Easy-to-Attach/Detach Solubilizing-Tag-Aided Chemical Synthesis of an Aggregative Capsid Protein.

A solubilizing Trt-K10 tag was developed for the effective chemical preparation of peptides/proteins with low solubility. The Trt-K10 tag comprises a hydrophilic oligo-Lys sequence and a trityl anchor, and can be selectively introduced to a side chain thiol of Cys of deprotected peptides/proteins with a trityl alcohol-type introducing reagent Trt(OH)-K10 under acidic conditions. Significantly, the ligation product in the reaction mixture of a thiol-additive-free native chemical ligation can be modified directly in a one-pot manner to facilitate the isolation of the product by high-performance liquid chromatography. Finally, the Trt-K10 tag can be readily removed with a standard trifluoroacetic acid cocktail. Using this easy-to-attach/detach tag-aided method, a hepatitis B virus capsid protein that is usually difficult to handle was synthesized successfully.

Total chemical synthesis of dengue 2 virus capsid protein via native chemical ligation: role of the conserved salt-bridge.

The dengue capsid protein C is a highly basic alpha-helical protein of ~100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55'. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C ((WT)DEN2C) and its salt bridge-void analog (E87A)DEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.

In vivo encapsulation of nucleic acids using an engineered nonviral protein capsid.

In Nature, protein capsids function as molecular containers for a wide variety of molecular cargoes. Such containers have great potential for applications in nanotechnology, which often require encapsulation of non-native guest molecules. Charge complementarity represents a potentially powerful strategy for engineering novel encapsulation systems. In an effort to explore the generality of this approach, we engineered a nonviral, 60-subunit capsid, lumazine synthase from Aquifex aeolicus (AaLS), to act as a container for nucleic acid. Four mutations were introduced per subunit to increase the positive charge at the inner surface of the capsid. Characterization of the mutant (AaLS-pos) revealed that the positive charges lead to the uptake of cellular RNA during production and assembly of the capsid in vivo. Surprisingly, AaLS-pos capsids were found to be enriched with RNA molecules approximately 200-350 bases in length, suggesting that this simple charge complementarity approach to RNA encapsulation leads to both high affinity and a degree of selectivity. The ability to control loading of RNA by tuning the charge at the inner surface of a protein capsid could illuminate aspects of genome recognition by viruses and pave the way for the development of improved RNA delivery systems.

Interaction of synthetic HPV-16 capsid peptides with heparin: thermodynamic parameters and binding mechanism.

Capsid proteins binding cell surface proteoglycans is a key early event in human papillomavirus (HPV) infection. The positively charged sequences at the C-terminus of the L1 protein and the N- and C-termini of the L2 protein of HPV-16 can efficiently bind to heparin receptors, which were characterized in the present study by quantitative isothermal titration calorimetry experiments primarily, fluorescence spectroscopy, and static right-angle light scattering. The binding constant, K, was at an order of magnitude of 10(7) M(-1) for the two peptides at the N- and C-termini of HPV-16 L2 and segment b at the C-terminus of HPV-16 L1, while that for other L1 analogues were of a smaller order, illustrating that the heparin binding is a typical sequence-specific and -dependent phenomenon. These results suggest that, in addition to L1, the L2 protein may participate in cell surface attachment during HPV infection. Furthermore, the calorimetry results demonstrated that hydrophobic interactions and hydrogen bonding are involved in peptide binding to heparin in addition to the essential electrostatic interactions. Meanwhile, circular dichroism spectroscopy revealed that binding to heparin does not induce obvious secondary structural changes in the peptides.

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

[Features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modeling antigenic determinants of hepatitis A virus].

AIM: To study features of antigen-antibody interaction during use of linear synthetic peptides and multipeptide antigen modelling antigenic determinants of hepatitis A virus (HAV) and to evaluate perspectives for use of heterogeneous tetrameric multipeptide antigens for detection of HAV serological markers. MATERIALS AND METHODS: Linear peptides VP1 and VP3 were synthesized by fluorenylmethyloxycarbonyl (Fmoc)-polyamide solid phase method. MAP4 (VP1+VP3) was synthesized according to 9-Fmoc strategy. Interaction of these peptides with anti-HAV IgM positive sera from patients with HA was studied by noncompetitive and competitive methods of immunoenzyme assay. RESULTS. Using immunoenzyme assay, high heterogeneity of immune response in patients with HA (62 and 67% in two groups) was shown. MAP4 (VP1+VP3), unlike the combination of linear peptides VP1 and VP3, interacted with anti-HAV IgM in 41 - 45% of sera and, at the same time, did not lead to false positive results. CONCLUSION: Population of HAV is not so uniform which is usually assumed. It could be reasonable to use heterogenous multipeptide antigens, including those containing VP1 (11 - 25 a.r.) and VP3 (110 - 121 a.r.), for the development of new assays for HA diagnostics.

