bluuebunny/crossref_metadata_2025 huggingface dataset:
An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations
Authors: Makita Noriko , Sato Junichiro , Manaka Katsunori , Shoji Yuki , Oishi Atsuro , Hashimoto Makiko , Fujita Toshiro , Iiri Taroh
Published: March 2007

The seven-spanning calcium-sensing receptor (CaSR) activates multiple G proteins including Gq and Gi, and thereby activates a variety of second messengers and inhibits parathyroid hormone (PTH) secretion. However, the exact signaling mechanisms underlying the functional activity of CaSR are not yet fully understood. The heterozygous inactivation of CaSR or its inhibition by antibody blocking results in either familial hypocalciuric hypercalcemia or acquired hypocalciuric hypercalcemia (AHH), respectively. Here, we report the identification of a unique CaSR autoantibody in an AHH patient. Paradoxically, we find that this autoantibody potentiates the Ca
2+
/Gq-dependent accumulation of inositol phosphates by slightly shifting the dose dependence curve of the Ca
2+
mediated activation of phosphatidylinositol turnover to the left, whereas it inhibits the Ca
2+
/Gi-dependent phosphorylation of ERK1/2 in HEK293 cells stably expressing human CaSR. Treatment of these same cells with a calcimimetic, NPS-R-568, augments the CaSR response to Ca
2+
, increasing phosphatidylinositol turnover and ERK1/2 phosphorylation, and overcoming the autoantibody effects. Our observations thus indicate that a calcium-stimulated CaSR primed by a specific autoantibody adopts a unique conformation that activates Gq but not Gi. Our findings also suggest that CaSR signaling may act via both Gq and Gi to inhibit PTH secretion. This is the first report of a disease-related autoantibody that functions as an allosteric modulator and maintains G protein-coupled receptors (GPCRs) in a unique active conformation with its agonist. We thus speculate that physiological modulators may exist that enable an agonist to specifically activate only one signaling pathway via a GPCR that activates multiple signaling pathways.
Wnt/β-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency
Authors: Miyabayashi Tomoyuki , Teo Jia-Ling , Yamamoto Masashi , McMillan Michael , Nguyen Cu , Kahn Michael
Published: March 2007

Embryonic stem cells (ESCs) represent an important research tool and a potential resource for regenerative medicine. Generally, ESCs are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts, which maintain the ESCs' capacity for self-renewal and block spontaneous differentiation. These cumbersome conditions, as well as the risk of xenobiotic contamination of human ESCs grown on murine embryonic fibroblasts, make it a priority to develop chemically defined methods that can be safely used for the expansion of ESCs. Using a high-throughput, cell-based assay, we identified the small molecule IQ-1 that allows for the Wnt/β-catenin-driven long-term expansion of mouse ESCs and prevents spontaneous differentiation. We demonstrate that IQ-1, by targeting the PR72/130 subunit of the serine/threonine phosphatase PP2A, prevents β-catenin from switching coactivator usage from CBP to p300. The increase in β-catenin/CBP-mediated transcription at the expense of β-catenin/p300-mediated transcription is critical for the maintenance of murine stem cell pluripotency.
Essential role of PDK1 in regulating endothelial cell migration
Authors: Primo Luca , di Blasio Laura , Roca Cristina , Droetto Sara , Piva Roberto , Schaffhausen Brian , Bussolino Federico
Published: March 2007

The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1−/− embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.
Rap1 controls cell adhesion and cell motility through the regulation of myosin II
Authors: Jeon Taeck J. , Lee Dai-Jen , Merlot Sylvain , Weeks Gerald , Firtel Richard A.
Published: March 2007

We have investigated the role of Rap1 in controlling chemotaxis and cell adhesion in Dictyostelium discoideum. Rap1 is activated rapidly in response to chemoattractant stimulation, and activated Rap1 is preferentially found at the leading edge of chemotaxing cells. Cells expressing constitutively active Rap1 are highly adhesive and exhibit strong chemotaxis defects, which are partially caused by an inability to spatially and temporally regulate myosin assembly and disassembly. We demonstrate that the kinase Phg2, a putative Rap1 effector, colocalizes with Rap1–guanosine triphosphate at the leading edge and is required in an in vitro assay for myosin II phosphorylation, which disassembles myosin II and facilitates filamentous actin–mediated leading edge protrusion. We suggest that Rap1/Phg2 plays a role in controlling leading edge myosin II disassembly while passively allowing myosin II assembly along the lateral sides and posterior of the cell.
Inhibition of caveolar uptake, SV40 infection, and β1-integrin signaling by a nonnatural glycosphingolipid stereoisomer
Authors: Singh Raman Deep , Holicky Eileen L. , Cheng Zhi-jie , Kim Seong-Youl , Wheatley Christine L. , Marks David L. , Bittman Robert , Pagano Richard E.
Published: March 2007

