John P. , Barnes Peter G. , Fon Gerald T.
Published: January 2005

The clinical assessment of groin pain in athletes is difficult, with the lack of specific clinical tests being in part responsible. Three pain provocation tests used in the diagnosis of chronic groin pain are described and their relationship to defined clinical and MRI criteria has been assessed.

Eighty‐nine Australian Rules football players with and without groin symptoms underwent clinical examination followed by history. Three pain provocation tests were performed (named as the Single Adductor, Squeeze and Bilateral Adductor tests). All athletes subsequently underwent MRI of their groin region for the presence of significant pubic bone marrow oedema (BMO).

Of the 89, 47 were defined as having chronic groin pain, and 46 had significant BMO with 37 having both chronic groin pain and BMO. The three pain provocation tests demonstrated only moderate sensitivity (range 30–65%). Positive predictive values were moderate to high (67–93%) depending upon the individual test. The Bilateral Adductor test was the most sensitive test with the highest positive predictive values. The high specificity (88–93%) demonstrated should be treated with caution due to the study methodology.

Three pain provocation tests of potential value in assessing chronic groin pain in athletes are described. If positive, all three pain provocation tests demonstrated a high likelihood for the athlete having MR‐detected parasymphyseal pubic BMO. Further research is required on assessing the clinical usefulness of these tests.
Injury incidence and distribution in elite football—a prospective study of the Danish and the Swedish top divisions
Authors: Hägglund Martin , Waldén Markus , Ekstrand Jan
Published: January 2005

The Danish and Swedish male top football divisions were studied prospectively from January to June 2001. Exposure to football and injury incidence, severity and distribution were compared between the countries.

Swedish players had greater exposure to training (171 vs. 123 h per season, P<0.001), whereas exposure to matches did not differ between the countries. There was a higher risk for injury during training in Denmark than in Sweden (11.8 vs. 6.0 per 1000 h, P<0.01), whereas for match play there was no difference (28.2 vs. 26.2 per 1000 h). The risk for incurring a major injury (absence from football more than 4 weeks) was greater in Denmark (1.8 vs. 0.7 per 1000 h, P=0.002). The distribution of injuries according to type and location was similar in both countries. Of all injuries in Denmark and Sweden, overuse injury accounted for 39% and 38% (NS), and re‐injury for 30% and 24% (P=0.032), respectively.

The greater training exposure and the long pre‐season period in Sweden may explain some of the reported differences.
The human diving response, its function, and its control
Authors: Foster G. E. , Sheel A. W.
Published: January 2005

The purpose of this review is to outline the physiological responses associated with the diving response, its functional significance, and its cardiorespiratory control. This review is separated into four major sections. Section one outlines the diving response and its physiology. Section two provides support for the hypothesis that the primary role of the diving response is the conservation of oxygen. The third section describes how the diving response is controlled and provides a model that illustrates the cardiorespiratory interaction. Finally, the fourth section illustrates potential adaptations that result after regular exposure to an asphyxic environment. The cardiovascular and endocrine responses associated with the diving response and apnea are bradycardia, vasoconstriction, and an increase in secretion of suprarenal catecholamines. These responses require the integration of both the cardiovascular system and the respiratory system. The primary role of the diving response is likely to conserve oxygen for sensitive brain and heart tissue and to lengthen the time before the onset of serious hypoxic damage. We suggest that future research should be focused towards understanding the role of altered ventilatory responses in human breath‐hold athletes as well as in patients suffering from sleep‐disordered breathing.
Description of an as Yet Unclassified DNA Virus from Diseased
Cyprinus carpio
Species
Authors: Hutoran Marina , Ronen Ariel , Perelberg Ayana , Ilouze Maya , Dishon Arnon , Bejerano Izhak , Chen Nissim , Kotler Moshe
Published: January 2005

