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231.
Déborah Harrus Neveen Ahmed-El-Sayed Philip C. Simister Steve Miller Martine Triconnet Curt H. Hagedorn Kathleen Mahias Félix A. Rey Thérèse Astier-Gin Stéphane Bressanelli 《The Journal of biological chemistry》2010,285(43):32906-32918
The hepatitis C virus (HCV) NS5b protein is an RNA-dependent RNA polymerase
essential for replication of the viral RNA genome. In vitro and
presumably in vivo, NS5b initiates RNA synthesis by a
de novo mechanism. Different structural elements of NS5b
have been reported to participate in RNA synthesis, especially a so-called
“β-flap” and a C-terminal segment (designated
“linker”) that connects the catalytic core of NS5b to a
transmembrane anchor. High concentrations of GTP have also been shown to
stimulate de novo RNA synthesis by HCV NS5b. Here we describe a
combined structural and functional analysis of genotype 1 HCV-NS5b of strains
H77 (subtype 1a), for which no structure has been previously reported, and J4
(subtype 1b). Our results highlight the linker as directly involved in lifting
the first boundary to processive RNA synthesis, the formation of the first
dinucleotide primer. The transition from this first dinucleotide primer state to
processive RNA synthesis requires removal of the linker and of the
β-flap with which it is shown to strongly interact in crystal
structures of HCV NS5b. We find that GTP specifically stimulates this transition
irrespective of its incorporation in neosynthesized RNA. 相似文献
232.
Anna Tranell Eva Maria Feny? Stefan Schwartz 《The Journal of biological chemistry》2010,285(41):31537-31547
HIV-1 non-coding exon 3 can either be spliced to exons 4, 4a, 4b, 4c, and 5 to generate tat, rev, and nef mRNAs or remain unspliced to produce the 13a7 vpr mRNA. Here we show that serine- and arginine-rich proteins 55 and 75 (SRp55 and SRp75) inhibit splicing from the 5′-splice site of exon 3 thereby causing an accumulation of the partially unspliced 13a7 vpr mRNA. In contrast, serine- and arginine-rich protein 40 (SRp40) induces splicing from exon 3 to exon 4, thereby promoting the production of the 1347 tat mRNA. We demonstrate that SRp55 stimulates vpr mRNA production by interacting with the previously identified HIV-1 splicing enhancer named GAR and inhibiting its function. This inhibition requires both serine arginine-rich and RNA-binding domains of SRp55, indicating that production of HIV-1 vpr mRNA depends on the interaction of SRp55 with an unknown factor. 相似文献
233.
234.
Yeon-Kye Kim 《Carbohydrate research》2010,345(8):1065-1007
Waxy maize starch (100% amylopectin) granules were modified by reaction of the granules with glucoamylase in a minimum amount of water to give 29% (w/w) d-glucose inside the granules [Kim, Y.-K.; Robyt, J. F. Carbohydr. Res.1999, 318, 129−134]. These granules were made into beads by dropping an ethanol slurry of starch and different amounts of Eudragit L100-55 in a constant ratio of 100:1 from a pipette onto Whatman 3MM filter paper. The starch beads were air dried and then repeatedly sprayed 0-12 times with 2.0% (w/v) Eudragit L100-55 in ethanol, with drying between each spraying, to coat the surface of the starch beads, giving different amounts of Eudragit L100-55 coating. Seven different kinds of beads, with different amounts of Eudragit L100-55 binding and coating agent, were obtained. The rates of release of d-glucose into water from the seven kinds of beads were inversely proportional to the amount of binding and coating agent. Bead type I, which was without any binding and coating gave a fast 100% release of d-glucose in 30 min. Beads II and III also gave a fast 100% release in 60 min and 90 min, respectively. Bead IV gave a near linear release of 97% d-glucose in 150 min; Bead V gave a 50% release in 120 min followed by the remaining 50% in 60 min; and Beads VI and VII gave a slow release of 10% and 4%, respectively, from 0 to 120 min, followed by a rapid 100% release from 120 to 180 min. 相似文献
235.
Yukie Iwabu Masanobu Kinomoto Masashi Tatsumi Hideaki Fujita Mari Shimura Yoshitaka Tanaka Yukihito Ishizaka David Nolan Simon Mallal Tetsutaro Sata Kenzo Tokunaga 《The Journal of biological chemistry》2010,285(46):35350-35358
Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo. 相似文献
236.
Albin JS LaRue RS Weaver JA Brown WL Shindo K Harjes E Matsuo H Harris RS 《The Journal of biological chemistry》2010,285(52):40785-40792
Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu(324) as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu(324) is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins. 相似文献
237.
238.
Yubin Zhou Wen-Pin Tzeng Hing-Cheung Wong Yiming Ye Jie Jiang Yanyi Chen Yun Huang Suganthi Suppiah Teryl K. Frey Jenny J. Yang 《The Journal of biological chemistry》2010,285(12):8855-8868
The rubella virus (RUBV) nonstructural (NS) protease domain, a Ca2+- and Zn2+-binding papain-like cysteine protease domain within the nonstructural replicase polyprotein precursor, is responsible for the self-cleavage of the precursor into two mature products, P150 and P90, that compose the replication complex that mediates viral RNA replication; the NS protease resides at the C terminus of P150. Here we report the Ca2+-dependent, stoichiometric association of calmodulin (CaM) with the RUBV NS protease. Co-immunoprecipitation and pulldown assays coupled with site-directed mutagenesis demonstrated that both the P150 protein and a 110-residue minidomain within NS protease interacted directly with Ca2+/CaM. The specific interaction was mapped to a putative CaM-binding domain. A 32-mer peptide (residues 1152–1183, denoted as RUBpep) containing the putative CaM-binding domain was used to investigate the association of RUBV NS protease with CaM or its N- and C-terminal subdomains. We found that RUBpep bound to Ca2+/CaM with a dissociation constant of 100–300 nm. The C-terminal subdomain of CaM preferentially bound to RUBpep with an affinity 12.5-fold stronger than the N-terminal subdomain. Fluorescence, circular dichroism and NMR spectroscopic studies revealed a “wrapping around” mode of interaction between RUBpep and Ca2+/CaM with substantially more helical structure in RUBpep and a global structural change in CaM upon complex formation. Using a site-directed mutagenesis approach, we further demonstrated that association of CaM with the CaM-binding domain in the RUBV NS protease was necessary for NS protease activity and infectivity. 相似文献
239.
Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro
Dorothee Andres Christin Hanke Ulrich Baxa Ana?t Seul Stefanie Barbirz Robert Seckler 《The Journal of biological chemistry》2010,285(47):36768-36775
Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins. 相似文献
240.
Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro. 相似文献