Báo cáo khoa học: Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein doc

Prevalence of intrinsic disorder in the hepatitis C virus ARFP/Core+1/S protein ´ Anissa Boumlic1,2,*, Yves Nomine1,*, Sebastian Charbonnier1, Georgia Dalagiorgou2, Niki ´ Vassilaki2, Bruno Kieffer3, Gilles Trave1, Penelope Mavromara2 and Georges Orfanoudakis1 ´ ´ Oncoproteins Group, Universite de Strasbourg, CNRS FRE 3211, Ecole Superieure de Biotechnologie de Strasbourg, Illkirch, France Molecular Virology Laboratory, Hellenic Pasteur Institute, Athens, Greece ´ ´ ´ Biomolecular NMR Group, UMR CNRS 7104, Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France Keywords ARFP/Core+1/S; hepatitis C virus (HCV); intrinsic disorder; IUP/IDP; NMR Correspondence G Orfanoudakis, Oncoproteins Group, ´ Universite de Strasbourg, CNRS FRE 3211, ´ Ecole Superieure de Biotechnologie de Strasbourg, Illkirch, France Fax: +33 68 85 47 70 Tel: +33 68 85 47 65 E-mail: georges.orfanoudakis@unistra.fr *These authors contributed equally to this work (Received 25 October 2009, revised 30 November 2009, accepted December 2009) doi:10.1111/j.1742-4658.2009.07527.x The hepatitis C virus (HCV) Core+1/S polypeptide, also known as alternative reading frame protein (ARFP)/S, is an ARFP expressed from the Core coding region of the viral genome Core+1/S is expressed as a result of internal initiation at AUG codons (85–87) located downstream of the polyprotein initiator codon, and corresponds to the C-terminal part of most ARFPs Core+1/S is a highly basic polypeptide, and its function still remains unclear In this work, untagged recombinant Core+1/S was expressed and purified from Escherichia coli in native conditions, and was shown to react with sera of HCV-positive patients We subsequently undertook the biochemical and biophysical characterization of Core+1/S The conformation and oligomeric state of Core+1/S were investigated using size exclusion chromatography, dynamic light scattering, fluorescence, CD, and NMR Consistent with sequence-based disorder predictions, Core+1/S lacks significant secondary structure in vitro, which might be relevant for the recognition of diverse molecular partners and/or for the assembly of Core+1/S This study is the first reported structural characterization of an HCV ARFP/Core+1 protein, and provides evidence that ARFP/Core+1/ S is highly disordered under native conditions, with a tendency for selfassociation Introduction Hepatitis C virus (HCV) is the major etiological agent of chronic hepatitis, with more than 170 million people being infected worldwide [1,2] Persistent HCV infection progresses, in 20% of cases, to liver cirrhosis within 20 years of infection, with the possible development of hepatocellular carcinoma (HCC) in 1–4% of cases [3] No prophylactic vaccine against HCV exists, and the efficiency of therapies is hindered by the extreme heterogeneity of the HCV genome [4,5] HCV, a Hepacivirus genus member of the Flaviviridae family, is a small, enveloped RNA virus [6] Its genome is a positive, single-stranded 9.6 kb RNA containing 5¢-UTRs and 3¢-UTRs involved in viral protein translation and viral replication [7–9] The genome encodes a large precursor polyprotein that undergoes proteolysis, generating HCV structural proteins (Core, E1, and E2) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) An alternative reading frame (Core+1 ORF) overlapping the Core protein gene in the +1 frame was recently reported [10–13] Abbreviations ARFP, alternative reading frame protein; DLS, dynamic light scattering; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HSQC, heteronuclear single quantum coherence; IDP, intrinsically disordered protein; IMAC, immobilized metal ion affinity chromatography; MBP, maltose-binding protein; OG, n-octyl-b-D-glucoside; SSP, secondary structure propensity; TEV, tobacco etch virus 774 FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS A Boumlic et al This ORF is responsible for the expression of various alternative reading frame proteins (ARFPs), also named Core+1 proteins, resulting from mechanisms such as ribosomal frame shifting and internal initiation at alternative AUG or non-AUG codons [10–12,14– 17] Core+1 proteins were recently shown not to be required for HCV replication [18,19] However, the presence of specific antibodies and T-cell-mediated immune responses in serum from HCV-infected patients suggests the expression of the Core+1 ORF during HCV infection [10–12,20,21] Furthermore, Core+1 proteins were found to interfere with apoptosis and cell cycle regulation [22,23], suggesting a possible role of these proteins in HCV pathogenesis One remarkable ARFP is Core+1/S, a small polypeptide with a length varying from 38 to 76 residues among HCV genotypes Core+1/S corresponds to the C-terminal fragment of the Core+1 ORF, and to date is the shortest ARFP form described Its translation results from internal initiation at alternative AUG codons (85–87) located downstream of the polyprotein codon initiator Recently, two different groups observed that Core+1/S is the predominant alternative form when the Core+1 ORF is introduced into mammalian expression systems [16,24] In addition, Core+1/S was found to be downregulated by the Core protein and degraded in a proteasome-dependent manner [25,26] In order to further our understanding of these proteins, we undertook biochemical and biophysical studies of the Core+1/S proteins derived from HCV-1a and HCV-1b isolates The Core+1/S proteins were produced in bacteria and purified in native conditions ELISA experiments using the purified recombinant Core+1/S of HCV-1b demonstrated the ability of the protein to react with sera from HCV-infected patients We subsequently investigated the biophysical features of HCV-1a and HCV-1b Core+1/S proteins using sequence analysis and complementary biophysical approaches [fluorescence, CD, dynamic light scattering (DLS), and NMR] We provide evidence that ARFP/ Core+1/S is highly disordered under native conditions, with a tendency for self-association Results Sequence analysis of Core+1/S predicts the largely disordered character of the protein Sequence alignments were performed to analyze the degree of Core+1/S amino acid conservation among reference sequences of different HCV genotypes (Fig 1A) [5] The N-terminal sequence is well conserved Biophysical characterization of HCV ARFP/Core+1/S and exhibits hydrophobic patches, encompassing residues 1–6, 14–25, and 32–35 In contrast, considerable variability was observed in the location of the stop codon on the RNA