Numerous deaths of koi and common carp (
Cyprinus carpio
) were observed on many farms throughout Israel, resulting in severe financial losses. The lethal viral disease observed is highly contagious and extremely virulent, but morbidity and mortality are restricted to koi and common carp populations. Diseased fish exhibit fatigue and gasping movements in shallow water. Infected fish had interstitial nephritis and gill necrosis as well as petechial hemorrhages in the liver and other symptoms that were not consistent with viral disease, suggesting a secondary infection. Here we report the isolation of carp nephritis and gill necrosis virus (CNGV), which is the etiologic agent of this disease. The virus propagates and induces severe cytopathic effects by 5 days postinfection in fresh koi or carp fin cell cultures (KFC and CFC, respectively), but not in epithelioma papillosum cyprini cells. The virus harvested from KFC cultures induced the same clinical signs, with a mortality of 75 to 95%, upon inoculation into naive koi and common carp. Using PCR, we provide final proof that the isolated virus is indeed the etiologic agent of food and ornamental carp mortalities in fish husbandry. Electron microscopy revealed viral cores with icosahedral morphology of 100 to 110 nm that resembled herpesviruses. Electron micrographs of purified pelleted CNGV sections, together with viral sensitivities to ether and Triton X-100, suggested that it is an enveloped virus. However, the genome of the isolated virus is a double-stranded DNA (dsDNA) molecule of 270 to 290 kbp, which is larger than known herpesviruses. The viral DNA seems highly divergent and bears only small fragments (16 to 45 bp) that are similar to the genomes of several DNA viruses. Nevertheless, amino acid sequences encoded by CNGV DNA fragments bear similarities primarily to members of the
Poxviridae
and
Herpesviridae
and to other large dsDNA viruses. We suggest, therefore, that the etiologic agent of this disease may represent an as yet unclassified virus species that is endemic in
C. carpio
(carp).
Identification of a Novel Coronavirus in Bats
Authors: Poon L. L. M. , Chu D. K. W. , Chan K. H. , Wong O. K. , Ellis T. M. , Leung Y. H. C. , Lau S. K. P. , Woo P. C. Y. , Suen K. Y. , Yuen K. Y. , Guan Y. , Peiris J. S. M.
Published: January 2005

Exotic wildlife can act as reservoirs of diseases that are endemic in the area or can be the source of new emerging diseases through interspecies transmission. The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlights the importance of virus surveillance in wild animals. Here, we report the identification of a novel bat coronavirus through surveillance of coronaviruses in wildlife. Analyses of the RNA sequence from the ORF1b and S-gene regions indicated that the virus is a group 1 coronavirus. The virus was detected in fecal and respiratory samples from three bat species (
Miniopterus
spp.). In particular, 63% (12 of 19) of fecal samples from
Miniopterus pusillus
were positive for the virus. These findings suggest that this virus might be commonly circulating in
M
.
pusillus
in Hong Kong.
Formulation with CpG Oligodeoxynucleotides Prevents Induction of Pulmonary Immunopathology following Priming with Formalin-Inactivated or Commercial Killed Bovine Respiratory Syncytial Virus Vaccine
Authors: Oumouna M. , Mapletoft J. W. , Karvonen B. C. , Babiuk L. A. , van Drunen Littel-van den Hurk S.
Published: January 2005

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-γ production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.
Persistent Borna Disease Virus Infection Confers Instability of HSP70 mRNA in Glial Cells during Heat Stress
Authors: Yamashita Makiko , Kamitani Wataru , Yanai Hideyuki , Ohtaki Naohiro , Watanabe Yohei , Lee Byeong-Jae , Tsuji Shoutaro , Ikuta Kazuyoshi , Tomonaga Keizo
Published: January 2005

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes neurological disorders in many vertebrate species. Although BDV readily establishes lasting persistence, persistently infected cells maintain an apparently normal cell phenotype in terms of morphology, viability, and proliferation. In this study, to understand the regulation of stress responses in BDV infection, we investigated the expression of heat shock proteins (HSPs) in glial cells persistently infected with BDV. Interestingly, we found that BDV persistence did not upregulate HSP70 expression even in cells treated with heat stress. Furthermore, BDV-infected glial cells exhibited rapid rounding and detachment from the culture plate under various stressful conditions. Immunofluorescence analysis demonstrated that heat stress rapidly disrupts the cell cytoskeleton only in persistently infected cells, suggesting a lack of thermotolerance. Intriguingly, we found that although persistently infected glial cells expressed HSP70 mRNA after heat stress, its expression rapidly disappeared during the recovery period. These observations indicated that persistent BDV infection may affect the stability of HSP70 mRNA. Finally, we found that the double-stranded RNA-dependent protein kinase (PKR) is expressed at a constant level in persistently infected cells with or without heat shock. Considering the interrelationship between HSP70 and PKR production, our data suggest that BDV infection disturbs the cellular stress responses to abolish antiviral activities and maintain persistence.
Antibody Neutralization Escape Mediated by Point Mutations in the Intracytoplasmic Tail of Human Immunodeficiency Virus Type 1 gp41
Authors: Kalia Vandana , Sarkar Surojit , Gupta Phalguni , Montelaro Ronald C.
Published: January 2005