Genome-free viral capsids as carriers for positron emission tomography radiolabels.

PURPOSE: We have developed a modular synthetic strategy to append imaging agents to a viral capsid. PROCEDURES: The hollow protein shell of bacteriophage MS2 (mtMS2) was labeled on its inside surface with [18F]fluorobenzaldehyde through a multistep bioconjugation strategy. An aldehyde functional group was first attached to interior tyrosine residues through a diazonium coupling reaction. The aldehyde was further elaborated to an alkoxyamine functional group, which was then condensed with n.c.a. [18F]fluorobenzaldehyde. Biodistribution of the radioactive MS2 conjugates was subsequently evaluated in Sprague-Dawley rats. RESULTS: Relative to fluorobenzaldehyde, fluorine-18-labeled MS2 exhibited prolonged blood circulation time and a significantly altered excretion profile. It was also observed that additional small molecule cargo installed inside the capsids did not alter the biodistribution. CONCLUSIONS: These studies provide further insight into the pharmacokinetic behavior of nanomaterials and serve as a platform for the future development of targeted imaging and therapeutic agents based on mtMS2.

Identification of an HLA-A*0201-restricted cytotoxic T-lymphocyte epitope in rotavirus VP6 protein.

The function of cytotoxic T lymphocytes (CTLs) in rotavirus (RV) infection in humans is poorly understood. To date, no RV-specific human leukocyte antigen (HLA) class I-restricted T-cell epitopes have been described. In this study, four peptides derived from human RV Wa strain VP6 protein were predicted by computer algorithms and verified by an HLA*0201-binding assay. Two peptides with high affinity for HLA-A*0201 molecules were further assessed. The CTLs induced in vitro by P340-348 (TLLANVTAV)-loaded autologous dendritic cells from peripheral blood lymphocytes of HLA-A*0201-matched healthy donors released gamma interferon specifically upon stimulation with P340-348-loaded T2 cells. The CTLs lysed both P340-348-loaded T2 cells and human RV Wa strain-infected HLA-A*0201(+) Caco-2 cells in an antigen-specific and HLA-A*0201-restricted manner. At the same time, P340-348 was shown to be immunogenic in vivo in HLA-A*0201/Kb transgenic mice. It is proposed that P340-348 is an HLA-A*0201-restricted CTL epitope.

SUBPOL: a novel SUcrose-Based Polymer support for solid-phase peptide synthesis and affinity chromatography applications.

A novel SUcrose-Based Polymer support (SUBPOL) with tailored morphology suitable for the use in solid-phase peptide synthesis (SPPS) is described, and its application as a hydrophilic affinity matrix for the specific removal of fibrinogen from human plasma is demonstrated. After suspension polymerization of partly methacrylated 2,1':4,6-di-O-isopropylidene sucrose and subsequent removal of the protecting groups, hydrophilic spherical polymer beads were obtained. The morphology of the resulting resin was controlled by variation of the porogen as well as the average degree of substitution with respect to the methacryloyl groups of the monomer mixture. After introduction of amino groups for a permanent attachment of immobilized peptide ligands, prevention of unintended esterification during SPPS was achieved by silylation of remaining hydroxy groups. Alternatively, a Rink amide linker was introduced prior to SPPS to allow cleavage and subsequent analysis of the peptide assembled on the SUBPOL resins. Two hexapeptides of sequence kwiivw and hffflw, consisting of d-amino acids, as well as a 19-mer peptide corresponding to the sequence GSGVRGDFGSLAPRVARQL of the VP1 protein from the foot-and-mouse disease virus (FMDV) were successfully prepared both manually or in a semi-automated process on SUBPOL resins according to the Fmoc/tBu strategy. Yields and purities were comparable to peptides prepared on commercially available polystyrene resins. A specific affinity adsorbent containing the fibrinogen-binding pentapeptide GPRPK was prepared by SPPS on SUBPOL resins of different morphology, and the strong impact of the affinity matrix on adsorption performance was demonstrated.