Caveolar endocytosis is an important mechanism for the uptake of certain pathogens and toxins and also plays a role in the internalization of some plasma membrane (PM) lipids and proteins. However, the regulation of caveolar endocytosis is not well understood. We previously demonstrated that caveolar endocytosis and β1-integrin signaling are stimulated by exogenous glycosphingolipids (GSLs). In this study, we show that a synthetic GSL with nonnatural stereochemistry, β-d-lactosyl-N-octanoyl-l-threo-sphingosine, (1) selectively inhibits caveolar endocytosis and SV40 virus infection, (2) blocks the clustering of lipids and proteins into GSLs and cholesterol-enriched microdomains (rafts) at the PM, and (3) inhibits β1-integrin activation and downstream signaling. Finally, we show that small interfering RNA knockdown of β1 integrin in human skin fibroblasts blocks caveolar endocytosis and the stimulation of signaling by a GSL with natural stereochemistry. These experiments identify a new compound that can interfere with biological processes by inhibiting microdomain formation and also identify β1 integrin as a potential mediator of signaling by GSLs.
The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis
Authors: Yu Hong-Guo , Koshland Douglas
Published: March 2007

Homologue segregation during the first meiotic division requires the proper spatial regulation of sister chromatid cohesion and its dissolution along chromosome arms, but its protection at centromeric regions. This protection requires the conserved MEI-S332/Sgo1 proteins that localize to centromeric regions and also recruit the PP2A phosphatase by binding its regulatory subunit, Rts1. Centromeric Rts1/PP2A then locally prevents cohesion dissolution possibly by dephosphorylating the protein complex cohesin. We show that Aurora B kinase in Saccharomyces cerevisiae (Ipl1) is also essential for the protection of meiotic centromeric cohesion. Coupled with a previous study in Drosophila melanogaster, this meiotic function of Aurora B kinase appears to be conserved among eukaryotes. Furthermore, we show that Sgo1 recruits Ipl1 to centromeric regions. In the absence of Ipl1, Rts1 can initially bind to centromeric regions but disappears from these regions after anaphase I onset. We suggest that centromeric Ipl1 ensures the continued centromeric presence of active Rts1/PP2A, which in turn locally protects cohesin and cohesion.
Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain–like mechanism
Authors: Mattila Pieta K. , Pykäläinen Anette , Saarikangas Juha , Paavilainen Ville O. , Vihinen Helena , Jokitalo Eija , Lappalainen Pekka
Published: March 2007

The actin cytoskeleton plays a fundamental role in various motile and morphogenetic processes involving membrane dynamics. We show that actin-binding proteins MIM (missing-in-metastasis) and IRSp53 directly bind PI(4,5)P2-rich membranes and deform them into tubular structures. This activity resides in the N-terminal IRSp53/MIM domain (IMD) of these proteins, which is structurally related to membrane-tubulating BAR (Bin/amphiphysin/Rvs) domains. We found that because of a difference in the geometry of the PI(4,5)P2-binding site, IMDs induce a membrane curvature opposite that of BAR domains and deform membranes by binding to the interior of the tubule. This explains why IMD proteins induce plasma membrane protrusions rather than invaginations. We also provide evidence that the membrane-deforming activity of IMDs, instead of the previously proposed F-actin–bundling or GTPase-binding activities, is critical for the induction of the filopodia/microspikes in cultured mammalian cells. Together, these data reveal that interplay between actin dynamics and a novel membrane-deformation activity promotes cell motility and morphogenesis.
Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo
Authors: Hirai Maretoshi , Ohbayashi Tetsuya , Horiguchi Masahito , Okawa Katsuya , Hagiwara Akari , Chien Kenneth R. , Kita Toru , Nakamura Tomoyuki
Published: March 2007

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.
The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x
Authors: Paronetto Maria Paola , Achsel Tilman , Massiello Autumn , Chalfant Charles E. , Sette Claudio
Published: March 2007

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.
CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells
Authors: Schneider Martin A. , Meingassner Josef G. , Lipp Martin , Moore Henrietta D. , Rot Antal
Published: March 2007