The persistence of human immunodeficiency virus type 1 (HIV-1) infection in the presence of robust host immunity has been associated in part with variation in viral envelope proteins leading to antigenic variation and escape from neutralizing antibodies. Previous studies of natural neutralization escape mutants have predominantly focused on gp120 and gp41 ectodomain sequence variations that alter antibody binding via changes in conformation or glycosylation pattern of the Env, likely due to the immune pressure exerted on the exposed ectodomain component of the glycoprotein. Here, we show for the first time a novel mechanism by which point mutations in the intracytoplasmic tail of the transmembrane component (gp41) of envelope can render the virus resistant to neutralization by monoclonal antibodies and broadly neutralizing polyclonal serum antibodies. Point mutations in a highly conserved structural motif within the intracytoplasmic tail resulted in decreased binding of neutralizing antibodies to the Env ectodomain, evidently due to allosteric changes both in the gp41 ectodomain and in gp120. While receptor binding and infectivity of the mutant virus remained unaltered, the changes in Env antigenicity were associated with an increase in neutralization resistance of the mutant virus. These studies demonstrate the structurally integrated nature of gp120 and gp41 and underscore a previously unrecognized potentially critical role for even minor sequence variation of the intracytoplasmic tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.
Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand
Authors: Yuan Ji , Cheung Paul K. M. , Zhang Huifang M. , Chau David , Yang Decheng
Published: January 2005

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3′ end than the 5′ end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.
Complex Formation by Glycoproteins M and N of Human Cytomegalovirus: Structural and Functional Aspects
Authors: Mach Michael , Kropff Barbara , Kryzaniak Magdalena , Britt William
Published: January 2005

The genomes of herpesviruses contain a number of genes which are conserved throughout the family of
Herpesviridae
, indicating that the proteins may serve important functions in the replication of these viruses. Among these are several envelope glycoproteins, including glycoprotein M (gM) and gN, which form a complex that is covalently linked via disulfide bonds in some herpesviruses. However, deletion of gM and/or gN from most alphaherpesviruses has limited effects on replication of the respective viruses in vitro. In contrast, insertional inactivation of the gM gene of the betaherpesvirus human cytomegalovirus (HCMV) results in a replication-incompetent virus. We have started to analyze the structural and functional aspects of the interaction between gM and gN of HCMV. We show that large parts of gM are dispensable for the formation of a gM/gN complex that is transported to distal parts of the cellular secretory pathway. In addition, we demonstrate that the disulfide bond is between the cysteine at position 44 in gM and cysteine 90 in gN. However, disulfide linkage is not a prerequisite for modification and transport of the gM/gN complex. Moreover, mutant viruses that lack a disulfide bridge between gM and gN replicate with efficiencies similar to that of wild-type viruses.
Cell Biological and Functional Characterization of the Vaccinia Virus F10 Kinase: Implications for the Mechanism of Virion Morphogenesis
Authors: Punjabi Almira , Traktman Paula
Published: January 2005

The vaccinia virus F10 protein is one of two virally encoded protein kinases. A phenotypic analysis of infections involving a tetracycline-inducible recombinant (vΔiF10) indicated that F10 is involved in the early stages of virion morphogenesis, as previously reported for the mutants
ts
28 and
ts
15. The proteins encoded by
ts
28 and
ts
15 have primary defects in enzymatic activity and thermostability, respectively. Using a transient complementation assay, we demonstrated that the enzymatic activity of F10 is essential for its biological function and that both its enzymatic and biological functions depend upon N-terminal sequences that precede the catalytic domain. An execution point analysis indicated that in addition to its role at the onset of morphogenesis, F10 is also required at later stages, when membrane crescents surround virosomal contents and develop into immature virions. The F10 protein is phosphorylated in vivo, appears to be tightly associated with intracellular membranes, and can bind to specific phosphoinositides in vitro. When F10 is repressed or impaired, the phosphorylation of several cellular and viral proteins appears to increase in intensity, suggesting that F10 may normally intersect with cellular signaling cascades via the activation of a phosphatase or the inhibition of another kinase. These cascades may drive the F10-induced remodeling of membranes that accompanies virion biogenesis. Upon the release of
ts
28-infected cultures from a 40°C-induced block, a synchronous resumption of morphogenesis that culminates in the production of infectious virus can be observed. The pharmacological agents H89 and cerulenin, which are inhibitors of endoplasmic reticulum exit site formation and de novo lipid synthesis, respectively, block this recovery.
Hepatitis C Virus Core Protein Induces an Anergic State Characterized by Decreased Interleukin-2 Production and Perturbation of Mitogen-Activated Protein Kinase Responses
Authors: Sundström Sara , Ota Seisuke , Dimberg Lina Y. , Masucci Maria G. , Bergqvist Anders
Published: January 2005

Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infections, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Here we demonstrate that the expression of the HCV core (C) protein in stably transfected T cells correlates with a selective reduction of interleukin-2 (IL-2) promoter activity and IL-2 production in response to T-cell receptor triggering, whereas the activation of IL-4, IL-10, gamma interferon, and tumor necrosis factor alpha was moderately increased. This altered cytokine expression profile was associated with a perturbation of mitogen-activated protein (MAP) kinase responses. Extracellular regulated kinase and p38 were constitutively phosphorylated in C-expressing cells, while triggering of the costimulatory c-Jun N-terminal kinase (JNK) signaling cascade and activation of the CD28 response element within the IL-2 promoter appeared to be impaired. The perturbations of MAP kinase phosphorylation could be eliminated by cyclosporine A-mediated inhibition of nuclear factor of activated T cells, suggesting that the inactivation of JNK signaling and hyporesponsiveness to IL-2 induction were downstream consequences of C-induced Ca2+flux in a manner that mimics the induction of clonal anergy.
Genetic Variability of the Major Histocompatibility Complex Class I Homologue Encoded by Human Cytomegalovirus Leads to Differential Binding to the Inhibitory Receptor ILT2
Authors: Valés-Gómez Mar , Shiroishi Mitsunori , Maenaka Katsumi , Reyburn Hugh T.
Published: January 2005

Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with β2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted α1, α2, and α3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.
Genome Packaging Sense Is Controlled by the Efficiency of the Nick Site in the Right-End Replication Origin of Parvoviruses Minute Virus of Mice and LuIII
Authors: Cotmore Susan F. , Tattersall Peter
Published: January 2005

The parvovirus minute virus of mice (MVM) packages predominantly negative-sense single strands, while its close relative LuIII encapsidates strands of both polarities with equal efficiency. Using genomic chimeras and mutagenesis, we show that the ability to package positive strands maps not, as originally postulated, to divergent untranslated regions downstream of the capsid gene but to the viral hairpins and predominantly to the nick site of OriR, the right-end replication origin. In MVM, the sequence of this site is 5′-CTAT
▾
TCA-3′, while in LuIII a two-base insertion (underlined) changes it to 5′-CTAT

AT

▾
TCA-3′. Matched LuIII genomes differing only at this position (designated LuIII and LuΔ2) packaged 47 and <8% positive-sense strands, respectively. OriR sequences from these viruses were both able to support NS1-mediated nicking in vitro, but initiation efficiency was consistently two- to threefold higher for LuΔ2 derivatives, suggesting that LuIII's ability to package positive strands is determined by a suboptimal right-end origin rather than by strand-specific packaging sequences. These observations support a mathematical “kinetic hairpin transfer” model, previously described by Chen and colleagues (K. C. Chen, J. J. Tyson, M. Lederman, E. R. Stout, and R. C. Bates, J. Mol. Biol. 208:283-296, 1989), that postulates that preferential excision of particular strands is solely responsible for packaging specificity. By analyzing replicative-form (RF) DNA generated in vivo during LuIII and LuΔ2 infections, we extend this model, showing that positive-sense strands do accumulate in LuΔ2 infections as part of duplex RF DNA, but these do not support packaging. However, replication is biphasic, so that accumulation of positive-sense strands is ultimately suppressed, probably because the onset of packaging removes newly displaced single strands from the replicating pool.
The Adenovirus E4-6/7 Protein Directs Nuclear Localization of E2F-4 via an Arginine-Rich Motif
Authors: Schaley Joel E. , Polonskaia Marina , Hearing Patrick
Published: January 2005

