1Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which 2 are involved in viral attachment and entry into target cells. We have obtained in 3 insect cells infected by recombinant baculovirus a chimeric secreted recombinant 4 protein, E1 341 E2 661, containing the ectodomains of E1 and E2. The described 5 procedure allows the purification of approximately 2 mg of protein from 1 L of 6 culture media. Sedimentation velocity experiments and SDS-PAGE in the absence 7 of reducing agents indicate that the protein has a high tendency to self-associate, the 8 dimer being the main species observed. All the oligomeric forms observed maintain 9 a conformation which is recognized by the conformation-dependent monoclonal 10 antibody H53 directed against the E2 ectodomain. The spectroscopic properties of 11 E1 341 E2 661 are those of a three-dimensionally structured protein. Moreover, the 12 chimeric protein is able to bind to human antibodies present in HCV-positive human 13 sera. Accordingly, this chimeric soluble polypeptide chain may be a valuable tool to 14 study the structure-function relationship of HCV envelope proteins. 15 16

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The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14-104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pK(a) determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO(-) ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species.

In this report it is described for the first time the expression and purification of large quantities of a soluble and correctly folded chimeric recombinant protein, E2 661 E1 340, containing the permuted Hepatitis C virus (HCV) glycoprotein ectodomains E1 (aminoacids 192-340) and E2 (aminoacids 384-661). Using the baculovirus/insect cell expression system, 8 mg of secreted protein were purified from 1 L of culture media, a yield 4 times higher than the described for its counterpart E1 341 E2 661 . This permuted chimeric protein is glycosylated and possesses a high tendency to selfassociate. The fluorescence emission spectrum indicates that Trp residues occupy a relatively low hydrophobic environment. The secondary structure was determined by deconvolution of the far-UV circular dichroism spectrum yielding 13% -helix structure, 49% extended structure and 38% non-ordered structure. E2 661 E1 340 binds to antibodies present in human sera from HCV-positive patients, a binding that is blocked at different levels by a rabbit anti-E2 661 antibody. All these structural and antigenic features of E2 661 E1 340 are very similar to those described for E1 340 E2 661 , Thus, this high-yield isolated chimeric protein may be a valuable tool to study the first steps of the HCV infection.

We have used an isolated chimeric protein E1 340 E2 661 that includes the ectodomains of the envelope proteins of hepatitis C virus to study its interaction with model membranes. E1 340 E2 661 has some of the membrane destabilization properties, vesicle aggregation, lipid mixing and the release of internal aqueous content, which have previously been ascribed to fusion proteins. The effects are preferentially produced on vesicles of acidic phospholipids which would indicate the importance of the electrostatic interactions. In fact, an increase of the ionic strength of the buffer induced a considerable decrease of the destabilizing properties. Moreover, fluorescence polarization studies show that the recombinant protein reduces the amplitude of the thermal transition of dimyristoylphosphatidylglycerol vesicles and increases the transition temperature at pH 5.0 in a dose-dependent manner, indicating its insertion into the bilayer. Furthermore, a decrease of the pH induces a conformational change in the protein structure as evidenced by fluorescence of tryptophan residues and 4,4 0 -bis(1-anilinonaphthalene-8-sulfonate). A model for the fusion of hepatitis C virus with the host cell membrane can be postulated. The dissociation of E1E2 dimers would uncover the fusion peptides which can then interact with the polar lipid heads of the outer leaflet of the lipid bilayer and next insert into the hydrophobic moiety producing the destabilization of the bilayer which finally leads to fusion.

BackgroundSome functions of 4.1R in non-erythroid cells are directly related with its distinct sub-cellular localisation during cell cycle phases. During mitosis, 4.1R is implicated in cell cycle progression and spindle pole formation, and co-localizes with NuMA1. However, during interphase 4.1R is located in the nucleus and only partially co-localizes with NuMA1.ResultsWe have characterized by NMR the structural features of the C-terminal domain of 4.1R and those of the minimal region (the last 64 residues) involved in the interaction with NuMA1. This subdomain behaves as an intrinsically unfolded protein containing a central region with helical tendency. The specific residues implicated in the interaction with NuMA1 have been mapped by NMR titrations and involve the N-terminal and central helical regions. The segment of NuMA1 that interacts with 4.1R is phosphorylated during mitosis. Interestingly, NMR data indicates that the phosphorylation of NuMA1 interacting peptide provokes a change in the interaction mechanism. In this case, the recognition occurs through the central helical region as well as through the C-terminal region of the subdomain meanwhile the N-terminal region do not interact.ConclusionsThese changes in the interaction derived from the phosphorylation state of NuMA1 suggest that phosphorylation can act as subtle mechanism of temporal and spatial regulation of the complex 4.1R-NuMA1 and therefore of the processes where both proteins play a role.

The steps leading from hepatitis C virus (HCV) attachment to the hepatocytes to the fusion of viral and cellular membranes remain uncharacterized. In this regard, we have studied the mechanism underlying the HCV fusion process using liposomes and a truncated form of E2 protein lacking the transmembrane region, E2 (amino acids 384-661). E2 has been previously obtained by using the baculovirus expression system and shown to behave as an independent folding domain (M. Rodriguez-Rodriguez, D. Tello, B. Yelamos, J. Gomez-Gutierrez, B. Pacheco, S. Ortega, A.G. Serrano, D.L. Peterson, F. Gavilanes, Structural properties of the ectodomain of hepatitis C virus E2 envelope protein, Virus Res. 139 (2009) 91-99). This form has been used in lipid-protein interaction studies with different model vesicles, at different pHs and by employing a variety of fluorescent assays. The obtained results indicate that E2 induces vesicle aggregation, lipid mixing and liposome leakage, reaching higher values in the presence of negatively charged phospholipids and cholesterol at acidic pH. Therefore, the results of these studies would be indicative of an HCV infection process through receptor mediated endocytosis. Accordingly, E2 might be important in the HCV initial infective steps, interacting with the target membranes and giving rise to their subsequent destabilization.

In this protocol, we describe the production and purification of the ectodomain of the E2661 envelope protein (amino acids 384-661) of the Hepatitis C virus, which plays a fundamental role in the entry of the virus into the host cell. This protein has been expressed in both prokaryotic and eukaryotic systems but in small quantities or without native protein characteristics. In our case, we use the Baculovirus expression system in insect cells. E2661 is secreted into the extracellular medium and purified by means of affinity chromatography a Ni-NTA-column because the protein has a tag of six histidines at its amino terminal end. The purified protein possesses a native-like conformation and it is produced in large quantities, around 5-6 mg per liter.