CCR7-mediated migration of naive T cells into the secondary lymphoid organs is a prerequisite for their encounter with mature dendritic cells, the productive presentation of cognate antigen, and consequent T cell proliferation and effector differentiation. Therefore, CCR7 was suggested to play an important role in the initiation of adaptive immune responses. In this study, we show that primary immunity can also develop in the absence of CCR7. Moreover, CCR7-deficient knockout (KO) mice display augmented immune responses. Our data cumulatively suggest that enhanced immunity in CCR7 KO mice is caused by the defective lymph node (LN) positioning of FoxP3+ CD4+ CD25+ regulatory T cells (T reg cells) and the consequent impediment of their function. The FoxP3+ T reg cells express CCR7 and, after their adoptive transfer, migrate into the LNs of wild-type mice. Here, they proliferate in situ upon antigen stimulation and inhibit the generation of antigen-specific T cells. Conversely, transferred CCR7-deficient T reg cells fail to migrate into the LNs and suppress antigen-induced T cell responses. The transfer of combinations of naive and T reg cells from wild-type and CCR7 KO mice into syngeneic severe combined immunodeficient mice directly demonstrates that CCR7-deficient T reg cells are less effective than their wild-type counterparts in preventing the development of inflammatory bowel disease.
Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue
Authors: Kocks Jessica R. , Davalos-Misslitz Ana Clara Marques , Hintzen Gabriele , Ohl Lars , Förster Reinhold
Published: March 2007

Presence and extent of bronchus-associated lymphoid tissue (BALT) is subject to considerable variations between species and is only occasionally observed in lungs of mice. Here we demonstrate that mice deficient for the chemokine receptor CCR7 regularly develop highly organized BALT. These structures were not present at birth but were detectable from day 5 onwards. Analyzing CCR7−/−/wild-type bone marrow chimeras, we demonstrate that the development of BALT is caused by alterations of the hematopoietic system in CCR7-deficient mice. These observations together with the finding that CCR7-deficient mice posses dramatically reduced numbers of regulatory T cells (T reg cells) in the lung-draining bronchial lymph node suggest that BALT formation might be caused by disabled in situ function of T reg cells. Indeed, although adoptive transfer of wild-type T reg cells to CCR7-deficient recipients resulted in a profound reduction of BALT formation, neither naive wild-type T cells nor T reg cells from CCR7−/− donors impair BALT generation. Furthermore, we provide evidence that CCR7-deficient T reg cells, although strongly impaired in homing to peripheral lymph nodes, are fully effective in vitro. Thus our data reveal a CCR7-dependent homing of T reg cells to peripheral lymph nodes in conjunction with a role for these cells in controlling BALT formation.
Diacylglycerol kinase ζ regulates microbial recognition and host resistance toToxoplasma gondii
Authors: Liu Cheng-Hu , Machado Fabiana S. , Guo Rishu , Nichols Kim E. , Burks A. Wesley , Aliberti Julio C. , Zhong Xiao-Ping
Published: March 2007

Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol (DAG) kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting DAG to phosphatidic acid (PA). We demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mφ) and dendritic cells. DGKζ deficiency results in impaired interleukin (IL) 12 and tumor necrosis factor α production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the phosphatidylinositol 3–kinase (PI3K)–Akt pathway and that inhibition of PI3K activity or treatment with PA can restore lipopolysaccharide-induced IL-12 production by DGKζ-deficient Mφ. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.
Immunological differences between susceptible and resistant sheep during the preclinical phase of scrapie infection
Authors: Eaton S. L. , Rocchi M. , González L. , Hamilton S. , Finlayson J. , Sales J. , Jeffrey M. , Steele P. J. , Dagleish M. P. , Rodger S. M. , Reid H. W. , Chianini F.
Published: March 2007

In order to investigate the relationship between the immune response to scrapie infection and genetic susceptibility to the disease in sheep, immune cell subsets and prion protein (PrP) expression were determined in susceptible and resistant Suffolk sheep in the preclinical phase of infection. At 6 months of age, 12 ARQ/ARQ (susceptible) and nine ARR/ARR (resistant) scrapie-free Suffolk lambs were challenged subcutaneously with scrapie inoculum. Prefemoral lymphadenectomies were carried out at 14 and 180 days post-inoculation (p.i.) and serial bleeds were collected at monthly intervals for up to 1 year p.i. An indirect double-labelling procedure was carried out on peripheral blood mononuclear cells (PBMCs) and lymph node cell preparations and analysed using flow cytometry. Prior to scrapie challenge, significantly more PrP+cells were detected in PBMCs from the susceptible sheep. Furthermore, following challenge, significantly more CD8+andγΔ+T cells were detected in the PBMCs of the resistant sheep. However, at both 14 and 180 days p.i, CD21+cell expression was significantly higher in the lymph node preparations of the susceptible sheep. In contrast, more CD4+cells were detected in the lymph nodes of the resistant sheep at both time points. It was concluded that significant differences in immune cell subsets and PrP expression occur between ARQ/ARQ and ARR/ARR Suffolk sheep in the preclinical phase of infection.
Molecular analysis of bovine spongiform encephalopathy infection by cDNA arrays
Authors: Sawiris G. Peter , Becker Kevin G. , Elliott Ellen J. , Moulden Robert , Rohwer Robert G.
Published: March 2007

