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Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.

International audience; Hepatitis C virus (HCV) E1 and E2 envelope glycoproteins (GPs) displayed on retroviral cores (HCVpp) are a powerful and highly versatile model system to investigate wild-type HCV entry. To further characterize this model system, the cellular site of HCVpp assembly and the respective roles of the HCV GPs in this process were investigated. By using a combination of biochemical methods with confocal and electron microscopic techniques, it was shown that, in cells producing HCVpp, both E1 and E2 colocalized with retroviral core proteins intracellularly, presumably in multivesicular bodies, but not at the cell surface. When E1 and E2 were expressed individually with retroviral core proteins, only E2 colocalized with and was incorporated on retroviral cores. Conversely, the colocalization of E1 with retroviral core proteins and its efficient incorporation occurred only upon co-expression of E2. Moreover, HCVpp infectivity correlated strictly with the presence of both E1 and E2 on retroviral cores. Altogether, these results confirm that the E1E2 heterodimer constitutes the prebudding form of functional HCV GPs and, more specifically, show that dimerization with E2 is a prerequisite for efficient E1 incorporation onto particles.Hepatitis C virus (HCV) E1 and E2 envelope glycoproteins (GPs) displayed on retroviral cores (HCVpp) are a powerful and highly versatile model system to investigate wild-type HCV entry. To further characterize this model system, the cellular site of HCVpp assembly and the respective roles of the HCV GPs in this process were investigated. By using a combination of biochemical methods with confocal and electron microscopic techniques, it was shown that, in cells producing HCVpp, both E1 and E2 colocalized with retroviral core proteins intracellularly, presumably in multivesicular bodies, but not at the cell surface. When E1 and E2 were expressed individually with retroviral core proteins, only E2 colocalized with and was incorporated on retroviral cores. Conversely, the colocalization of E1 with retroviral core proteins and its efficient incorporation occurred only upon co-expression of E2. Moreover, HCVpp infectivity correlated strictly with the presence of both E1 and E2 on retroviral cores. Altogether, these results confirm that the E1E2 heterodimer constitutes the prebudding form of functional HCV GPs and, more specifically, show that dimerization with E2 is a prerequisite for efficient E1 incorporation onto particles.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.

International audience; Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic alpha-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention.

International audience; The Bacillus subtilis catabolite repression HPr (Crh) exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system. We report here that Crh preparations contain a mixture of monomers and homodimers, whereas HPr is known to be monomeric in solution. The dissociation rate of dimers is very slow (t1/2 of about 10 hours), and the percentage of dimers in Crh preparations increases with rising temperature or protein concentration. However, at temperatures above 25 degrees C and a protein concentration of 10 mg/ml, Crh dimers slowly aggregate. Typically, NMR spectra recorded at 25 degrees C showed the coexistence of both forms of Crh, while in Crh solutions kept at 35 degrees C, almost exclusively Crh monomers could be detected. Circular dichroism analysis revealed that the monomeric and dimeric forms of Crh are well folded and exhibit the same overall structure. The physiological significance of the slow Crh monomer/dimer equilibrium remains enigmatic.The Bacillus subtilis catabolite repression HPr (Crh) exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system. We report here that Crh preparations contain a mixture of monomers and homodimers, whereas HPr is known to be monomeric in solution. The dissociation rate of dimers is very slow (t1/2 of about 10 hours), and the percentage of dimers in Crh preparations increases with rising temperature or protein concentration. However, at temperatures above 25 degrees C and a protein concentration of 10 mg/ml, Crh dimers slowly aggregate. Typically, NMR spectra recorded at 25 degrees C showed the coexistence of both forms of Crh, while in Crh solutions kept at 35 degrees C, almost exclusively Crh monomers could be detected. Circular dichroism analysis revealed that the monomeric and dimeric forms of Crh are well folded and exhibit the same overall structure. The physiological significance of the slow Crh monomer/dimer equilibrium remains enigmatic.