E2F transcription factors are key participants in the regulation of proliferation, apoptosis, and differentiation in mammalian cells. E2Fs are negatively regulated by members of the retinoblastoma protein (pRb) family. During adenovirus (Ad) infection, viral proteins that displace pRb family members from E2Fs and recruit E2F complexes to viral and cellular promoter regions are expressed. This recruitment of E2F involves the induction of stable E2F binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters and induces both viral and cellular gene expression. E2F-4 has abundant E2F activity within cells, and the majority of E2F-4 in asynchronous cells is found in the cytoplasm. Upon expression of the adenovirus E4-6/7 protein, a significant portion of E2F-4 is translocated to the nucleus, and its activity constitutes the majority of Ad-induced nuclear E2F DNA binding activity. This redirection of E2F-4 from cytoplasm to the nucleus requires an N-terminal arginine-rich nuclear localization sequence within E4-6/7. The directed targeting of E4-6/7 to the nucleus is important for the function of this protein in the context of viral infection. This function of E4-6/7 has a redundant component as well as nonredundant components in cooperation with the adenovirus E1A oncoproteins to deregulate and usurp host cell E2F function.
Ribavirin Resistance in Hepatitis C Virus Replicon-Containing Cell Lines Conferred by Changes in the Cell Line or Mutations in the Replicon RNA
Authors: Pfeiffer Julie K. , Kirkegaard Karla
Published: January 2005

Ribavirin (RBV), used in combination with alpha interferon to treat hepatitis C virus (HCV) infections, is a guanosine nucleotide analog that can increase the error rate of viral RNA-dependent RNA polymerases, imbalance intracellular nucleotide pools, and cause toxicity in many cell types. To determine potential mechanisms of RBV resistance during HCV RNA replication, we passaged HCV replicon-containing cell lines in the presence of increasing concentrations of RBV. RBV-resistant, HCV replicon-containing cell lines were generated, and the majority of RBV resistance was found to be conferred by changes in the cell lines. The resistant cell lines were defective in RBV import, as measured by [
3
H]RBV uptake experiments. These cell lines displayed reduced RBV toxicity and reduced error accumulation during infection with poliovirus, whose replication is known to be sensitive to RBV-induced error. For one RBV-resistant isolate, two mutations in the replicon RNA contributed to the observed phenotype. Two responsible mutations resided in the C-terminal region of NS5A, G404S, and E442G and were each sufficient for low-level RBV resistance. Therefore, RBV resistance in HCV replicon cell lines can be conferred by changes in the cell line or by mutations in the HCV replicon.
Structural and Functional Analysis of the RNA Transport Element, a Member of an Extensive Family Present in the Mouse Genome
Authors: Smulevitch Sergey , Michalowski Daniel , Zolotukhin Andrei S. , Schneider Ralf , Bear Jenifer , Roth Patricia , Pavlakis George N. , Felber Barbara K.
Published: January 2005

We previously identified an RNA transport element (RTE), present in a subclass of rodent intracisternal A particle retroelements (F. Nappi, R. Schneider, A. Zolotukhin, S. Smulevitch, D. Michalowski, J. Bear, B. Felber, and G. Pavlakis, J. Virol.
75:
4558-4569, 2001), that is able to replace Rev-responsive element regulation in human immunodeficiency virus type 1. RTE-directed mRNA export is mediated by a still-unknown cellular factor(s), is independent of the CRM1 nuclear export receptor, and is conserved among vertebrates. Here we show that this RTE folds into an extended RNA secondary structure and thus does not resemble any known RTEs. Computer searches revealed the presence of 105 identical elements and more than 3,000 related elements which share at least 70% sequence identity with the RTE and which are found on all mouse chromosomes. These related elements are predicted to fold into RTE-like structures. Comparison of the sequences and structures revealed that the RTE and related elements can be divided into four groups. Mutagenesis of the RTE revealed that the minimal element contains four internal stem-loops, which are indispensable for function in mammalian cells. In contrast, only part of the element is essential to mediate RNA transport in microinjected
Xenopus laevis
oocyte nuclei. Importantly, the minimal RTE able to promote RNA transport has key structural features which are preserved in all the RTE-related elements, further supporting their functional importance. Therefore, RTE function depends on a complex secondary structure that is important for the interaction with the cellular export factor(s).
Roles of the ITAM and PY Motifs of Epstein-Barr Virus Latent Membrane Protein 2A in the Inhibition of Epithelial Cell Differentiation and Activation of β-Catenin Signaling
Authors: Morrison Jennifer A. , Raab-Traub Nancy
Published: January 2005