Here, the first cDNA array analysis of differential gene expression in bovine spongiform encephalopathy (BSE) is reported, using a spotted cDNA array platform representing nearly 17 000 mouse genes. Array analysis identified 296 gene candidates for differential expression in brain tissue from VM mice in late-stage infection with the 301V strain of BSE, compared with brain tissue from normal, age-matched VM mice. Real-time PCR confirmed differential expression of 25 of 31 genes analysed. Some of the genes identified by array analysis as being expressed differentially are associated with ubiquitin/proteasome function, lysosomal function, molecular chaperoning of protein folding or apoptosis. Other genes are involved in calcium ion binding/homeostasis, zinc ion binding/homeostasis or regulation of transcription. Principal-component analysis shows that the global gene-expression profiles of the BSE-infected samples have gene-expression signatures that are markedly different from, and completely non-overlapping with, those obtained from the normal controls.
Differential onset of apoptosis in influenza A virus H5N1- and H1N1-infected human blood macrophages
Authors: Mok Chris K. P. , Lee Davy C. W. , Cheung Chung-Yan , Peiris Malik , Lau Allan S. Y.
Published: March 2007

Pathogenesis of the highly pathogenic avian influenza virus A/Hong Kong/483/97 (H5N1/97) remains to be investigated. It was demonstrated recently that H5N1 dysregulation of proinflammatory cytokines in human macrophages is a p38-kinase-dependent process. The results indicated that macrophages may play a role in disease severity. To investigate cellular responses to H5N1 infection further, apoptosis and its related pathways were studied in primary blood macrophages. Here, it is shown that the H5N1/97 virus triggered apoptosis, including caspases and PARP activation, in infected macrophages with a delayed onset compared with H1N1 counterparts. Similar results were also found in human macrophages infected by precursors of the H5N1/97 virus. Thus, these results showed that the delay in apoptosis onset in macrophages infected by H5N1/97 and its related precursor subtypes may be a means for the pathogens to have longer survival in the cells; this may contribute to the pathogenesis of H5N1 disease in humans.
Mapping and structural analysis of B-cell epitopes on the morbillivirus nucleoprotein amino terminus
Authors: Bodjo S. C. , Kwiatek O. , Diallo A. , Albina E. , Libeau G.
Published: March 2007

By analysing the antigenic structure of the morbillivirus nucleoprotein (N) using a competitive-binding assay of monoclonal antibodies (mAbs), six different antigenic sites were identified previously. By using Pepscan methodology complemented by analysis of truncated N proteins, a better characterization of five of these antigenic sites was provided: I, II, III, IV and VI. mAbs specific toRinderpest virus, defining antigenic sites II, III and IV, and those common to four morbilliviruses, delineating sites I and VI, were analysed in the present study. It was found that all but one mapped to the same region, between aa 120 and 149 of N. However, the mAb 3-1 epitope was located in the carboxy-terminal region (aa 421–525). This result may indicate the high immunogenicity of the amino-terminal variable region, at least in the mouse. It was surprising that the epitope of mAb 33-4, antigenic site VI, which recognized all morbilliviruses so far tested, was located in one of the two non-conserved regions between morbillivirus N proteins. It is shown that the conserved amino acid motif126EAD128----131F-------148EN149is critical for epitope constitution and recognition.
Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus
Authors: Tamberg Nele , Lulla Valeria , Fragkoudis Rennos , Lulla Aleksei , Fazakerley John K. , Merits Andres
Published: March 2007

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.
A reverse-genetics system for Influenza A virus using T7 RNA polymerase
Authors: de Wit Emmie , Spronken Monique I. J. , Vervaet Gaby , Rimmelzwaan Guus F. , Osterhaus Albert D. M. E. , Fouchier Ron A. M.
Published: March 2007

The currently available reverse-genetics systems forInfluenza A virusare all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue ofInfluenza A virushas been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.
Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors
Authors: Chapel Cynthia , Garcia Céline , Bartosch Birke , Roingeard Philippe , Zitzmann Nicole , Cosset François-Loïc , Dubuisson Jean , Dwek Raymond A. , Trépo Christian , Zoulim Fabien , Durantel David
Published: March 2007