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Collagen XIV is known to bind to the dermatan sulfate chain of decorin and to the heparan sulfate chain of perlecan. To study its possible interaction with glycosaminoglycans, the NC1 domain of chicken collagen XIV was overproduced in E. coli. Purified NC1*(6-119)* appears poorly organized (the asterisks indicate the presence of extension sequences), but V8-protease generated fragments containing the 84-108 basic sequence tend to fold into alpha-helix. These fragments interact specifically with heparin, which induces an alpha-helical fold with a maximum effect for equimolar heparin/peptide ratio. These data demonstrate the existence of a glycosaminoglycan binding site in NC1.

The spectral densities of the backbone and arginine side chain NH bonds of the DNA binding domain of the fructose repressor (FruR) were extensively analyzed in order to extract reliable motions parameters. An accurate measurement of 15N NMR relaxation rates allowed their calculation at three frequencies, zero, omegaN, and omegaH + omegaN, using a reduced matrix approach. Linear correlations were found between J(omegaN) and J(0) and between and J(0). The analysis of the compatibility between the motions parameters obtained independently from the two correlation lines allowed further development of the linear correlation approach proposed recently [Lefevre, J. F., Dayie, K. T., Peng, J. W., and Wagner, G. (1996) Biochemistry 35, 2674-2686]. The results demonstrate (i) the existence of a concerted motion along the whole backbone with a global correlation time equal to 5.95 ns.rad-1, and (ii) the presence of complex internal movements at an intermediate time scale around 1 ns. The extracted motion parameters have been related to those obtained with the extended Lipari and Szabo approach but are incompatible with those obtained using the usual simple Lipari and Szabo approach. They were correlated to the features of the NMR structure of FruR(1-57)*. Some residues in the turns and in the third helix experience slow motions in the micro- to millisecond time scale. Side-chain motions are not correlated to the backbone dynamics. A direct examination of spectral densities reveals a higher flexibility for the side chains of arginines that are not involved in ionic bridges.The spectral densities of the backbone and arginine side chain NH bonds of the DNA binding domain of the fructose repressor (FruR) were extensively analyzed in order to extract reliable motions parameters. An accurate measurement of 15N NMR relaxation rates allowed their calculation at three frequencies, zero, omegaN, and omegaH + omegaN, using a reduced matrix approach. Linear correlations were found between J(omegaN) and J(0) and between and J(0). The analysis of the compatibility between the motions parameters obtained independently from the two correlation lines allowed further development of the linear correlation approach proposed recently [Lefevre, J. F., Dayie, K. T., Peng, J. W., and Wagner, G. (1996) Biochemistry 35, 2674-2686]. The results demonstrate (i) the existence of a concerted motion along the whole backbone with a global correlation time equal to 5.95 ns.rad-1, and (ii) the presence of complex internal movements at an intermediate time scale around 1 ns. The extracted motion parameters have been related to those obtained with the extended Lipari and Szabo approach but are incompatible with those obtained using the usual simple Lipari and Szabo approach. They were correlated to the features of the NMR structure of FruR(1-57)*. Some residues in the turns and in the third helix experience slow motions in the micro- to millisecond time scale. Side-chain motions are not correlated to the backbone dynamics. A direct examination of spectral densities reveals a higher flexibility for the side chains of arginines that are not involved in ionic bridges.