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is important for maintenance of latency in infected B lymphocytes. Through its immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs, LMP2A is able to block B-cell receptor (BCR) signaling, bind BCR-associated kinases, and manipulate the turnover of itself and these kinases via a PY-mediated interaction with the Nedd4 family of ubiquitin ligases. In epithelial cells, LMP2A has been shown to activate the phosphatidylinositol 3′-OH kinase/Akt and β-catenin signaling pathways. In the present study, the biological consequences of LMP2A expression in the normal human foreskin keratinocyte (HFK) cell line were investigated and the importance of the ITAM and PY motifs for LMP2A signaling effects in HFK cells was ascertained. The ITAM was essential for the activation of Akt by LMP2A in HFK cells, while both the ITAM and PY motifs contributed to LMP2A-mediated accumulation and nuclear translocation of the oncoprotein β-catenin. LMP2A inhibited induction of differentiation in an assay conducted with semisolid methylcellulose medium, and the PY motifs were critical for this inhibition. LMP2A is expressed in the EBV-associated epithelial malignancies nasopharyngeal carcinoma and gastric carcinoma, and these data indicate that LMP2A affects cellular processes that likely contribute to carcinogenesis.
Identification of Specific Cellular Genes Up-Regulated Late in Adenovirus Type 12 Infection
Authors: Dorn Andreas , Zhao Hongxing , Granberg Frederik , Hösel Marianna , Webb Dennis , Svensson Catharina , Pettersson Ulf , Doerfler Walter
Published: January 2005

The infection of human cells by adenoviruses leads to a gradual reduction in the activity of host cell functions while viral gene expression progresses in a regulated way. We used the DNA microarray technique to determine the transcriptional activity profiles of cellular genes upon infection with adenovirus type 12 (Ad12). The microarray data were validated by quantitative real-time PCR for genes which showed significant alterations after Ad12 infection. At 12 h postinfection, there is a striking up-regulation between 10- and 30-fold in the expression of the G1P2, IFIT1, and IFIT2 cellular immune response genes compared to mock-infected cells. At later stages of infection, when the majority of regulated cellular genes has been turned down, a limited number of cellular genes exhibit increased activities by factors of 3 or less. These genes belong to the signal transduction or transcriptional regulator classes or are active in protein degradation, like ANPEP, an aminopeptidase. The SCD and CYP2S1 genes function in lipid metabolism. The eucaryotic translation initiation factor 4 is up-regulated, and one of the major histocompatibility complex genes is diminished in activity. For two of the genes, one up-regulated (CTSF gene) and one down-regulated (CYR61 gene), alterations in gene activity were confirmed at the protein level by Western blotting experiments. Increased genetic activity of cellular genes late in adenovirus infection has not been reported previously and demonstrates that Ad12 has a sustained control of host cell gene expression well into the late phase of infection.
Role of Ebola Virus Secreted Glycoproteins and Virus-Like Particles in Activation of Human Macrophages
Authors: Wahl-Jensen Victoria , Kurz Sabine K. , Hazelton Paul R. , Schnittler Hans-Joachim , Ströher Ute , Burton Dennis R. , Feldmann Heinz
Published: January 2005

Ebola virus, a member of the family
Filoviridae
, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Δ-peptide), GP
1
, and GP
1,2Δ
. While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP
1,2
in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.
Adenovirus Protein VII Functions throughout Early Phase and Interacts with Cellular Proteins SET and pp32
Authors: Xue Yuming , Johnson Jeffrey S. , Ornelles David A. , Lieberman Judy , Engel Daniel A.
Published: January 2005

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. “Input” protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.
Genetic Analyses of DNA-Binding Mutants in the Catalytic Core Domain of Human Immunodeficiency Virus Type 1 Integrase
Authors: Lu Richard , Limón Ana , Ghory Hina Z. , Engelman Alan
Published: January 2005