sequence (data not shown), leading to variation in the lengths of protein sequences Amino acid sequences were analyzed using the disorder prediction tools globplot and pondr globplot evaluates the sum of the disorder propensity for each amino acid among the sequence, and pondr analyzes the mean net charge and hydrophobicity of the polypeptide chain This combination of properties seems to be a prerequisite for the absence of compact structure in native conditions [27] globplot predicted disordered regions encompassing amino acids 6–28 and 42–52 for HCV-1a Core+1/S, and amino acids 6–28 and 42–58 for HCV1b Core+1/S, whereas pondr suggested that most of the Core+1/S sequence is disordered In order to assess whether the disorder prediction is also confirmed by the absence of secondary structure, four algorithms (phd, gor4, simpa96, and sopma) were used to predict the secondary structure contents of both HCV-1a and HCV-1b Core+1/S proteins (Fig 1B) A consensus is drawn for residues with at least three out of four identical secondary structure predictions Such a consensus suggested that the majority of residues are not embedded in secondary structure elements, with the exception of short residue stretches mainly located in the second and third hydrophobic patches The combination of secondary structure and disordered region predictions strongly suggests that the N-terminal and C-terminal regions of HCV Core+1 proteins are largely unstructured and highly disordered (Fig 1A) These predictions are supported by the high degree of conservation of several disorder-promoting residues, such as alanines, arginines, glycines, and serines (Fig 1B) [28] Expression and purification of Core+1/S proteins in native conditions We cloned and expressed the HCV-1a and HCV-1b Core+1/S proteins encompassing residues 85–160 and 85–142 of the full Core+1 ORF, respectively These constructs were fused to the C-terminus of either His6, His6–maltose-binding protein (MBP) (Fig S1), or His6–NusA (Fig 2A) Screenings of optimal yield and solubility conditions were first performed on the three constructs of HCV-1a Core+1/S by varying the induction temperatures between 37 and 22 °C Analysis on Tris/Tricine SDS gels showed expression of proteins at the expected molecular mass (Fig 2B; Fig S1), with an optimum induction temperature at 22 °C However, both His-tagged and MBP-tagged HCV-1a Core+1/S FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 775 Biophysical characterization of HCV ARFP/Core+1/S A Boumlic et al A B Fig Sequence analysis of Core+1/S proteins (A) Alignment of 17 Core+1/S amino acid reference sequences for different HCV genotypes Protein sequences were obtained after translation of the Core+1 ORF nucleotide sequences retrieved from the GenBank database (accession numbers are given in parentheses) Core+1/S amino acid sequences were aligned using CLUSTALW Similarity percentages are indicated on the right, according to CLUSTALW calculations Hydrophobic residues are boxed (B) Disorder and structure predictions Disorder predictions were made using GLOBPLOT and PONDR Disordered and ordered regions are indicated by ‘D’ and ‘.’, respectively Secondary structure predictions were performed with GOR4, SOPMA, SIMPA96 and PHD, using both HCV-1a and HCV-1b Core+1/S amino acid sequences as inputs (see Experimental procedures) Structure predictions for each residue position are indicated as a-helix (H), extended strand (E), b-turn (T), or random coil (C) Uppercase letters indicate a prediction rate higher than 80% A consensus was reported when three or more predictions over the four algorithms provide identical secondary structure prediction Residues are numbered from the start of Core+1/S and correspond to residues 85–161 and 85–144 of the Core+1 ORF, and nucleotides 599–827 and 599–776 of the Core/Core+1 RNA sequence, for HCV-1a and HCV-1b, respectively proteins were found largely in the insoluble fractions after cell lysis (Fig S1), even after incubation at low temperatures In contrast, NusA-tagged HCV-1a Core+1/S was largely soluble even after tobacco etch virus (TEV) protease cleavage (Fig 2C) The solubilizing properties of NusA have already been described in the literature [29] However, it was surprising to observe MBP fusion proteins in the insoluble fractions, as the MBP carrier is also a well-known protein solubilizer Despite its small size, Core+1/S seems, therefore, to promote aggregation of the fusion protein when fused to the MBP carrier As NusA solubilized 776 HCV-1a Core+1/S, we fused the same carrier protein to the HCV-1b Core+1/S After TEV protease-mediated proteolysis, both HCV Core+1/S proteins remained soluble (Fig 2C) When Core+1/S production was scaled up, the use of the optimal expression and purification conditions as described above led to protein aggregation In order to prevent this, we lowered the expression temperature to 15 °C and systematically supplemented the purification buffer with l-arginine and l-glutamic acid at a final concentration of 50 mm each These additives are known to prevent protein aggregation [30] Finally, FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS A Boumlic et al Biophysical characterization of HCV ARFP/Core+1/S 6xHis A B kDa 72 55 37 TEV Core+1/S NusA T7 28 22 37 T (°C) kDa P S P S P S NusAHCV-1a Core+1/S 72 55 28 22 T (°C) NusAHCV-1b Core+1/S 28 17 11 28 P S P S P S 17 11 C kDa NusAHCV-1a Core+1/S NusA TEV kDa 72 55 NusAHCV-1b Core+1/S NusA 28 28 TEV 17 11 17 11 HCV-1b Core+1/S 72 55 HCV-1a Core+1/S Fig Expression and purification screenings of native NusA–HCV Core+1/S proteins (A) Cloning strategy for expression of Core+1/S The sequence His6–NusA is fused at the 5¢-terminus of the Core+1/S DNA sequence (B) Pellet/supernatant assays After transformation, expression of recombinant proteins was monitored for 2, h or overnight at 37, 28 or 22 °C, respectively Fifty microliters of bacterial culture was sonicated and centrifuged for 15 at 16 000 g Supernatants (S) and pellets (P) were analyzed by Tris/Tricine SDS/PAGE (C) IMAC purification of NusA–Core+1/S proteins followed by TEV protease digestion Labeled or unlabeled His6-NusA–Core+1/S proteins were expressed under optimized conditions, and purified on Ni2+–nitrilotriacetic acid resin in the presence of arginine and glutamic acid (50 mM each) After IMAC purification, fusion proteins were desalted and subjected to TEV protease cleavage to release Core+1/S proteins Lane 1: bacterial lysate Lane 2: IMAC elution at 250 mM imidazole Lane 3: desalted NusA–HCV-1a Core+1/S before TEV