In this study we have investigated, using three different human melanoma cell lines (M1Do., M3Da., M4Be.). the varying capacity of melanoma cells to induce platelet aggregation in the presence or absence of inhibitors of ADP or thrombin. The expression levels of different integrins (alpha v, beta 3, alpha v beta 3, alpha IIb, alpha v beta 3) were evaluated by immunoprecipitation, binding and flow cytometry studies. The level of ADP in supernatants of melanoma cells were quantified by ADP bioassay and HPLC. Platelets were irreversibly aggregated by M3Da, as shown by electron microscopy, in contrast to M1Do, which induced a slow reversible aggregation. M4Be. did not induce platelet aggregation. In both cases, with M3Da. or M1Do., apyrase but not PPACK inhibited platelet induced aggregation. An anti-alpha v beta 3 monoclonal antibody (LYP18) or polyclonal antibody inhibited platelet aggregation. A similar number of LYP18 molecules bound to the surface of M1Do., M3Da. and M4Be. cell lines. Biological HPLC assays of ADP present in the supernatant of tumour cell lines showed the highest concentration of ADP to be secreted by M3Da., followed by M1Do., and none detected for M4Be. These results show that differences in in vitro aggregating potential of the three human melanoma cell lines are not related to low integrin expression levels but to their ability to generate ADP. Generation of ADP by human melanoma cells may act as important modulator of melanoma-platelet interactions.In this study we have investigated, using three different human melanoma cell lines (M1Do., M3Da., M4Be.). the varying capacity of melanoma cells to induce platelet aggregation in the presence or absence of inhibitors of ADP or thrombin. The expression levels of different integrins (alpha v, beta 3, alpha v beta 3, alpha IIb, alpha v beta 3) were evaluated by immunoprecipitation, binding and flow cytometry studies. The level of ADP in supernatants of melanoma cells were quantified by ADP bioassay and HPLC. Platelets were irreversibly aggregated by M3Da, as shown by electron microscopy, in contrast to M1Do, which induced a slow reversible aggregation. M4Be. did not induce platelet aggregation. In both cases, with M3Da. or M1Do., apyrase but not PPACK inhibited platelet induced aggregation. An anti-alpha v beta 3 monoclonal antibody (LYP18) or polyclonal antibody inhibited platelet aggregation. A similar number of LYP18 molecules bound to the surface of M1Do., M3Da. and M4Be. cell lines. Biological HPLC assays of ADP present in the supernatant of tumour cell lines showed the highest concentration of ADP to be secreted by M3Da., followed by M1Do., and none detected for M4Be. These results show that differences in in vitro aggregating potential of the three human melanoma cell lines are not related to low integrin expression levels but to their ability to generate ADP. Generation of ADP by human melanoma cells may act as important modulator of melanoma-platelet interactions.

CD spectra have been recorded with subunit delta from chloroplast CF0CF1 and with OSCP from mitochondrial MF0MF1. These subunits are supposed to act similarly at the interface between proton transport through the F0-portion and ATP-synthesis in the F1-portion of their respective F0F1-ATPase. Evaluation of the data for both proteins revealed a very high alpha-helix content of approximately 85% and practically no beta-sheets. Despite their low homology on the primary structure level (23% identity) and their different electrostatic properties (pI-values differ by 3 units), spinach delta and porcine OSCP are indistinguishable with respect to their secondary structure as measured by CD. Prediction and analysis of consensual alpha-helices even in poorly conserved regions indicate a high degree of structural similarity between chloroplast delta and OSCP. In view of the topology and function of delta and OSCP in intact F0F1 these findings are interpreted to indicate the dominance of secondary and tertiary structure over the primary structure in their supposed function between proton flow and ATP-synthesis.CD spectra have been recorded with subunit delta from chloroplast CF0CF1 and with OSCP from mitochondrial MF0MF1. These subunits are supposed to act similarly at the interface between proton transport through the F0-portion and ATP-synthesis in the F1-portion of their respective F0F1-ATPase. Evaluation of the data for both proteins revealed a very high alpha-helix content of approximately 85% and practically no beta-sheets. Despite their low homology on the primary structure level (23% identity) and their different electrostatic properties (pI-values differ by 3 units), spinach delta and porcine OSCP are indistinguishable with respect to their secondary structure as measured by CD. Prediction and analysis of consensual alpha-helices even in poorly conserved regions indicate a high degree of structural similarity between chloroplast delta and OSCP. In view of the topology and function of delta and OSCP in intact F0F1 these findings are interpreted to indicate the dominance of secondary and tertiary structure over the primary structure in their supposed function between proton flow and ATP-synthesis.

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.