The Experts below are selected from a list of 225 Experts worldwide ranked by ideXlab platform
Marlyn P. Langford - One of the best experts on this subject based on the ideXlab platform.
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Analysis of neutralizing antibodies to Enterovirus 70 and Coxsackievirus A24 variant, levels of immunoglobulins and total protein in tears of patients with acute hemorrhagic conjunctivitis
Ocular Immunology and Inflammation, 2009Co-Authors: Marlyn P. Langford, Jeffrey B Robertson, Rogelio OrillacAbstract:Tear samples were collected from 37 residents of the Dominican Republic 3.5 U/ml) and anti-Coxsackievirus A24 variant (CA24v) TNA (10
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caspase 3 and 7 mediate apoptosis of human chang s conjunctival cells induced by Enterovirus 70
Virology, 2006Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, Youping Deng, Thomas B Redens, Marlyn P. LangfordAbstract:Abstract Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl–Ala–Ala–Pro–Val–chloromethylketone (MPCMK), but not actinomycin D and guanidine·HCl (although guanidine·HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.
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induction of caspase dependant apoptosis by Enterovirus 70 in human lens epithelial sra01 04 cells
American Journal of Infectious Diseases, 2005Co-Authors: Dequan Chen, Donald E Texada, Youping Deng, Christopher Duggan, Chanping Liang, Thomas B Reden, Marlyn P. LangfordAbstract:Enterovirus 70 (EV70), an etiological agent of acute hemorrhagic conjunctivitis (AHC), induces apoptosis in human Chang’s conjunctival (HCC) cells. EV70 is also able to infect and replicate in some other human cells such as human lens epithelial SRA01/04 cells and U937 cells. EV70 infection was found to induce apoptosis in SRA01/04 cells. During EV70-induced apoptosis of SRA01/04 cells, the expression levels of procaspase-3, 6 and 7 (all 32kD) were not increased, but procaspase-3 and 7 were activated (cleaved) while the constitutively activated caspase-6 decreased. Z-VD-FMK inhibited EV70-induced apoptosis and extracellular EV70 release, but not intracellular viral production. UV- or heat-inactivated EV70 did not induce apoptosis. Guanidine⋅HCl inhibited EV70 induced apoptosis in a multiple-step viral growth experiment but not in a one-step viral growth experiment. Cycloheximide and high concentration of methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (0.5 mM) or elastatinal (Ela) (125 mM) also inhibited EV70-induced apoptosis. The results suggest that infectious EV70 is capable of inducing caspase-dependent (especially caspases-3 and 7-dependent) apoptosis in human lens SRA01/04 cells that requires the syntheses of viral proteins, but not viral genome RNA. The results suggest that (a) EV70 protease(s) (2A protease and probably 3C protease) produced during viral replication initiates EV70 induced apoptosis of SRA01/04 cells and (b) EV70 may use similar or same mechanism(s) to induce apoptosis in different human cells such as SRA01/04 and HCC cells.
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immunogenicity of Enterovirus 70 capsid protein vp1 and its non overlapping n and c terminal fragments
Antiviral Research, 2005Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, Lakshmana M Kooragayala, Thomas B Reden, Marlyn P. LangfordAbstract:Abstract Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by Enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1–138 aa) and C170 (141–310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000–10,000 units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20–320 units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.