protease cleavage Lane 4: NusA–HCV-1a Core+1/S after TEV protease cleavage Lane 5: desalted NusA–HCV-1b Core+1/S before TEV protease cleavage Lane 6: NusA–HCV-1b Core+1/S after TEV protease cleavage Arrows on the right indicate the bands for soluble NusA-HCV-Core+1/S, NusA, TEV and Core+1/S proteins buffers were routinely supplemented with dithiothreitol and argon to reduce protein oxidation [31] Final yields were approximately mg of expressed protein per liter of bacterial culture Upon size exclusion chromatography, both HCV Core+1/S proteins eluted as monomers, according to column calibration (Fig 3A) MS analysis of the purified proteins gave experimental masses of 7630.7 ± 0.8 and 6076.0 ± 0.1 Da for HCV-1a and HCV-1b Core+1/S, respectively The mass of HCV-1b Core+1/ S corresponds to the calculated value (6075.9 Da), whereas that of HCV-1a Core+1/S showed loss of the GA sequence that is usually left after TEV protease proteolysis and the N-terminal methionine Purified recombinant Core+/1S proteins were also verified through SDS/PAGE (Fig 3B), and were specifically recognized by polyclonal antibodies against the Core+1 ORF in western blot experiments (Fig 3C) Sera from HCV-1-infected patients are reactive against native HCV-1b Core+1/S HCV-1b Core+1/S was used in ELISA to test the reactivity of sera from patients positive for HCV genotype Figure shows a high prevalence ( 60%) of Core+1 antibodies in patient sera as compared with the cutoff value, defined as the average of the negative controls plus two standard deviations The presence of antibodies against Core+1/S indicates that the purified recombinant untagged protein remains immunoreactive, and suggests that the protein is present in patients infected with HCV of genotype Intrinsic fluorescence of Core+1/S proteins HCV-1a and HCV-1b Core+1/S proteins contain tryptophans at positions 34, 49, 66, and 74, and positions 6, 34, and 49, respectively Intrinsic fluorescence spectroscopy was therefore used to evaluate the solvent accessibility of these residues As all tryptophans are simultaneously excited, the emission spectrum results from the sum of the signals of individual emitters The maxima of fluorescence emission for HCV-1a and HCV-1b Core+1/S proteins were observed at wavelength of 354 and 353 nm, respectively (Fig 5A) These values are close to that of soluble tryptophan in aqueous solution (355 nm) [32], indicating that all tryptophans of Core+1/S proteins are exposed to the sol- FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 777 Biophysical characterization of HCV ARFP/Core+1/S kDa Absorbance at 280 nm (a.u.) A 67 43 29 A Boumlic et al B kDa (a) 13.7 6.5 NusA 55 28 12 11 (b) 55 28 HCV-1a Core+1/S 11 (c) 55 28 20 40 60 80 a 100 HCV-1b Core+1/S 11 b c Elution volume (mL) C kDa Ponceau Red 28 Anti-Core+1 Anti-Core+1 HCV-1a HCV-1b 17 HPV E6 HCV-1b Core+1/S HPV E6 HCV-1b Core+1/S HCV-1a Core+1/S HPV E6 HCV-1b Core+1/S HCV-1a Core+1/S 11 vent In a second step, an HCV-1b Core+1/S sample was subjected to a 20 heat pulse 16 h prior to fluorescence analysis (Fig 5B) No change in either the wavelength or the intensity of the maximum fluorescence emission was observed This observation indicates an absence of precipitation, suggesting resistance of the protein to heat treatment, a feature that is often associated with disordered proteins [33] Self-assembly of HCV Core+1/S proteins DLS allows the oligomeric status of proteins in solution to be evaluated Hydrodynamic radius distributions were derived from DLS data recorded for each protein sample under various conditions, assuming a coil model as implemented in dynals (Fig 6) In the absence of any treatment or additive (Fig 6, upper panels), the average hydrodynamic radii (Rh) were 4.5 ± 2.4 and 2.5 ± 1.2 nm for purified HCV-1a and HCV-1b Core+1/S proteins, respectively Assuming a coil model, these radii are equivalent to particles of nearly 15 and five monomers for HCV-1a and HCV1b, respectively The radius distribution indicates the polydisperse character of both isoforms As the 778 Fig Biochemical analysis of purified native HCV Core+1/S proteins (A) Size exclusion chromatography of HCV Core+1/S proteins After TEV protease proteolysis, proteins were injected onto a Hiload 16/60 Superdex 75 column in the presence of arginine and glutamic acid (50 mM each) The mass distribution in the eluant is indicated at the top Both HCV-1a Core+1/S (dotted line) and HCV-1b Core+1/S (bold line) eluted as monomers, according to the column calibration (B) Coomassie blue staining of purified proteins by Tris/Tricine SDS/PAGE Molecular masses are given on the left, and arrows indicate the expected expression products (C) Western blot analysis of purified Core+1/S proteins After purification and concentration, Core+1/S proteins were analysed by western blotting using antiHCV-1a Core+1 or anti-HCV-1b serum Left panel: Ponceau staining of HCV Core+1/S proteins and HPV16 E6 Middle panel: HCV1a Core+1/S revealed by anti-HCV-1a Core+1 serum Right panel: HCV-1b Core+1/S revealed by anti-HCV-1b Core+1 serum Molecular masses are indicated on the left proteins eluted as monomers in a size exclusion chromatography column, it appears that multimerization occurs during and/or after concentration We previously showed that HCV-1a Core+1/S is localized in the endoplasmic reticulum membranes [24] Under the hypothesis that HCV-1b Core+1/S contains membrane localization determinants, we added octyl glucoside [n-octyl-b-d-glucoside (OG)], a nonionic detergent that is frequently used to solubilize integral membrane proteins The presence of OG in Core+1/S proteins sharpened the size distributions as observed with DLS, and thus lowered the polydispersity in particle sizes, although the average hydrodynamic radii were not significantly altered (Fig 6, middle panels) When the proteins were subjected to a heat pulse, the average hydrodynamic radii shifted from 4.5 ± 2.4 to 1.8 ± 0.6 nm for HCV-1a Core+1/S, and from 2.5 ± 1.2 to 1.6 ± 0.6 nm for HCV-1b Core+1/S (Fig 6, lower panels), suggesting a transition to lower-size oligomers In addition, the polydispersity significantly decreased Thus, high temperature is able to disrupt Core+1/S multimers without leading to protein precipitation FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS A Boumlic et al Biophysical characterization of HCV ARFP/Core+1/S structure is consistent with disorder prediction, a significant amount of b-sheet content seems to be present The presence of such a signal might be due to the presence of intrinsic b-sheet structure