Folding and assembly into complexes of some viral glycoproteins are exquisitely sensitive to endoplasmic reticulum (ER)α-glucosidase inhibition, which prevents the trimming of glucose fromN-linked glycans. Derivatives of deoxynojirimycin (DNJ) iminosugars, which are potentα-glucosidase inhibitors, were shown to have antiviral activity against bovine viral diarrhea virus, a pestivirus related to hepatitis C virus (HCV). The aim of this study was to determine whether these inhibitors would affect HCV infectivity and to provide novel insights on their mechanism of action. The overall antiviral activity of glucosidase inhibitors was shown by using the two most relevant models currently available: the cell-culture model enabling complete replication of the HCV JFH1 strain in Huh7.5 cells, and infectious HCV pseudotyped particles (HCVpp) produced in HEK-293T cells that display functional E1–E2 glycoprotein complexes. By using the latter model, it is shown that the inhibition ofα-glucosidases by iminosugars results in the misfolding and misassembly of HCV glycoprotein pre-budding complexes. This inhibition of the assembly of E1–E2 in the ER of transfected HEK-293T cells leads to a reduction in the incorporation of E1–E2 complexes into HCVpp. More importantly, it is demonstrated that the infectivity of HCVpp that are released under treatment is reduced and that this reduction in infectivity is due to the incorporation of misfolded envelope glycoproteins in secreted particles. These properties suggest the potential usefulness of DNJ derivatives in combating HCV infection.
Stability of recombinant plant viruses containing genes of unrelated plant viruses
Authors: Chung Bong-Nam , Canto Tomas , Palukaitis Peter
Published: March 2007

The stability of hybrid plant viruses that might arise by recombination in transgenic plants was examined using hybrid viruses derived from the viral expression vectors potato virus X (PVX) and tobacco rattle virus (TRV). The potato virus Y (PVY) NIb and HCPro open reading frames (ORFs) were introduced into PVX to generate PVX-NIb and PVX-HCPro, while the PVY NIb ORF was introduced into a vector derived from TRV RNA2 to generate TRV-NIb. All three viruses were unstable and most of the progeny viruses had lost the inserted sequences between 2 and 4 weeks post-inoculation. There was some variation in the rate of loss of part or all of the inserted sequence and the number of plants containing the deleted viruses, depending on the sequence, the host (Nicotiana tabacumvsNicotiana benthamiana) or the vector, although none of these factors was associated consistently with the preferential loss of the inserted sequences. PVX-NIb was unable to accumulate in NIb-transgenic tobacco resistant to infection by PVY and also showed loss of the NIb insert from PVX-NIb in some NIb-transgenic tobacco plants susceptible to infection by PVY. These data indicate that such hybrid viruses, formed in resistant transgenic plants from a transgene and an unrelated virus, would be at a selective disadvantage, first by being targeted by the resistance mechanism and second by not being competitive with the parental virus.
Polymorphisms of the prion protein gene coding region in born-after-the-reinforced-ban (BARB) bovine spongiform encephalopathy cattle in Great Britain
Authors: Saunders G. C. , Griffiths P. C. , Cawthraw S. , Tout A. C. , Wiener P. , Woolliams J. A. , Williams J. L. , Windl O.
Published: March 2007

Polymorphisms of the prion protein gene are associated with differing susceptibilities to transmissible spongiform encephalopathy diseases, as shown for variant Creutzfeldt–Jakob disease in humans and scrapie in sheep, but not yet in cattle. Imposition of control measures in the UK, including a reinforced ruminant feed ban in 1996, has led to a reduction in the incidence of bovine spongiform encephalopathy (BSE). BSE-affected cattle born after 1996 in Great Britain have been termed born-after-the-reinforced-ban (BARB) cases. In this study, the PrP gene coding region from 100 BARB BSE cases and 66 matched healthy-control cattle was sequenced to investigate whether this would reveal a genetic basis to their origin. Polymorphisms identified were not found to be associated with increased susceptibility to BSE in the BARB cases. Analysis of BARB cases grouped either by clinical status or by whether they formed an isolated or clustered case was also undertaken, but differences were not found to be significant.
Isolation and characterization of hantavirus carried by Apodemus peninsulae in Jilin, China
Authors: Zhang Yong-Zhen , Zou Yang , Yao Lai-Shun , Hu Guang-Wei , Du Zhan-Shen , Jin Long-Zhe , Liu Yao-Yuan , Wang Hong-Xia , Chen Xian , Chen Hua-Xin , Fu Zhen F.
Published: March 2007

To provide a better understanding of hantavirus epidemiology in China, Korean field mice (Apodemus peninsulae) and striped field mice (Apodemus agrarius) were captured in Jilin province, China, where haemorrhagic fever with renal syndrome (HFRS) is endemic. Hantavirus antigens were detected in eight of the 130 A. peninsulae individuals and in four of the 193 A. agrarius individuals by using an immunofluorescence assay. Partial S and M segments were amplified from all of the antigen-positive samples. Furthermore, two hantaviruses (CJAp89 and CJAp93) were isolated successfully in cell culture and the entire S and M segments were amplified from one of them (CJAp93). Phylogenetic analysis of these sequences (partial or complete) showed that hantaviruses carried by A. peninsulae and A. agrarius form two distinct lineages, although viruses carried by A. peninsulae are similar to those isolated previously from A. agrarius in China and from HFRS patients in Russia. However, the viruses detected in A. peninsulae in China are genetically different from those detected in A. peninsulae in other countries. These data suggest that A. peninsulae is also a natural host for HTNV in north-eastern China.
West Nile virus strain Kunjin NS5 polymerase is a phosphoprotein localized at the cytoplasmic site of viral RNA synthesis
Authors: Mackenzie Jason M. , Kenney Mark T. , Westaway Edwin G.
Published: March 2007