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Induction of Caspase-dependant Apoptosis by Enterovirus 70 in Human Lens Epithelial SRA01/04 Cells
American Journal of Infectious Diseases, 2005Co-Authors: Dequan Chen, Donald E Texada, Youping Deng, Christopher Duggan, Chanping Liang, Thomas B Reden, Marlyn P. LangfordAbstract:Enterovirus 70 (EV70), an etiological agent of acute hemorrhagic conjunctivitis (AHC), induces apoptosis in human Chang’s conjunctival (HCC) cells. EV70 is also able to infect and replicate in some other human cells such as human lens epithelial SRA01/04 cells and U937 cells. EV70 infection was found to induce apoptosis in SRA01/04 cells. During EV70-induced apoptosis of SRA01/04 cells, the expression levels of procaspase-3, 6 and 7 (all 32kD) were not increased, but procaspase-3 and 7 were activated (cleaved) while the constitutively activated caspase-6 decreased. Z-VD-FMK inhibited EV70-induced apoptosis and extracellular EV70 release, but not intracellular viral production. UV- or heat-inactivated EV70 did not induce apoptosis. Guanidine⋅HCl inhibited EV70 induced apoptosis in a multiple-step viral growth experiment but not in a one-step viral growth experiment. Cycloheximide and high concentration of methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (0.5 mM) or elastatinal (Ela) (125 mM) also inhibited EV70-induced apoptosis. The results suggest that infectious EV70 is capable of inducing caspase-dependent (especially caspases-3 and 7-dependent) apoptosis in human lens SRA01/04 cells that requires the syntheses of viral proteins, but not viral genome RNA. The results suggest that (a) EV70 protease(s) (2A protease and probably 3C protease) produced during viral replication initiates EV70 induced apoptosis of SRA01/04 cells and (b) EV70 may use similar or same mechanism(s) to induce apoptosis in different human cells such as SRA01/04 and HCC cells.
Kenneth Dimock - One of the best experts on this subject based on the ideXlab platform.
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Enterovirus 70 binds to different glycoconjugates containing α2 3 linked sialic acid on different cell lines
Journal of Virology, 2005Co-Authors: Reza M Nokhbeh, David A Alexander, Samir Hazra, Ahmar Khan, Morgan Mcallister, Erik J Suuronen, May Griffith, Kenneth DimockAbstract:Enterovirus 70 (EV70), the causative agent of acute hemorrhagic conjunctivitis, exhibits a restricted tropism for conjunctival and corneal cells in vivo but infects a wide spectrum of mammalian cells in culture. Previously, we demonstrated that human CD55 is a receptor for EV70 on HeLa cells but that EV70 also binds to sialic acid-containing receptors on a variety of other human cell lines. Virus recognition of sialic acid attached to underlying glycans by a particular glycosidic linkage may contribute to host range, tissue tropism, and pathogenesis. Therefore, we tested the possibility that EV70 binds to α2,3-linked sialic acid, like other viruses associated with ocular infections. Through the use of linkage-specific sialidases, sialyltransferases, and lectins, we show that EV70 recognizes α2,3-linked sialic acid on human corneal epithelial cells and on U-937 cells. Virus attachment to both cell lines is CD55 independent and sensitive to benzyl N-acetyl-α-d-galactosaminide, an inhibitor of O-linked glycosylation. Virus binding to corneal cells, but not U-937 cells, is inhibited by proteinase K, but not by phosphatidylinositol-specific phospholipase C treatment. These results are consistent with the idea that a major EV70 receptor on corneal epithelial cells is an O-glycosylated, non-glycosyl phosphatidylinositol-anchored membrane glycoprotein containing α2,3-linked sialic acid, while sialylated receptors on U-937 cells are not proteinaceous.
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binding to decay accelerating factor is not required for infection of human leukocyte cell lines by Enterovirus 70
Journal of Virology, 2004Co-Authors: Alain Haddad, Sandra Dawe, David A Alexander, Reza M Nokhbeh, Christine Grise, Naveed Gulzar, Kenneth DimockAbstract:Enterovirus 70 (EV70) is one of several human Enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic Enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic Enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other Enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-α-d-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.
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sialic acid functions in Enterovirus 70 binding and infection
Journal of Virology, 2002Co-Authors: David A Alexander, Kenneth DimockAbstract:The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for Enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.
