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A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.  相似文献   
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The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.  相似文献   
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Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i). mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii). mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.  相似文献   
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Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway.  相似文献   
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Interaction of hepatitis C virus proteins with host cell membranes and lipids   总被引:15,自引:0,他引:15  
For replication, viruses depend on specific components and energy supplies from the host cell. The main steps in the lifecycle of positive-strand RNA viruses depend on cellular membranes. Interest is increasing in studying the interactions between host cell membranes and viral proteins to understand how such viruses replicate their genome and produce infectious particles. These studies should also lead to a better knowledge of the different mechanisms underlying membrane-protein associations. The various molecular interactions of hepatitis C virus proteins with the membranes and lipids of the infected cell highlight how a virus can exploit the diversity of interactions that occur between proteins and membranes or lipid structures.  相似文献   
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Oligomerization of viral envelope proteins is essential to control virus assembly and fusion. The transmembrane domains (TMDs) of hepatitis C virus envelope glycoproteins E1 and E2 have been shown to play multiple functions during the biogenesis of E1E2 heterodimer. This makes them very unique among known transmembrane sequences. In this report, we used alanine scanning insertion mutagenesis in the TMDs of E1 and E2 to examine their role in the assembly of E1E2 heterodimer. Alanine insertion within the center of the TMDs of E1 or E2 or in the N-terminal part of the TMD of E1 dramatically reduced heterodimerization, demonstrating the essential role played by these domains in the assembly of hepatitis C virus envelope glycoproteins. To better understand the alanine scanning data obtained for the TMD of E1 which contains GXXXG motifs, we analyzed by circular dichroism and nuclear magnetic resonance the three-dimensional structure of the E1-(350-370) peptide encompassing the N-terminal sequence of the TMD of E1 involved in heterodimerization. Alanine scanning results and the three-dimensional molecular model we obtained provide the first framework for a molecular level understanding of the mechanism of hepatitis C virus envelope glycoprotein heterodimerization.  相似文献   
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Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.  相似文献   
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Malaria remains the most important of the tropical diseases, widespread throughout the tropics, but also occurring in many temperate regions. The disease causes a heavy toll of illness and death, especially among children in endemic areas. It also poses a risk to business travellers, tourists and inmigrants and imported cases of malaria are increasingly seen in non-endemic areas. We discuss here how microscopical diagnosis is essential for identifyingPlasmodium species responsible of the infection and discarding possible mixed infections. Thus, a correct treatment can be administered in 30 min, avoiding secondary stays and saving important amounts of money. Problems of drug resistance have to be distinguished from those arising due to erroneous diagnosis.  相似文献   
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