in Core+1/ S protein Alternatively, it might also correspond to b-sheet structure formed at the interface of Core+1/ S monomers upon multimerization, as it has been shown that b-sheet structure is predominant in aggregates and is often associated with intrinsically disordered proteins [36] Finally, the CD spectrum recorded for an HCV-1b Core+1/S sample subjected to a heat pulse was slightly different from that of an unheated sample (Fig 7D) In contrast, the addition of OG induced drastic changes in the CD spectrum as compared with the untreated sample spectrum for both Core+1/S proteins (Fig 7A,B), suggesting an effect of OG on the conformation of HCV-1b Core+1/S However, the addition of OG prevented the recording of data at wavelengths below 206 nm, hindering the deconvolution of data HCV/HCC Controls 0.3 0.2 0.1 0.0 1b 1a Fig Reactivity of sera from genotype HCV-infected patients against HCV-1b Core+1/S The sera from HCV-infected patients were tested by enzyme immunoassay, using the native HCV-1b Core+1/S Controls correspond to HCV-negative patient sera A450 nm values of the different sera are represented The cutoff was determined as the average of HCV-negative sera absorbance plus two standard deviations CD analysis of potential secondary structure of Core+1/S proteins CD spectra were recorded for both proteins in the far-UV region Globally, CD spectra for HCV-1a (Fig 7A) and HCV-1b (Fig 7B) Core+1/S proteins did not show the characteristics of a full random coil conformation (a strong negative minimum at 195– 198 nm, and a weak negative signal at 220 nm) [34] Instead, we observed a maximum at 195 nm and a minimum at 220 nm, suggesting the existence of bsheet secondary structure Deconvolution of the CD data was performed using three sets of reference proteins and the algorithms provided by the cdpro suite [35] As selcon3 failed several times to fit the CD data, this program was not used for data analysis However, both cdsstr and contin/ll gave consistent results, and allowed the contributions of structural elements to be estimated The percentages of a-helix (a), b-sheet (b) and unordered (U) structures were  5%,  30% and  65%, respectively, with a typical range of variation of 10–20% (Fig 7C) Although the high content of unordered Fig Intrinsic fluorescence of Core+1/S proteins UV fluorescence emission spectra of Core+1/S proteins were recorded in 20 mM sodium phosphate buffer (pH 6.8, mM) (A) Fluorescence emission spectra of HCV-1a and HCV-1b Core+1/S proteins in buffer (B) Fluorescence emission spectra of HCV-1b Core+1/S proteins were recorded after boiling the protein for 20 and cooling to room temperature Fluorescence intensity (normalized) A NMR analysis of HCV-1b Core+1/S In order to further investigate the structural properties of Core+1/S proteins, NMR 1H–15N heteronuclear single quantum coherence (HSQC) experiments were performed for both HCV-1b Core+1/S (Fig 8A) and HCV-1a Core+1/S (Fig S2) Both spectra exhibit a rather narrow amide proton chemical shift dispersion, limited to 0.7 p.p.m Such a range is characteristic of a lack of structural organization of the backbone [37] The spectrum recorded for HCV-1a Core+1/S showed a high number of overlapping peaks, impeding the accurate counting of peaks In contrast, the HSQC spectrum of HCV-1b Core+1/S allows the counting of a number of peaks consistent with that expected from the protein sequence In order to assign backbone frequencies of the polypeptide, three-dimensional NMR experiments were performed on a 15N,13C-labeled HCV-1b Core+1/S B HCV-1a Core+1/S Fluorescence intensity (normalized) OD (450 nm) 0.4 HCV-1b Core+1/S 305 325 345 365 385 Wavelength (nm) FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 400 HCV-1b Core+1/S Heated 305 Unheated 325 345 365 385 400 Wavelength (nm) 779 Biophysical characterization of HCV ARFP/Core+1/S HCV-1a Core+1/S 0.4 HCV-1b Core+1/S Rave: 4.5 nm s: 2.4 nm Rave: 2.5 nm s: 1.2 nm 0.4 0.0 0.8 0.0 Rave: 4.0 nm s: n/a Rave: 2.6 nm s: n/a 0.4 0.0 0.0 Rave: 1.8 nm s: 0.6 nm Rave: 1.6 nm s: 0.6 nm Heat pulse 0.2 0.0 0.0 2.0 4.0 6.0 8.0 10.0 Hydrodynamic radius (nm) 0.0 0.8 0.4 0.0 2.0 4.0 6.0 8.0 10.0 Hydrodynamic radius (nm) sample Near-complete 1HN,15N-backbone and 13C-resonance assignment could be achieved for HCV-1b Core+1/S (Fig 8A), with the exception of His14 and Ser38, as well as the first two residues (Gly-Ala) remaining from the TEV protease site The lack of His14 resonances might be due to protonation–deprotonation equilibrium of the imidazole ring [38] The absence of Ser38 resonances needs to be further investigated We used experimental carbon chemical shifts to probe the presence of helical or b-sheet secondary structures For all residues of HCV-1b Core+1/S, Ca secondary chemical shifts were below 1.0 p.p.m (positive or negative) (Fig 8B), confirming the absence of stable secondary structure elements in HCV-1b Core+1/S However, a consensus was observed for residues encompassing the region between residues 32 and 35, suggesting that this region might have a tendency to b-sheet character Interestingly, the same region was predicted to contain b-sheet elements by the majority of the secondary structure prediction methods, and also corresponds to a nondisordered region according to globplot analysis (Fig 1B) Methods based on chemical shifts are often used to depict secondary structure elements, but quantitative interpretation of secondary chemical shifts alone remains difficult, because the expected values for fully formed secondary structures vary for different amino acids [39] In order to quickly visualize the fractional deviation of the experimental chemical shifts from pure a-helix or b-sheet secondary shifts, residue-specific secondary structure propensity (SSP) scores of HCV-1b Core+1/S were calculated on the basis of ssp software recommendations [40] ssp combines chemical shifts 780 0.8 OG 0.4 0.4 0.8 control 0.2 Mass distribution (%) A Boumlic et al Fig Size distribution histograms of HCV1a and HCV-1b Core+1/S proteins determined by DLS Twenty microliters of 80–100 lM protein samples in 20 mM sodium phosphate buffer (pH 6.8, 400 mM NaCl) were directly analyzed, incubated with OG, or subjected to a heat pulse prior to analysis Samples were analyzed by DLS, and the hydrodynamic radius distributions of Core+1 proteins were determined using DYNALS, assuming a coil model Solid lines are the three-parameter nonlinear least squares fits of the size distribution profiles using a Gaussian model, yielding average radii (Rave) and widths at the half-height (s) When the profile exhibits only two values, an average radius was determined by weight averaging of the intensities from different nuclei weighted by their sensitivity to a-helix or b-sheet structures into a single SSP score varying between and 1, or and )1, for a-helix and b-sheet structures, respectively These scores represent the expected fraction of a-helix or b-sheet secondary structure for each residue Calculated scores of HCV1b Core+1/S are very close to zero values, indicating an overall low SSP In particular, the SSP profile shows almost no propensity to adopt a helical conformation along the protein sequence Although a mild propensity to adopt a b-sheet conformation is visible for residues encompassing the regions between and 8, 32 and 35, and 41 and 44, it is very limited as compared to the maximal amplitude expected for a full b-sheet conformation Finally, the 1H–15N-HSQC NMR spectrum recorded for HCV-1b Core+1/S in the presence of 6% OG (Fig 8D) showed a few notable changes for Val21, Ile33, Trp34, Val35, Thr47, and five glycines distributed all over the sequence (Gly7, Gly8, Gly22, Gly30, and Gly50) These results suggest a possible weak interaction of HCV-1b Core+1/S with OG Discussion HCV Core+1/S proteins are intrinsically disordered Core+1/S proteins correspond to the C-terminal parts of most of the described HCV ARFPS To date, neither biochemical nor biophysical data have been described for ARFPs Here, we succeeded in producing the Core+1/S proteins from HCV-1a and HCV-1b genotypes, using the standard Escherichia coli BL21 FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS A Boumlic et al OG –5 –10 –15 –20 –25 190 210 230 250 Wavelength (nm) HCV-1b Core+1/S 15 buffer 10 OG –5 –10 –15 –20 –25 190 210 230 Wavelength (nm) 100 250 α β U 50 HCV-1a Core+1/S C buffer 10 Secondary structure contents (%) θ[MRW] × 10–3 (deg·cm2·dmol–1) B HCV-1a Core+1/S 15 HCV-1b Core+1/S θ[MRW] × 10–3 (deg·cm2·dmol–1) A Biophysical characterization of HCV ARFP/Core+1/S Fig Far-UV CD analysis of HCV Core+1/S proteins Data are represented as molar ellipticity per residue Core+1/S proteins (4 lM) in 20 mM sodium phosphate buffer, 50 mM NaCl, and 0.15 mM dithiothreitol (A, B) CD spectra of HCV-1a and HCV-1b Core+1/S proteins in buffer (solid line), after incubation with 6% OG (C) Far-UV data were analyzed with the CDPro package, using two algorithms (CONTINLL, and CDSSR) and three protein databases (SP43, SMP56, and SDP48) a, a-helix; b, b-sheet; U, turns and unordered secondary structure bacterial system We optimized the expression and purification processes under native conditions, and obtained substantial amount of native, highly pure, untagged proteins We detected antibodies against recombinant HCV-1b Core+1/S in the sera of HCVinfected patients, suggesting that the protein might be expressed during HCV infection, either alone or as a part of a larger ARFP Combining the results of complementary biophysical techniques, our study showed that Core+1/S proteins lack secondary and tertiary structure 1H–15N-HSQC NMR experiments performed on both HCV-1a and HCV-1b Core+1/S constructs showed a limited chemical shift dispersion of amide proton resonances into a narrow range (0.7 p.p.m) This is indicative of a disordered state, as inherent flexibility and rapid interconversion between multiple conformations generally lead to a poor chemical shift dispersion Exceptions are the 15 N-backbone resonances in 1H–15N-HSQC spectra of Core+1/S proteins These resonances are influenced both by residue type and by the local amino acid sequence, and therefore remain well dispersed, even in fully unfolded states [41] In addition, the distribution of correlation peaks around 10 p.p.m in the HSQC spectrum, which are assigned to tryptophan side chains, indicates that these residues lie in a very similar environment, in agreement with fluorescence data indicative of solvent-exposed tryptophans Together with the absence of consensus in the backbone carbon chemical shift differences, these observations suggest a lack of secondary structure for HCV-1b Core+1/S This conclusion is further reinforced by the high content of unordered conformation ( 65%) determined by CD spectroscopy Finally, the HSQC spectrum recorded for HCV-1a Core+1/S also displays a poor proton chemical shift distribution, suggesting that this protein is also disordered When subjected to a heat pulse, folded proteins commonly unfold and precipitate, owing to solvent exposure of hydrophobic residues, whereas nonfolded peptides may remain in solution [33] We demonstrated that HCV-1b Core+1/S remains soluble after heat pulse treatment, as observed on fluorescence spectra Moreover, DLS shows that the mass distribution shifts to lower molecular masses This is confirmed by the observation in NMR spectra of more intense peaks following a heat pulse (data not shown) No significant change was observed in CD spectra after such treatment, indicating that this treatment does not influence the global conformation of the polypeptide Intrinsically disordered proteins (IDPs) are defined as proteins containing at least one disordered region, and were recently recognized as a new protein class [42] Disordered proteins are gaining considerable attention, owing to their capacity to perform numerous biological functions despite their lack of defined FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 781 A G28 G19 G11 G8 G50 15N (p.p.m.) Biophysical characterization of HCV ARFP/Core+1/S A Boumlic et al D G8 110 G50 G30 G30 G7 G7 T47 T51 T47 114 G22 G22 S41 Without OG S12 S45 S48 S54 S55 T26 V29 M1 Q9 M3 V16 W6 D10 R31V21 V32 M25 L20 R4 F52 R42 R53 V35 W49 S3 R36 A5 A44 I39 C13 A43 A23 I33 A2 W34 A56 With OG 6% 118 V21 122 V35 126 I33 130 130 10.0 8.4 A17 8.2 7.8 1H (p.p.m.) 8.4 8.2 8.0 7.8 C C0 –1 –2 C α –1 –2 C β –1 –2 11 1.0 0.5 SSP score Δδ (p.p.m.) B A17 10.0 130 8.0 W34 0.0 –0.5 –1.0 21 31 41 51 Amino acid sequence 11 21 31 41 51 Amino acid sequence structure [42–47] Under native conditions, Core+1/S proteins remain unstructured, and should therefore be classed as IDPs This character is also confirmed by disorder and structure predictions based on protein sequences This is not the first time that an HCV protein has been reported to be at least partially disordered Indeed, the first 82 amino acids of the N-terminal part of Core protein and domain of NS5A protein have already been classed as IDPs [48–50] Domain of NS5A is also natively unfolded [51] More generally, intrinsic disorder is commonly found in viruses For instance, among