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Short Consensus Repeat Domain 1 of Decay-Accelerating Factor Is Required for Enterovirus 70 Binding
Journal of Virology, 1998Co-Authors: Timothy Karnauchow, Sandra Dawe, Douglas M. Lublin, Kenneth DimockAbstract:Enterovirus 70 (EV70), like several other human Enteroviruses, can utilize decay-accelerating factor (DAF [CD55]) as an attachment protein. Using chimeric molecules composed of different combinations of the short consensus repeat domains (SCRs) of DAF and membrane cofactor protein (CD46), we show that sequences in SCR1 of DAF are essential for EV70 binding. Of the human Enteroviruses that can bind to DAF, only EV70 and coxsackievirus A21 require sequences in SCR1 for this interaction.
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the hela cell receptor for Enterovirus 70 is decay accelerating factor cd55
Journal of Virology, 1996Co-Authors: Timothy Karnauchow, Douglas M. Lublin, Douglas L Tolson, Blair A Harrison, Eleonora Altman, Kenneth DimockAbstract:Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human Enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several Enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.
Dequan Chen - One of the best experts on this subject based on the ideXlab platform.
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caspase 3 and 7 mediate apoptosis of human chang s conjunctival cells induced by Enterovirus 70
Virology, 2006Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, Youping Deng, Thomas B Redens, Marlyn P. LangfordAbstract:Abstract Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl–Ala–Ala–Pro–Val–chloromethylketone (MPCMK), but not actinomycin D and guanidine·HCl (although guanidine·HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.
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induction of caspase dependant apoptosis by Enterovirus 70 in human lens epithelial sra01 04 cells
American Journal of Infectious Diseases, 2005Co-Authors: Dequan Chen, Donald E Texada, Youping Deng, Christopher Duggan, Chanping Liang, Thomas B Reden, Marlyn P. LangfordAbstract:Enterovirus 70 (EV70), an etiological agent of acute hemorrhagic conjunctivitis (AHC), induces apoptosis in human Chang’s conjunctival (HCC) cells. EV70 is also able to infect and replicate in some other human cells such as human lens epithelial SRA01/04 cells and U937 cells. EV70 infection was found to induce apoptosis in SRA01/04 cells. During EV70-induced apoptosis of SRA01/04 cells, the expression levels of procaspase-3, 6 and 7 (all 32kD) were not increased, but procaspase-3 and 7 were activated (cleaved) while the constitutively activated caspase-6 decreased. Z-VD-FMK inhibited EV70-induced apoptosis and extracellular EV70 release, but not intracellular viral production. UV- or heat-inactivated EV70 did not induce apoptosis. Guanidine⋅HCl inhibited EV70 induced apoptosis in a multiple-step viral growth experiment but not in a one-step viral growth experiment. Cycloheximide and high concentration of methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (0.5 mM) or elastatinal (Ela) (125 mM) also inhibited EV70-induced apoptosis. The results suggest that infectious EV70 is capable of inducing caspase-dependent (especially caspases-3 and 7-dependent) apoptosis in human lens SRA01/04 cells that requires the syntheses of viral proteins, but not viral genome RNA. The results suggest that (a) EV70 protease(s) (2A protease and probably 3C protease) produced during viral replication initiates EV70 induced apoptosis of SRA01/04 cells and (b) EV70 may use similar or same mechanism(s) to induce apoptosis in different human cells such as SRA01/04 and HCC cells.
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immunogenicity of Enterovirus 70 capsid protein vp1 and its non overlapping n and c terminal fragments
Antiviral Research, 2005Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, Lakshmana M Kooragayala, Thomas B Reden, Marlyn P. LangfordAbstract:Abstract Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by Enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1–138 aa) and C170 (141–310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000–10,000 units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20–320 units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.
