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The proteins of vaccinia virus associated with plasma membrane infected cells BHK-21, p60, p45, p42, p40,p35,p34,p28,p23 were isolated from plasma membranes using affinity chromatography, gel-electrophoresis and passive elution. An immunochemical characterization was carried out using specific antiserum to these proteins. Investigation of temporal regulation of proteins synthesis in infected cells showed that proteins p60, p45, p42, p40, p28 were late, and p35, p34, p23--early-late proteins. Immunochemical analysis of vaccinia virus mRNA cell-free translational products was carried out using specific antiserum. The polypeptide-precursors of viral proteins were identified. 相似文献
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Multiple receptor interactions trigger release of membrane and intracellular calcium stores critical for herpes simplex virus entry 总被引:4,自引:0,他引:4 下载免费PDF全文
Herpes simplex viruses (HSV) harness cellular calcium signaling pathways to facilitate viral entry. Confocal microscopy and small interfering RNA (siRNA) were used to identify the source of the calcium and to dissect the requisite viral-cell interactions. Binding of HSV to human epithelial cells induced no calcium response, but shifting the cells to temperatures permissive for penetration triggered increases in plasma membrane calcium followed by a global release of intracellular calcium. Transfection with siRNA targeting the proteoglycan syndecan-2 blocked viral binding and abrogated any calcium response. Transfection with siRNA targeting nectin-1, a glycoprotein D receptor, also prevented both membrane and intracellular calcium responses. In contrast, the membrane response was preserved after transfection with siRNA targeting integrinalphav, a novel glycoprotein H receptor. The membrane response, however, was not sufficient for viral entry, which required interactions with integrinalphav and release of inositol-triphosphate receptor-dependent intracellular calcium stores. Thus, calcium plays a critical, complex role in HSV entry. 相似文献
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Theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry 下载免费PDF全文
Yasin B Wang W Pang M Cheshenko N Hong T Waring AJ Herold BC Wagar EA Lehrer RI 《Journal of virology》2004,78(10):5147-5156
We tested the ability of 20 synthetic theta defensins to protect cells from infection by type 1 and type 2 herpes simplex viruses (HSV-1 and -2, respectively). The peptides included rhesus theta defensins (RTDs) 1 to 3, originally isolated from rhesus macaque leukocytes, and three peptides (retrocyclins 1 to 3) whose sequences were inferred from human theta-defensin (DEFT) pseudogenes. We also tested 14 retrocyclin analogues, including the retro, enantio, and retroenantio forms of retrocyclin 1. Retrocyclins 1 and 2 and RTD 3 protected cervical epithelial cells from infection by both HSV serotypes, but only retrocyclin 2 did so without causing cytotoxicity or requiring preincubation with the virus. Surface plasmon resonance studies revealed that retrocyclin 2 bound to immobilized HSV-2 glycoprotein B (gB2) with high affinity (K(d), 13.3 nM) and that it did not bind to enzymatically deglycosylated gB2. Temperature shift experiments indicated that retrocyclin 2 and human alpha defensins human neutrophil peptide 1 (HNP 1) to HNP 3 protected human cells from HSV-2 by different mechanisms. Retrocyclin 2 blocked viral attachment, and its addition during the binding or penetration phases of HSV-2 infection markedly diminished nuclear translocation of VP16 and expression of ICP4. In contrast, HNPs 1 to 3 had little effect on binding but reduced both VP16 transport and ICP4 expression if added during the postbinding (penetration) period. We recently reported that theta defensins are miniature lectins that bind gp120 of human immunodeficiency virus type 1 (HIV-1) with high affinity and inhibit the entry of R5 and X4 isolates of HIV-1. Given its small size (18 residues), minimal cytotoxicity, lack of activity against vaginal lactobacilli, and effectiveness against both HSV-2 and HIV-1, retrocyclin 2 provides an intriguing prototype for future topical microbicide development. 相似文献
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A V Kachko T B Cheusova A V Sorokin E I Kazachinskaia I O Cheshenko E F Belanov A A Bukreev A V Ivanova I A Razumov E I Riabchikova S V Netesov 《Molekuliarnaia biologiia》2001,35(3):492-499
The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP. 相似文献
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Cheshenko N Del Rosario B Woda C Marcellino D Satlin LM Herold BC 《The Journal of cell biology》2003,163(2):283-293
The cellular pathways required for herpes simplex virus (HSV) invasion have not been defined. To test the hypothesis that HSV entry triggers activation of Ca2+-signaling pathways, the effects on intracellular calcium concentration ([Ca2+]i) after exposure of cells to HSV were examined. Exposure to virus results in a rapid and transient increase in [Ca2+]i. Pretreatment of cells with pharmacological agents that block release of inositol 1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum stores abrogates the response. Moreover, treatment of cells with these pharmacological agents inhibits HSV infection and prevents focal adhesion kinase (FAK) phosphorylation, which occurs within 5 min after viral infection. Viruses deleted in glycoprotein L or glycoprotein D, which bind but do not penetrate, fail to induce a [Ca2+]i response or trigger FAK phosphorylation. Together, these results support a model for HSV infection that requires activation of IP3-responsive Ca2+-signaling pathways and that is associated with FAK phosphorylation. Defining the pathway of viral invasion may lead to new targets for anti-viral therapy. 相似文献
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Molecular umbrellas: a novel class of candidate topical microbicides to prevent human immunodeficiency virus and herpes simplex virus infections 总被引:1,自引:0,他引:1 下载免费PDF全文
Madan RP Mesquita PM Cheshenko N Jing B Shende V Guzman E Heald T Keller MJ Regen SL Shattock RJ Herold BC 《Journal of virology》2007,81(14):7636-7646