Using West Nile virus strain Kunjin virus (WNVKUN) as a model system for flavivirus replication, we showed that the virus replication complex (RC) is associated with the dsRNA template located in induced membranes only in the cytoplasm. In this report we established for the first time that the RNA-dependent RNA polymerase NS5 is located in flavivirus-induced membranes, including the site of viral RNA replication. We found no evidence for nuclear localization of the essential RC components NS5 and its dsRNA template for WNVKUN or the closely related WNV strain Sarafend, by immuno-electron microscopy or by immunofluorescence. Metabolic radiolabelling with [32P]orthophosphate revealed that WNVKUN NS5 was phosphorylated and this was confirmed by Western blotting with antibodies specific for phosphorylated serine and threonine only. These observations of a cytoplasmic location for the WNV polymerase and its phosphorylation state correspond to the characteristics of the hepatitis C virus RNA polymerase NS5B.
Mutagenesis of a conserved fusion peptide-like motif and membrane-proximal heptad-repeat region of hepatitis C virus glycoprotein E1
Authors: Drummer Heidi E. , Boo Irene , Poumbourios Pantelis
Published: March 2007

The E1E2 glycoprotein heterodimer of Hepatitis C virus mediates viral entry. E2 attaches the virus to cellular receptors; however, the function of E1 is unknown. We tested the hypothesis that E1 is a truncated class II fusion protein. We mutated amino acids within a predicted fusion peptide (residues 276–286) and a truncated C-terminal stem-like motif, containing a membrane-proximal heptad-repeat sequence (residues 330–347). The fusion peptide mutation F285A abolished viral entry, while mutation of other hydrophobic residues had no effect. Alanine replacement of heptad-repeat residues blocked entry in three of five cases, whereas substitution with the helix breaker, Pro, led to loss of entry function in all cases. The mutations did not affect glycoprotein expression, heterodimerization with E2 or global folding, in contrast to the effects of mutations in the fusion motifs of prototypical class II fusion proteins. Our data suggest that E1 is unlikely to function in an analogous manner to other class II fusion glycoproteins.
Expression of the alternative reading frame protein of Hepatitis C virus induces cytokines involved in hepatic injuries
Authors: Fiorucci Marc , Boulant Steeve , Fournillier Anne , Abraham Jean Daniel , Lavergne Jean Pierre , Paranhos-Baccala Glaucia , Inchauspé Geneviève , Bain Christine
Published: March 2007

Hepatitis C virus (HCV) Core has been implicated in immune-mediated mechanisms associated with the development of chronic hepatic diseases. Discovery of different alternative reading frame proteins (ARFPs) expressed from the HCV Core coding sequence challenges properties assigned to Core. This study was designed to evaluate the immunomodulatory functions of Core and ARFPs in monocytes, dendritic cells (DCs), macrophages (Mφ) and hepatocytes, cells that are all capable of supporting HCV replication. THP-1 cells, monocyte-derived Mφ and DCs, and Huh7 cells were infected by using adenoviruses (Ad) encoding Core, CE1E2 and a Core sequence modified so that the Core protein is wild type, but no ARFPs are expressed (CΔARFP). THP-1 cells and DCs infected with Ad encoding Core or CE1E2 produced significant levels of interleukin-6 (IL-6), IL-8, MCP-1 and MIP-1β, whereas production of these chemokines with AdCΔARFP was reduced or abolished. Similar effects on IL-8 production were observed in Huh7 cells and on IL-6 and MIP-1β in Mφ. Wild-type Core sequence, but not CΔARFP, could trans-activate the IL-8 promoter and this activation was not associated with activation of p38/p42–44MAPK. This study illustrates, for the first time, the critical importance of ARFP expression in immunomodulatory functions attributed to Core expression and suggests a potential involvement of ARFP in mechanisms associated with HCV pathogenesis.
Measles virus M and F proteins associate with detergent-resistant membrane fractions and promote formation of virus-like particles
Authors: Pohl Christine , Duprex W. Paul , Krohne Georg , Rima Bertus K. , Schneider-Schaulies Sibylle
Published: March 2007