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Induction of Caspase-dependant Apoptosis by Enterovirus 70 in Human Lens Epithelial SRA01/04 Cells
American Journal of Infectious Diseases, 2005Co-Authors: Dequan Chen, Donald E Texada, Youping Deng, Christopher Duggan, Chanping Liang, Thomas B Reden, Marlyn P. LangfordAbstract:Enterovirus 70 (EV70), an etiological agent of acute hemorrhagic conjunctivitis (AHC), induces apoptosis in human Chang’s conjunctival (HCC) cells. EV70 is also able to infect and replicate in some other human cells such as human lens epithelial SRA01/04 cells and U937 cells. EV70 infection was found to induce apoptosis in SRA01/04 cells. During EV70-induced apoptosis of SRA01/04 cells, the expression levels of procaspase-3, 6 and 7 (all 32kD) were not increased, but procaspase-3 and 7 were activated (cleaved) while the constitutively activated caspase-6 decreased. Z-VD-FMK inhibited EV70-induced apoptosis and extracellular EV70 release, but not intracellular viral production. UV- or heat-inactivated EV70 did not induce apoptosis. Guanidine⋅HCl inhibited EV70 induced apoptosis in a multiple-step viral growth experiment but not in a one-step viral growth experiment. Cycloheximide and high concentration of methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (0.5 mM) or elastatinal (Ela) (125 mM) also inhibited EV70-induced apoptosis. The results suggest that infectious EV70 is capable of inducing caspase-dependent (especially caspases-3 and 7-dependent) apoptosis in human lens SRA01/04 cells that requires the syntheses of viral proteins, but not viral genome RNA. The results suggest that (a) EV70 protease(s) (2A protease and probably 3C protease) produced during viral replication initiates EV70 induced apoptosis of SRA01/04 cells and (b) EV70 may use similar or same mechanism(s) to induce apoptosis in different human cells such as SRA01/04 and HCC cells.
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expression of Enterovirus 70 capsid protein vp1 in escherichia coli
Protein Expression and Purification, 2004Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, J P Ganley, Lakshmana M Kooragayala, Tomas B Reden, Marlyn P. LangfordAbstract:Abstract The VP1 gene of Enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5′-UGUCUCCUUUUC-3′ flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21 ( DE3 ), Rosetta 2 ( DE3 ) or Rosetta (DE3)pLysS) . High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1–138 aa) and C170 (141–310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli , high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli .
Donald E Texada - One of the best experts on this subject based on the ideXlab platform.
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caspase 3 and 7 mediate apoptosis of human chang s conjunctival cells induced by Enterovirus 70
Virology, 2006Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, Youping Deng, Thomas B Redens, Marlyn P. LangfordAbstract:Abstract Enterovirus 70 (EV70) is the major etiological agent of acute hemorrhagic conjunctivitis (AHC). EV70 m.o.i.- (multiplicity of infection) and time-dependently induced apoptosis in human Chang's conjunctival (HCC) cells. UV- or heat-inactivated EV70 did not induce apoptosis. EV70-induced apoptosis was inhibited by cycloheximide and methoxysuccinyl–Ala–Ala–Pro–Val–chloromethylketone (MPCMK), but not actinomycin D and guanidine·HCl (although guanidine·HCl inhibited the apoptosis induced by EV70 infection at 0.5 PFU/cell for 18 h). EV70 infection induced activation of caspase-3 and -7 and degradation of the constitutively activated caspase-6. EV70-induced apoptotic DNA ladders and activated caspase-3 and -7, correlated with virus release. Caspase inhibitor IX (Z-VD-FMK) inhibited EV70-induced apoptosis and virus release, but not intracellular viral production. The results suggest that infectious virus and the syntheses of viral proteins especially EV70 proteases, but not viral genome RNA, are required for caspase-3 and -7-mediated EV70-induced apoptosis, and that apoptosis through cell lysis promotes EV70 release from HCC cells.