Flaviviridae, Dengue virus, West Nile virus and bovine viral diarrhea virus capsid proteins contain flexible, basic regions [52–54] Proteins from other virus families were also identified as being partially or completely 782 Fig NMR results for HCV-1b Core+1/S (A) Standard 2D 1H–15N-HSQC spectrum recorded at 600 MHz and 22 °C on a 100 lM sample of HCV-1b Core+1/S Each cross-peak corresponds to a correlation between an amide hydrogen atom and a nitrogen atom Assignments have been deposited in the BMRB (Ref 16487) (B) Differences between experimental carbon chemical shifts and random coil values as a function of sequence number (C) SSP of HCV-1b Core+1/S Carbon chemical shifts were used to calculate the residue-specific SSP scores of HCV-1b Core+1/S by following the SSP software recommendations Positive values ranging from to and negative values ranging from to )1 represent the propensities to form pure a-helix and b-sheet structures, respectively (D) Effects of the nonionic detergent OG on HCV-1b Core+1/S The superimposition of 2D H–15N-HSQC spectra of HCV-1b Core+1/S in the absence (blue) or presence (green) of 6% OG is shown disordered, such as the Nef protein of simian immunodeficiency virus [55], HIV tat protein [56], and the nucleoprotein and phosphoprotein of the measles virus [57,58] As virus genomes are restricted in molecular size, the flexible nature of disordered regions of proteins may allow efficient interaction with several targets [59] HCV Core+1/S proteins tend to self-associate The deconvolution of Core+1/S CD spectra suggested the presence of a significant proportion of b-sheet secondary structures (30%), in disagreement with the NMR-derived SSP A first hypothesis to explain this is the difference in concentration range used to obtain CD and NMR data However, the position and FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS A Boumlic et al bandwidth of peaks from HSQC spectra recorded with 30 or 400 lm HCV-1b Core+1/S samples are strictly identical (data not shown), suggesting the absence of a concentration effect, at least in this concentration range On the other hand, the fact that the NMR technique is a very powerful method, allowing recording of data at an atomic level, raises the question of potential problems with experimental CD data collection and/or inappropriate reference databases used to fit the CD data First, CD data were collected and analyzed following the key considerations well described by Greenfield [60], allowing us to reasonably rule out data collection issues, although they are not fully excluded Second, the reference databases are derived from globular soluble proteins, and include only a few disordered proteins For instance, the SDP48 reference database employed in the present study contains only five denaturated proteins in a total of 48 proteins Therefore, the use of these databases for nonglobular proteins is not really appropriate, as peptides or disordered proteins tend to adopt multiple conformations in equilibrium rather than a single structure Although the CD results might overestimate the b-sheet content, both CD and NMR data qualitatively indicate a b-sheet secondary structure propensity This observation suggests that the detected b-sheet signal could be due to partial oligomerization of the natively disordered HCV Core+1/S proteins This hypothesis is also supported by the DLS results, which reveal the existence of relatively high molecular mass particles in protein samples, although previously purified in a monomeric form by size exclusion chormatography The residues involved in such oligomerization might be located in the core of the protein between Ile33 and Val35, as suggested by the chemical shift deviations from random coil values Despite their lack of folded and globular structure, intrinsically disordered states of proteins often possess significant amounts of transient structure [47] Biophysical characterization of HCV ARFP/Core+1/S has been found to be associated with membranes [48] The influence on Core+1/S behavior of OG, a nonionic detergent known to solubilize integral membrane proteins, was therefore investigated further DLS showed that OG micelles reduce Core+1/S dispersity Moreover, the CD spectrum showed a change on the addition of the detergent Finally, HSQC experiments showed that only a few residues are affected by the presence of OG Taken together, these results are indicative of a possible weak interaction with the detergent, as is often observed for IDPs However, further experimental data on the structural characterization of a putative interaction between Core+1/S proteins and membranes and comparison with the membrane association properties of the Core protein would be required The presence of circulating antibodies against the HCV Core+1/S proteins suggests that their expression might occur at a certain stage of HCV infection Furthermore, the facts that Core+1/S proteins are disordered under native conditions, and that their ORFs are well conserved among HCV genotypes, support the hypothesis that the disordered nature of Core+1/S proteins might have some roles during HCV infection The disordered nature of the Core+1/ S proteins, which confers conformational and recognition plasticity to the proteins, may be required for the binding of different partners through the same region, as is typical for natively disordered proteins [59] This feature is often found for proteins involved in cell signaling and regulation [44] Our study contributes to the characterization of the Core+1/S proteins, providing new insights into their biophysical properties Further studies will be required to identify the cellular targets of Core+1/S proteins, enabling the characterization of the role of Core+1/S proteins in HCV pathogenicity Experimental procedures Biological roles of Core+1/S proteins Most HCV proteins contain membrane anchor domains [61] The presence of hydrophobic patches on Core+1/S protein sequences supports the hypothesis that the proteins might contain membrane association determinants, which may partially explain the polydisperse behavior of the protein in aqueous solution Interestingly, confocal microscopy and Triton X-100 cell fractionation have previously demonstrated that HCV-1a Core+1/S localizes in internal membranes and the endoplasmic reticulum of transiently transfected Huh7 cells [25] Furthermore, the Core protein itself Protein sequence analysis To analyze the degree of conservation of the Core+1/S amino acid sequence among HCV genotypes, Core+1/S amino acid sequences