The catalytic core domain (CCD) of human immunodeficiency virus type 1 (HIV-1) integrase (IN) harbors the enzyme active site and binds viral and chromosomal DNA during integration. Thirty-five CCD mutant viruses were constructed, paying particular attention to conserved residues in the Phe
139
-Gln
146
flexible loop and abutting Ser
147
-Val
165
amphipathic alpha helix that were implicated from previous in vitro work as important for DNA binding. Defective viruses were typed as class I mutants (specifically blocked at integration) or pleiotropic class II mutants (additional particle assembly and/or reverse transcription defects). Whereas HIV-1
P145A
and HIV-1
Q146K
grew like the wild type, HIV-1
N144K
and HIV-1
Q148L
were class I mutants, reinforcing previous results that Gln-148 is important for DNA binding and uncovering for the first time an important role for Asn-144 in integration. HIV-1
Q62K
, HIV-1
H67E
, HIV-1
N120K
, and HIV-1
N155K
were also class I mutants, supporting findings that Gln-62 and Asn-120 interact with viral and target DNA, respectively, and suggesting similar integration-specific roles for His-67 and Asn-155. Although results from complementation analyses established that IN functions as a multimer, the interplay between active-site and CCD DNA binding functions was unknown. By using Vpr-IN complementation, we determined that the CCD protomer that catalyzes integration also preferentially binds to viral and target DNA. We additionally characterized E138K as an intramolecular suppressor of Gln-62 mutant virus and IN. The results of these analyses highlight conserved CCD residues that are important for HIV-1 replication and integration and define the relationship between DNA binding and catalysis that occurs during integration in vivo.
Role of Nucleotides Immediately Flanking the Transcription-Regulating Sequence Core in Coronavirus Subgenomic mRNA Synthesis
Authors: Sola Isabel , Moreno José L. , Zúñiga Sonia , Alonso Sara , Enjuanes Luis
Published: January 2005

The generation of subgenomic mRNAs in coronavirus involves a discontinuous mechanism of transcription by which the common leader sequence, derived from the genome 5′ terminus, is fused to the 5′ end of the mRNA coding sequence (body). Transcription-regulating sequences (TRSs) precede each gene and include a conserved core sequence (CS) surrounded by relatively variable sequences (5′ TRS and 3′ TRS). Regulation of transcription in coronaviruses has been studied by reverse-genetics analysis of the sequences immediately flanking a unique CS in the
Transmissible gastroenteritis virus
genome (CS-S2), located inside the S gene, that does not lead to detectable amounts of the corresponding mRNA, in spite of its canonical sequence. The transcriptional inactivity of CS-S2 was genome position independent. The presence of a canonical CS was not sufficient to drive transcription, but subgenomic synthesis requires a minimum base pairing between the leader TRS (TRS-L) and the complement of the body TRS (cTRS-B) provided by the CS and its adjacent nucleotides. A good correlation was observed between the free energy of TRS-L and cTRS-B duplex formation and the levels of subgenomic mRNA S2, demonstrating that base pairing between the leader and body beyond the CS is a determinant regulation factor in coronavirus transcription. In TRS mutants with increasing complementarity between TRS-L and cTRS-B, a tendency to reach a plateau in Δ
G
values was observed, suggesting that a more precise definition of the TRS limits might be proposed, specifically that it consists of the central CS and around 4 nucleotides flanking 5′ and 3′ the CS. Sequences downstream of the CS exert a stronger influence on the template-switching decision according to a model of polymerase strand transfer and template switching during minus-strand synthesis.
Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes
Authors: Trinks Daniela , Rajeswaran R. , Shivaprasad P. V. , Akbergenov Rashid , Oakeley Edward J. , Veluthambi K. , Hohn Thomas , Pooggin Mikhail M.
Published: January 2005

Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from
Mungbean yellow mosaic virus-Vigna
(MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model
Nicotiana benthamiana
plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in
Arabidopsis
protoplasts, MYMV AC2 or its homologue from
African cassava mosaic geminivirus
coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from
Arabidopsis
were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.
Mutational Analysis of Narrow Pores at the Fivefold Symmetry Axes of Adeno-Associated Virus Type 2 Capsids Reveals a Dual Role in Genome Packaging and Activation of Phospholipase A2 Activity
Authors: Bleker Svenja , Sonntag Florian , Kleinschmidt Jürgen A.
Published: January 2005