Assembly and release of particles comprise a late step in virus–host cell interactions. Though it may share major biological properties with its orthologues in related viruses, trafficking and oligomerization of the matrix (M) protein of Measles virus (MV) and its relative contribution to assembly and budding of particles from particular host cells have not been addressed in more detail. Plasmid-driven expression of authentic and mutant M proteins revealed that the amino acid at position 89, an important adaptation determinant for growth of attenuated strains in Vero cells, influences the electrophoretic mobility but not the intracellular distribution of M proteins, nor their ability to oligomerize or migrate as a doublet band in SDS-PAGE. M proteins were found to co-float with detergent-resistant membrane fractions (DRM) and this was enhanced upon co-expression of the F protein. In contrast to their DRM association, the ability of M proteins to promote release of virus-like particles (VLPs) was not affected by the presence of F proteins, which on their own also efficiently promoted VLP production. Thus, DRM recruitment of MV F and M proteins and their ability to drive particle formation are not correlated.
Cross-reactive antibody responses to nsp1 and nsp2 of Porcine reproductive and respiratory syndrome virus
Authors: Johnson Craig R. , Yu Wanqin , Murtaugh Michael P.
Published: March 2007

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural proteins (nsps) play a key role in processing and maturation of the repertoire of structural and nsps of the virion, but little is known about the anti-nsp immune response. Here, it was hypothesized that pronounced antibody responses are generated to PRRSV nsp1 and nsp2, as they are present in infected cells and cytolytic infection releases viral proteins into interstitial spaces. Accordingly, nsp1 and nsp2 were cloned and expressed, and antibody responses in the sera of infected and vaccinated pigs were determined. Pigs mounted significant cross-reactive antibody responses that appeared equivalent to or greater than the response to nucleocapsid (N). Antibody reactivity to nsp1 and N was highly dependent on refolding of denatured proteins, suggesting that the porcine antibody response is directed primarily to conformational epitopes. The proteins reacted with sera from pigs infected with other PRRSV strains, indicating that multiple epitopes are conserved. Antibody responses to nsp1 and nsp2 were much higher than those to nsp4, which is encoded on the same RNA molecule and is equivalent in predicted antigenicity. These findings suggest either that nsp1 and nsp2 are highly immunogenic or that they are expressed at higher levels than nsp4 in PRRSV-infected cells, or both. Strong antibody responses to nsp1 and nsp2 may benefit the host by limiting potentially pathological consequences of viral protease activities encoded in these proteins that are released from dying cells. The identification of strain-specific antibody responses to a highly variable region of nsp2 may also provide the basis for immunoassays that differentiate serological responses of vaccines from field isolates.
An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene
Authors: van den Born Erwin , Posthuma Clara C. , Knoops Kèvin , Snijder Eric J.
Published: March 2007

Thus far, systems developed for heterologous gene expression from the genomes of nidoviruses (arteriviruses and coronaviruses) have relied mainly on the translation of foreign genes from subgenomic mRNAs, whose synthesis is a key feature of the nidovirus life cycle. In general, such expression vectors often suffered from relatively low and unpredictable expression levels, as well as genome instability. In an attempt to circumvent these disadvantages, the possibility to express a foreign gene [encoding enhanced green fluorescent protein (eGFP)] from within the nidovirus replicase gene, which encodes two large polyproteins that are processed proteolytically into the non-structural proteins (nsps) required for viral RNA synthesis, has now been explored. A viable recombinant of the arterivirus Equine arteritis virus, EAV-GFP2, was obtained, which contained the eGFP insert at the site specifying the junction between the two most N-proximal replicase-cleavage products, nsp1 and nsp2. EAV-GFP2 replication could be launched by transfection of cells with either in vitro-generated RNA transcripts or a DNA launch plasmid. EAV-GFP2 displayed growth characteristics similar to those of the wild-type virus and was found to maintain the insert stably for at least eight passages. It is proposed that EAV-GFP2 has potential for arterivirus vector development and as a tool in inhibitor screening. It can also be used for fundamental studies into EAV replication, which was illustrated by the fact that the eGFP signal of EAV-GFP2, which largely originated from an eGFP–nsp2 fusion protein, could be used to monitor the formation of the membrane-bound EAV replication complex in real time.
Discriminating between cellular and misfolded prion protein by using affinity to 9-aminoacridine compounds
Authors: Phuan Puay-Wah , Zorn Julie A. , Safar Jiri , Giles Kurt , Prusiner Stanley B. , Cohen Fred E. , May Barnaby C. H.
Published: March 2007

Quinacrine and related 9-aminoacridine compounds are effective in eliminating the alternatively folded prion protein, termed PrPSc, from scrapie-infected cultured cells. Clinical evaluations of quinacrine for the treatment of human prion diseases are progressing in the absence of a clear understanding of the molecular mechanism by which prion replication is blocked. Here, insight into the mode of action of 9-aminoacridine compounds was sought by using a chemical proteomics approach to target identification. Cellular macromolecules that bind 9-aminoacridine ligands were affinity-purified from tissue lysates by using a 9-aminoacridine-functionalized solid-phase matrix. Although the 9-aminoacridine matrix was conformationally selective for PrPSc, it was inefficient: approximately 5 % of PrPScwas bound under conditions that did not support binding of the cellular isoform, PrPC. Our findings suggest that 9-aminoacridine compounds may reduce the PrPScburden either by occluding epitopes necessary for templating on the surface of PrPScor by altering the stability of PrPScoligomers, where a one-to-one stoichiometry is not necessary.
In situ reactions of monoclonal antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 protein
Authors: Clark David C. , Lobigs Mario , Lee Eva , Howard Megan J. , Clark Kerri , Blitvich Bradley J. , Hall Roy A.
Published: March 2007