were deduced from the Core+1 ORFs of different HCV genotypes retrieved from the NCBI website (http://www.ncbi.nlm.nih.gov) [5] and aligned using clustalw [62] Prediction of intrinsic disorder in proteins was performed using globplot [63] and pondr [64] Secondary structure predictions were performed on HCV-1a and HCV-1b Core+1/S, using four algorithms (sopma, gor4, simpa96, and phd [65–68]) available on the IBCP website (http://pbil.ibcp.fr/) FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 783 Biophysical characterization of HCV ARFP/Core+1/S A Boumlic et al Plasmid construction Cloning was performed following standard methods, and plasmids were verified by sequencing For the cloning of Core+1/S proteins, the fragments corresponding to residues 85–161 of the HCV-1a Core+1 ORF and residues 85–144 of the HCV-1b Core+1 ORF were obtained by PCR, using as templates plasmid path 10/17-38 and pRSV/ AT (kindly provided by M J Beach, CDC, Atlanta, GA, ´ USA, and C Brechot, INSERM U785, Paris, France, respectively) [69], respectively Primers used were as follows: HCV-1a Core+1/S sense, 5¢-ATC CGG GGT CTC CCATG GCA ATG AGG GCT GCG GGT G-3¢; HCV-1b Core+1/S sense, 5¢-ATC CGG GGT CTC CCATG GCA ATG AGG GCC TGG GGT G-3¢; HCV-1a Core+1/S antisense, 5¢-AT CCG GGT CTC GGTACC TTA TCA CGC CGT C TT CCA GAA C-3¢; and HCV-1b Core+1/S antisense, 5¢-AT CCG GGT CTC GGTACC CTA GGG GGG CGCC G A CG-3¢ (italic indicates BsaI sites; underlined sequences correspond to NcoI sites for sense primers, and Acc65I sites for antisense primers) PCR fragments were digested with BsaI, and cloned into the NcoI and Acc65I sites of pETm-60 (a gift from G Stier, EMBL, Heidelberg, Germany) This vector is a modified pET24d expression vector (Novagen, Darmstadt, Germany) containing an N-terminal His6-NusA tag, followed by a TEV-protease sensitive linker For HCV-1b Core+1 antigen preparation, the cDNA fragment corresponding to residues 42–142 of the HCV-1b Core+1 ORF was amplified using a template plasmid ´ pRSV/BNT (kindly provided by C Brechot) [69] and the following primers: sense, 5¢-CAT GCC ATG GCA CCA ACC GCC GCC CAC A-3¢; and antisense, 5¢-CCC AAG CTT GGG GGG CGC CGA CAA GC-3¢ (underlined sequences indicate NcoI and HindIII sites, respectively) The PCR product was inserted into the NcoI–HindIII sites of the pET20b(+) expression vector (Novagen) fused to a His6 tag Production of unlabeled and 15N–13C-labeled Core+1/S proteins in native conditions E coli BL21(DE3) bacteria were transformed with the constructs corresponding to His6-NusA–Core+1/S of HCV-1a and HCV-1b Overnight cultures of freshly transformed cells were diluted 40-fold in L of LB or M9 medium containing antibiotics, and incubated at 37 °C until D600 nm reached 0.7 Expression was induced by the addition of 0.5 mm isopropyl thio-b-d-galactoside, and cells were incubated at 15 °C overnight Bacteria were harvested by centrifugation for 15 at 3000 g, and resuspended in buffer A (20 mm sodium phosphate, pH 6.8, 400 mm NaCl, 50 mm arginine, and 50 mm glutamic acid [30]) containing 2.5 lgỈmL)1 DNase I, 2.5 lgỈmL)1 RNase, and antiproteases All purification steps were performed at °C To minimize oxidation effects, all buffers were degassed using a vacuum pump, and then 784 bubbled extensively with argon Cells were sonicated on ice, and lysates were then centrifuged at 16 000 g and °C for 45 For purification by immobilized metal ion affinity chromatography (IMAC), the supernatants were filtered and loaded onto a column containing Ni2+–nitrilotriacetic acid resin (Qiagen, Courtaboeuf, France) pre-equilibrated with buffer A supplemented with 10 mm imidazole and one tenth of the antiprotease concentration recommended by the manufacturer The column was washed with buffer A supplemented with 10 mm imidazole, and then with buffer A supplemented with 20 mm imidazole The proteins were eluted with buffer A containing 250 mm imidazole After desalting, the protein was mixed with recombinant TEV protease at a ratio of 10)2 mol of TEV protease per mol of NusA fusion Incubation was performed at 20 °C for h to achieve cleavage of Core+1/S protein from NusA, leading to the addition of glycine and alanine residues upstream of the regular Core+1/S sequence To eliminate protein aggregates, protein solutions were centrifuged for 16 h at 160 000 g and 10 °C [70] Supernatants corresponding to soluble species were concentrated and loaded onto a Hiload 16/ 60 Superdex 75 size exclusion chromatography resin (GE Healthcare, Orsay, France) pre-equilibrated with buffer A Samples were concentrated using kDa cutoff concentrators (Sartorius, Goettingen, Germany), and protein concentrations were measured by absorbance at 280 nm using extinction coefficients of 23 500 and 16 500 m)1Ỉcm)1 for HCV-1a and HCV-1b Core+1/S, respectively Core+1/S samples were stored in buffer A supplemented with sodium azide (Sigma, Saint-Quentin Fallavier, France) and antiproteases All biophysical analyses were performed on freshly purified proteins When required, HCV-1a and HCV-1b Core+1/S proteins were incubated in the presence of OG (Sigma) The OG was used at a concentration 10 times higher than the critical micellar concentration, corresponding to a final concentration of 6% For chaotropic assays, samples were either incubated at 100 °C for 20 and then allowed to cool to room temperature overnight, or incubated in or 10 m urea prior to analysis Antisera The rabbit polyclonal antibody that specifically recognizes the C-terminal part of HCV-1a Core+1 was described previously [24] Similarly, rabbit polyclonal antibody directed against HCV-1b Core+1 was produced in rabbits, using HCV-1b Core+1 antigen conjugated to complete Freund’s adjuvant (Sigma), and used to immunize rabbits according to a classic immunization protocol [71] Antisera were collected weeks after the last booster, and used in western blot analysis SDS/PAGE and western blotting Proteins were separated on Tricine SDS gels [72] and stained with Coomassie blue For western immunoblotting, FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS A Boumlic et al purified proteins were subjected to Tricine SDS/PAGE and electrotransferred onto nitrocellulose membranes (Whatman, Maldstorm, UK) The membranes were incubated with blocking solution (7% nonfat dry milk and 0.05% Tween-20 in NaCl/Pi) for h at room temperature Subsequently, the membrane was incubated overnight at °C with the antibody against Core+1 (HCV-1a or HCV-1b) (1 : 500) in 1% semiskimmed dry milk and 0.05% Tween20 in · NaCl/Pi After three washes with NaCl/Pi/Tween, the