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induction of caspase dependant apoptosis by Enterovirus 70 in human lens epithelial sra01 04 cells
American Journal of Infectious Diseases, 2005Co-Authors: Dequan Chen, Donald E Texada, Youping Deng, Christopher Duggan, Chanping Liang, Thomas B Reden, Marlyn P. LangfordAbstract:Enterovirus 70 (EV70), an etiological agent of acute hemorrhagic conjunctivitis (AHC), induces apoptosis in human Chang’s conjunctival (HCC) cells. EV70 is also able to infect and replicate in some other human cells such as human lens epithelial SRA01/04 cells and U937 cells. EV70 infection was found to induce apoptosis in SRA01/04 cells. During EV70-induced apoptosis of SRA01/04 cells, the expression levels of procaspase-3, 6 and 7 (all 32kD) were not increased, but procaspase-3 and 7 were activated (cleaved) while the constitutively activated caspase-6 decreased. Z-VD-FMK inhibited EV70-induced apoptosis and extracellular EV70 release, but not intracellular viral production. UV- or heat-inactivated EV70 did not induce apoptosis. Guanidine⋅HCl inhibited EV70 induced apoptosis in a multiple-step viral growth experiment but not in a one-step viral growth experiment. Cycloheximide and high concentration of methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (0.5 mM) or elastatinal (Ela) (125 mM) also inhibited EV70-induced apoptosis. The results suggest that infectious EV70 is capable of inducing caspase-dependent (especially caspases-3 and 7-dependent) apoptosis in human lens SRA01/04 cells that requires the syntheses of viral proteins, but not viral genome RNA. The results suggest that (a) EV70 protease(s) (2A protease and probably 3C protease) produced during viral replication initiates EV70 induced apoptosis of SRA01/04 cells and (b) EV70 may use similar or same mechanism(s) to induce apoptosis in different human cells such as SRA01/04 and HCC cells.
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immunogenicity of Enterovirus 70 capsid protein vp1 and its non overlapping n and c terminal fragments
Antiviral Research, 2005Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, Lakshmana M Kooragayala, Thomas B Reden, Marlyn P. LangfordAbstract:Abstract Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by Enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1–138 aa) and C170 (141–310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000–10,000 units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20–320 units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.
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Induction of Caspase-dependant Apoptosis by Enterovirus 70 in Human Lens Epithelial SRA01/04 Cells
American Journal of Infectious Diseases, 2005Co-Authors: Dequan Chen, Donald E Texada, Youping Deng, Christopher Duggan, Chanping Liang, Thomas B Reden, Marlyn P. LangfordAbstract:Enterovirus 70 (EV70), an etiological agent of acute hemorrhagic conjunctivitis (AHC), induces apoptosis in human Chang’s conjunctival (HCC) cells. EV70 is also able to infect and replicate in some other human cells such as human lens epithelial SRA01/04 cells and U937 cells. EV70 infection was found to induce apoptosis in SRA01/04 cells. During EV70-induced apoptosis of SRA01/04 cells, the expression levels of procaspase-3, 6 and 7 (all 32kD) were not increased, but procaspase-3 and 7 were activated (cleaved) while the constitutively activated caspase-6 decreased. Z-VD-FMK inhibited EV70-induced apoptosis and extracellular EV70 release, but not intracellular viral production. UV- or heat-inactivated EV70 did not induce apoptosis. Guanidine⋅HCl inhibited EV70 induced apoptosis in a multiple-step viral growth experiment but not in a one-step viral growth experiment. Cycloheximide and high concentration of methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (0.5 mM) or elastatinal (Ela) (125 mM) also inhibited EV70-induced apoptosis. The results suggest that infectious EV70 is capable of inducing caspase-dependent (especially caspases-3 and 7-dependent) apoptosis in human lens SRA01/04 cells that requires the syntheses of viral proteins, but not viral genome RNA. The results suggest that (a) EV70 protease(s) (2A protease and probably 3C protease) produced during viral replication initiates EV70 induced apoptosis of SRA01/04 cells and (b) EV70 may use similar or same mechanism(s) to induce apoptosis in different human cells such as SRA01/04 and HCC cells.
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expression of Enterovirus 70 capsid protein vp1 in escherichia coli
Protein Expression and Purification, 2004Co-Authors: Dequan Chen, Donald E Texada, Chris Duggan, J P Ganley, Lakshmana M Kooragayala, Tomas B Reden, Marlyn P. LangfordAbstract:Abstract The VP1 gene of Enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5′-UGUCUCCUUUUC-3′ flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21 ( DE3 ), Rosetta 2 ( DE3 ) or Rosetta (DE3)pLysS) . High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1–138 aa) and C170 (141–310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli , high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli .
David A Alexander - One of the best experts on this subject based on the ideXlab platform.
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Enterovirus 70 binds to different glycoconjugates containing α2 3 linked sialic acid on different cell lines
Journal of Virology, 2005Co-Authors: Reza M Nokhbeh, David A Alexander, Samir Hazra, Ahmar Khan, Morgan Mcallister, Erik J Suuronen, May Griffith, Kenneth DimockAbstract:Enterovirus 70 (EV70), the causative agent of acute hemorrhagic conjunctivitis, exhibits a restricted tropism for conjunctival and corneal cells in vivo but infects a wide spectrum of mammalian cells in culture. Previously, we demonstrated that human CD55 is a receptor for EV70 on HeLa cells but that EV70 also binds to sialic acid-containing receptors on a variety of other human cell lines. Virus recognition of sialic acid attached to underlying glycans by a particular glycosidic linkage may contribute to host range, tissue tropism, and pathogenesis. Therefore, we tested the possibility that EV70 binds to α2,3-linked sialic acid, like other viruses associated with ocular infections. Through the use of linkage-specific sialidases, sialyltransferases, and lectins, we show that EV70 recognizes α2,3-linked sialic acid on human corneal epithelial cells and on U-937 cells. Virus attachment to both cell lines is CD55 independent and sensitive to benzyl N-acetyl-α-d-galactosaminide, an inhibitor of O-linked glycosylation. Virus binding to corneal cells, but not U-937 cells, is inhibited by proteinase K, but not by phosphatidylinositol-specific phospholipase C treatment. These results are consistent with the idea that a major EV70 receptor on corneal epithelial cells is an O-glycosylated, non-glycosyl phosphatidylinositol-anchored membrane glycoprotein containing α2,3-linked sialic acid, while sialylated receptors on U-937 cells are not proteinaceous.
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binding to decay accelerating factor is not required for infection of human leukocyte cell lines by Enterovirus 70
Journal of Virology, 2004Co-Authors: Alain Haddad, Sandra Dawe, David A Alexander, Reza M Nokhbeh, Christine Grise, Naveed Gulzar, Kenneth DimockAbstract:Enterovirus 70 (EV70) is one of several human Enteroviruses that exhibit a propensity for infecting the central nervous system (CNS). The mechanisms by which neurotropic Enteroviruses gain access to and invade the CNS are poorly understood. One possibility is that circulating leukocytes become infected and carry neurotropic Enteroviruses to the CNS. We examined the ability of EV70 to infect cell lines derived from lymphoid, myeloid, and monocytic lineages. Most leukocyte cell lines tested bound radiolabeled EV70 and were permissive for EV70 replication, suggesting that EV70, in contrast to other Enteroviruses, has an in vitro tropism that includes lymphoid, monocytic, and myeloid cell lines. For some of the cell lines, virus binding and infection correlated with surface expression of decay-accelerating factor (DAF), an attachment protein for EV70 on HeLa cells. However, EV70 also adsorbed to and infected cell lines that expressed little or no DAF. In contrast to what was observed for HeLa cells, neither DAF-specific monoclonal antibodies nor phosphatidylinositol-specific phospholipase C treatment inhibited EV70 binding to permissive leukocyte cell lines, and antibody blockade of DAF had little or no effect on EV70 replication. We also found that neither the human coxsackievirus-adenovirus receptor nor intercellular cell adhesion molecule 1, which mediate the entry of coxsackie B viruses and coxsackievirus A21, respectively, functions as a receptor for EV70. EV70 binding to all cell lines was sensitive to sialidase treatment and to inhibition of O glycosylation by benzyl N-acetyl-α-d-galactosaminide. Taken together, these results suggest that a sialylated molecule(s) other than DAF serves as a receptor for EV70 on permissive human leukocyte cell lines.
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sialic acid functions in Enterovirus 70 binding and infection
Journal of Virology, 2002Co-Authors: David A Alexander, Kenneth DimockAbstract:The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for Enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.