Studies on the NS1 protein of flaviviruses have concluded that formation of a stable homodimer is required for virus replication. However, previous work has reported that substitution of a conserved proline by leucine at residue 250 in NS1 of Kunjin virus (KUNV) eliminated dimerization, but allowed virus replication to continue. To assess whether this substitution has similar effects on other flaviviruses, it was introduced into an infectious clone of Murray Valley encephalitis virus (MVEV). Consistent with studies of KUNV, the mutant virus (MVEVNS1-250Leu) produced high levels of monomeric NS1 and the NS1 homodimer could not be detected. In contrast, wild-type MVEV cultures contained predominantly dimeric NS1. Retarded virus growth in Vero cells and loss of neuroinvasiveness for weanling mice revealed further similarities between MVEVNS1-250Leuand the corresponding KUNV mutant. To confirm that the lack of detection of dimeric NS1 in mutant virus samples was not due to denaturation of unstable dimers during Western blotting, a mAb (2E3) specific for the MVEV NS1 homodimer was produced. When NS1 protein was fixedin situin mammalian and arthropod cells infected with wild-type or mutant virus, 2E3 reacted strongly with the former, but not the latter.These results confirmed that Pro250in NS1 is important for dimerization and that substitution of this residue by leucine represents a conserved marker of attenuation for viruses of the Japanese encephalitis virus serocomplex. The inability to detect dimeric NS1 in supernatant or cell monolayers of cultures productively infected with mutant virus also suggests that dimerization of the protein may not be essential for virus replication.
PDTC inhibits picornavirus polyprotein processing and RNA replication by transporting zinc ions into cells
Authors: Lanke K. , Krenn B. M. , Melchers W. J. G. , Seipelt J. , van Kuppeveld F. J. M.
Published: March 2007

Previously, it was shown that pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. Here, it is shown that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a closely related virus that belongs to the genusEnterovirus, and mengovirus, an encephalomyocarditis virus strain that belongs to the genusCardiovirus, and that this inhibition is due to the dithiocarbamate moiety of the compound. Making use of subgenomic replicons, evidence is provided that PDTC inhibits replication of these two viruses by disturbing viral RNA synthesis. Furthermore, it is shown that PDTC transports zinc ions into cells and that these zinc ions play an important role in the antiviral activity mediated by PDTC. Finally, it is shown that PDTC interferes with proteolytic processing of the polyproteins of both CVB3 and mengovirus, but that the underlying mechanism between these two viruses differs. In CVB3-infected cells, PDTC interferes strongly with the proteolytic activity of 3CDpro, as shown by the impaired production of the mature capsid proteins as well as the autocleavage of 3CDprointo 3Cproand 3Dpol. In mengovirus-infected cells, however, PDTC had no effect on the proteolytic production of capsid proteins or the autocleavage of 3CDpro. Instead, PDTC caused the accumulation of a high-molecular-mass precursor protein, due to an impairment in the primary ‘break’ that normally occurs at the 2A–2B junction. Thus, PDTC disturbs polyprotein processing and replication of two groups of picornaviruses, enteroviruses and cardioviruses, but the underlying mechanism is different.
Molecular analysis of the S glycoprotein gene of bovine coronaviruses isolated in Japan from 1999 to 2006
Authors: Kanno Toru , Hatama Shinichi , Ishihara Ryoko , Uchida Ikuo
Published: March 2007

In total, 55 isolates of Bovine coronavirus (BCoV) were collected from cases of enteric and respiratory disease occurring between 1999 and 2006 in Japan. Phylogenetic analysis of the polymorphic region of the S glycoprotein gene of these isolates, together with those of other known strains, classified the BCoV strains and isolates into four clusters. Recent field isolates display distinctive genetic divergence from the prototype enteric BCoV strains – Mebus, Quebec, Kakegawa, F15 and LY138 – and have diverged in three different aspects over 8 years. These data suggested that the genetic divergence in the polymorphic region of the S glycoprotein has progressed considerably; thus, molecular analysis of this region should be useful in investigating the molecular epidemiology of BCoV. In addition, based on the differences in amino acids among the isolates, our study did not reveal the presence of certain genetic markers of pathogenicity and clinical symptoms in this polymorphic region.