membranes were incubated for h at room temperature with an enhanced chemiluminescence peroxidase-conjugated anti-rabbit secondary antibody (1 : 20 000; GE Healthcare) diluted in 1% nonfat dry milk and 1% NP40 in · NaCl/ Pi After three washes with NaCl/Pi/NP40 and three washes with NaCl/Pi, bound antibodies were detected using the enhanced chemiluminescence kit (GE Healthcare) according to the manufacturer’s protocol MS analysis Samples were diafiltered against 100 mm ammonium acetate at pH 7.0, and subsequently diluted in a : water/acetonitrile (v/v) mixture acidified with 1% formic acid to achieve a concentration of pmolỈmL)1 MS studies were performed on an ESI-TOF mass spectrometer fitted with a standard Z-spray source (LCT, Waters, MA, USA) Sample solutions were introduced into the mass spectrometer source with a syringe pump (Harvard Type 55 1111; Harvard Apparatus, South Natick, MA, USA) at a flow rate of lLỈmin)1 Calibration was achieved in the positive ion mode, using denaturated horse heart myoglobin (Sigma) Biophysical characterization of HCV ARFP/Core+1/S were placed in a quartz cuvette maintained at 20 °C Fluorescence was measured by exciting the sample at 280 nm and recording the emission spectrum from 300 to 400 nm Spectra were systematically corrected for fluctuations in lamp intensity and for background contributions (buffer without or with detergent) DLS DLS experiments were performed at 20 °C using a DynaPro instrument (Protein Solutions; Wyatt Technology Corporation, Santa Barbara, CA, USA) Solutions of purified HCV-1a and HCV-1b Core+1/S proteins were concentrated up to mgỈmL)1 by ultrafiltration (5 kDa cutoff) Aliquots were incubated with OG for h at 25 °C, or subjected to chaotropic agents Prior to measurement, samples were centrifuged for 15 at 16 000 g in a benchtop centrifuge At least 10 measurements, each of 10 s duration, were made for each sample Extreme care was taken to reduce contamination of samples by dust, and buffer alone was systematically measured to check the presence of dust To calculate hydrodynamic radii of particles, scattering data were analyzed using dynals, provided by the manufacturer The contribution of low molecular mass particles was filtered out Finally, the size distributions were fitted with a three-parameter Gaussian model, using matlab (The Mathworks Inc., Natick, MA, USA), in order to determine average hydrodynamic radii and polydispersities (defined as the ratio of the standard deviation to the average hydrodynamic radius) When dynals yielded only two particle sizes in the distribution, the fit was not performed, owing to the reduced number of populations In such a case, the weighted average only was calculated Human sera and ELISA Microplate wells were coated with lgỈmL)1 HCV-1b Core +1/S and incubated with 100 lL of diluted human serum (10 HCV-1-infected patient and 10 HCV-negative serum samples) at 37 °C for h The plates were washed and subsequently incubated with 100 lL of peroxidase-conjugated affinity-purified goat anti-human IgG (Dako Cytomation) at 37 °C for h The wells were washed again, and allowed to react with tetramethyl benzidine buffer (ThermoFisher, Illkirch, France) The reaction was then analyzed at 450 nm Intrinsic fluorescence spectroscopy Measurements were made using a SPEX Fluorolog-2 spectrofluorimeter (SPEX Industries, Inc., Edison, NJ, USA) equipped with a 450 W xenon lamp, a double-grating excitation monochromator, and a single-grating emission monochromator Data were acquired with a photoncounting photomultiplier (linear up to 107 counts per s), with high voltages fixed at 800 V Slit widths were adjusted to mm for both excitation and emission Samples of lm CD Far-UV CD measurements were performed with a JobinYvon spectropolarimeter, equipped with a temperaturecontrolled water bath and calibrated with ammonium d-10-camphorsulfonate Spectra were acquired at 20 °C, with a constant bandwidth of nm and a 3–5 s integration time Spectra were recorded using a quartz cell of path length 0.2 mm Protein concentrations were lm in 20 mm sodium phosphate (pH 6.8), 50 mm NaCl, and 0.15 mm dithiothreitol Spectra were averaged over three to six scans, and corrected for buffer contributions When possible, quantitative estimations of the secondary structure contents were performed using the cdpro program suite [35], which includes three methods (contin/ll, cdsstr, and selcon3) Three reference protein sets were used: SP43 (43 globular proteins), SMP56 (SP43 plus 13 membrane proteins), and SDP48 (SP43 plus five denatured proteins) In order to determine the variability of secondary structure content, several CD experiments were performed in duplicate or triplicate, and the data were FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 785 Biophysical characterization of HCV ARFP/Core+1/S A Boumlic et al then deconvoluted The typical range of variation was ± 10–20% NMR spectroscopy Samples for NMR spectroscopy were prepared in buffer A supplemented with 7% D2O Spectra were acquired at 600 MHz and 25 °C on a Bruker DRX600 spectrometer equipped with a z-gradient triple resonance cryoprobe Data were processed using nmrpipe, [73] and analyzed with cara [74] A 15N-labeled Core+1/S sample was used to record 2D 1H–15N-HSQC correlation spectra Core+1/S backbone and b-carbon resonances were assigned by using a 400 lm 15N,13C-labeled Core+1/S 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D402–D408 76 Wishart DS & Sykes BD (1994) The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data J Biomol NMR 4, 171–180 Biophysical characterization of HCV ARFP/Core+1/S Fig S2 2D NMR spectra of HCV-1a Core+1/S protein This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors Supporting information The following supplementary material is available: Fig S1 Expression and purification screenings of native NusA-HCV-1a Core+1/S proteins FEBS Journal 277 (2010) 774–789 ª 2010 The Authors Journal compilation ª 2010 FEBS 789 ... GGT CTC CCATG GCA ATG AGG GCT GCG GGT G-3¢; HCV-1b Core+1/S sense, 5¢-ATC CGG GGT CTC CCATG GCA ATG AGG GCC TGG GGT G-3¢; HCV-1a Core+1/S antisense, 5¢-AT CCG GGT CTC GGTACC TTA TCA CGC CGT C TT... Darbon H, Canard B, Finet S & Longhi S (2005) The intrinsically disordered C- terminal domain of the measles virus nucleoprotein interacts with the C- terminal domain of the phosphoprotein via two... antisense, 5¢-CCC AAG CTT GGG GGG CGC CGA CAA GC-3¢ (underlined sequences indicate NcoI and HindIII sites, respectively) The PCR product was inserted into the NcoI–HindIII sites of the pET20b(+)