Adeno-associated virus type 2 (AAV2) capsids show 12 pores at the fivefold axes of symmetry. We mutated amino acids which constitute these pores to investigate possible functions of these structures within the AAV2 life cycle. Mutants with alterations in conserved residues were impaired mainly in genome packaging or infectivity, whereas few mutants were affected in capsid assembly. The packaging phenotype was characterized by increased capsid-per-genome ratios. Analysis of capsid-associated DNA versus encapsidated DNA revealed that this observation was due to reduced and not partial DNA encapsidation. Most mutants with impaired infectivity showed a decreased capability to expose their VP1 N termini. As a consequence, the activation of phospholipase A2 (PLA2) activity, which is essential for efficient infection, was affected on intact capsids. In a few mutants, the exposure of VP1 N termini and the development of PLA2 activity were associated with enhanced capsid instability, which is obviously also deleterious for virus infection. Therefore, PLA2 activity seems to be required on intact capsids for efficient infection. In conclusion, these results suggest that the pores at the fivefold axes function not only as portals for AAV2 single-stranded DNA packaging but also as channels for presentation of the PLA2 domain on AAV2 virions during infection.
Molecular Epidemiology of Simian T-Cell Lymphotropic Virus Type 1 in Wild and Captive Sooty Mangabeys
Authors: Traina-Dorge Vicki L. , Lorino Rebecca , Gormus Bobby J. , Metzger Michael , Telfer Paul , Richardson David , Robertson David L. , Marx Preston A. , Apetrei Cristian
Published: January 2005

A study was conducted to evaluate the prevalence and diversity of simian T-cell lymphotropic virus (STLV) isolates within the long-established Tulane National Primate Research Center (TNPRC) colony of sooty mangabeys (SMs;
Cercocebus atys
). Serological analysis determined that 22 of 39 animals (56%) were positive for STLV type 1 (STLV-1). A second group of thirteen SM bush meat samples from Sierra Leone in Africa was also included and tested only by PCR. Twenty-two of 39 captive animals (56%) and 3 of 13 bush meat samples (23%) were positive for STLV-1, as shown by testing with PCR. Nucleotide sequencing and phylogenetic analysis of viral strains obtained demonstrated that STLV-1 strains from SMs (STLV-1sm strains) from the TNPRC colony and Sierra Leone formed a single cluster together with the previously reported STLV-1sm strain from the Yerkes National Primate Research Center. These data confirm that Africa is the origin for TNPRC STLV-1sm and suggest that Sierra Leone is the origin for the SM colonies in the United States. The TNPRC STLV-1sm strains further divided into two subclusters, suggesting STLV-1sm infection of two original founder SMs at the time of their importation into the United States. STLV-1sm diversity in the TNPRC colony matches the high diversity of SIVsm in the already reported colony. The lack of correlation between the lineage of the simian immunodeficiency virus from SMs (SIVsm) and the STLV-1sm subcluster distribution of the TNPRC strains suggests that intracolony transmissions of both viruses were independent events.
Interaction of the Tobacco Mosaic Virus Replicase Protein with the Aux/IAA Protein PAP1/IAA26 Is Associated with Disease Development
Authors: Padmanabhan Meenu S. , Goregaoker Sameer P. , Golem Sheetal , Shiferaw Haiymanot , Culver James N.
Published: January 2005

Virus-infected plants often display developmental abnormalities that include stunting, leaf curling, and the loss of apical dominance. In this study, the helicase domain of the
Tobacco mosaic virus
(TMV) 126- and/or 183-kDa replicase protein(s) was found to interact with the
Arabidopsis
Aux/IAA protein PAP1 (also named IAA26), a putative regulator of auxin response genes involved in plant development. To investigate the role of this interaction in the display of symptoms, a TMV mutant defective in the PAP1 interaction was identified. This mutant replicated and moved normally in
Arabidopsis
but induced attenuated developmental symptoms. Additionally, transgenic plants in which the accumulation of PAP1 mRNA was silenced exhibit symptoms like those of virus-infected plants. In uninfected tissues, ectopically expressed PAP1 accumulated and localized to the nucleus. However, in TMV-infected tissues, PAP1 failed to accumulate to significant levels and did not localize to the nucleus, suggesting that interaction with the TMV replicase protein disrupts PAP1 localization. The consequences of this interaction would affect PAP1's putative function as a transcriptional regulator of auxin response genes. This is supported by gene expression data indicating that ∼30% of the
Arabidopsis
genes displaying transcriptional alterations in response to TMV contain multiple auxin response promoter elements. Combined, these data indicate that the TMV replicase protein interferes with the plant's auxin response system to induce specific disease symptoms.