Synthesis and immunochemical properties of oligopeptides--fragments of capsid proteins of the hepatitis A virus]

The hepatitis A virus (HAV) capsid protein VP1, VP2 and VP3 are exposed at the virion surface and should therefore contain antigenic determinants. Algorithms for hydrophilicity, antigenicity and flexibility were used to predict probable antigenic sites. Synthesis of 7- to 23-membered overlapping peptides from seven sites, viz., 1-11, 1-17, 2-33, 11-25, 73-82, 76-86, 98-109, 98-112, 102-107, 102-108, 108-127, 113-123, 118-140, 276-298 from VP1, 42-62 from VP2, 76-85 from VP3, and 1-23 from VP4, was performed by various solid-phase methods. Free peptides and their conjugates with different carriers were used for immunization and study of antigenicity. The peptides did not interact with antibodies to the hepatitis A virus, whereas their conjugates did not induce the formation of anti-HAV-antibodies.     

Global Displacement of Canine Parvovirus by a Host-Adapted Variant: Structural Comparison between Pandemic Viruses with Distinct Host Ranges

Canine parvovirus type 2 (CPV-2) emerged in 1978 and spread worldwide within 2 years. Subsequently, CPV-2 was completely replaced by the variant CPV-2a, which is characterized by four specific capsid (VP2) mutations. The X-ray crystal structure of the CPV-2a capsid shows that each mutation confers small local changes. The loss of a hydrogen bond and introduction of a glycine residue likely introduce flexibility to sites that control interactions with the host receptor, antibodies, and sialic acids.     

猪水疱病病毒外壳蛋白的二级结构和抗原表位预测

以SVDV外壳蛋白基因序列为基础,采用Chou-Fasman法、Garnier-Robson 法和Karplus-Schulz法预测蛋白质的二级结构;按Kyte-Doolittle方案、Emini方案和Jameson-Wolf方案预测SVDV外壳蛋白的B细胞表位。预测结果表明,SVDV外壳蛋白的二级结构较为复杂,含有较多的转角和无规则卷曲等柔性区域以及α-螺旋和β-折叠区段;SVDV外壳蛋白的VP1、VP2和VP3上均有多个区域为B细胞优势表位,其中,VP1蛋白的B细胞表位优势区域比VP2和VP3蛋白的多,与已鉴定的B细胞表位相比较,该方法预测的结果有较高的准确度。为实验确定SVDV外壳蛋白的B细胞表位和反向疫苗学设计提供理论基础。     

A BASIC microcomputer program for prediction of B and T cell epitopes in proteins

A BASIC microcomputer program (EPIPLOT) has been developed forpredicting B and T cell antigenic sites in proteins from theirprimary structures. The program calculates and plots flexibility,hydrophilicity and antigenicity profiles using 13 differentscales, chosen as those yielding the best predictions on proteinswhose antigenic structures are known. T cell epitope predictionis basedon published algorithms focused on amphiphilic structuresand characteristic sequence patterns. The advantages of jointpredictions in locating T cell antigenic sites are also discussed. Received on December 12, 1989; accepted on January 31, 1990     

Structure and inherent properties of the bacteriophage lambda head shell. V. Amber mutants in gene E

A total of 940 amber mutants in gene E of bacteriophage lambda was isolated to study the structure-function relationship of the gene product, the major capsid protein. The mutants were mapped to 43 mutation sites, most of which have been located, albeit tentatively, at exact points in the known base sequence, by deletion mapping and by the specificity of mutagenesis and the patterns of suppression. The patterns of suppression were interpreted in terms of both the efficiency of insertion of amino acid residues by suppressors and the exchangeability of amino acid residues. The exchangeability seems to be related to the hydrophilicity of the residues themselves and their environment, as well as to the functional similarity between the replaced and the inserted amino acid residues. Suppression of two of the mutations resulted in the production of characteristic aberrant head-related structures, each showing a defect in a different functional site in the protein. This, together with the approximate positions of some specific missense mutations as determined in this study, revealed the distribution of the functional sites along the polypeptide chain of the gene E product.     

Structural and physico-chemical properties of amino acids of functionally important regions of proteins

A correlation of the localization of functionally important regions with places having low and high values on twelve profiles built on a basis of amino acid sequences was analysed using a broad set of proteins. The profiles of hydrophilicity, resemblance to the sequences of human proteins, flexibility, mutability and others were considered. The resemblance profile was plotted by the program fixing short similar fragments in the testing protein and 92 human ones. The active centres were shown to be located in the primary structure regions having relatively low values on the resemblance profiles. Similar effect was observed in the mutability and alpha-helicity profiles. The potential functionally important sites of the human leukocyte interferon and interleukin-2 isolated on the basis of the analysis of this profiles were in accord with the available literary data.     

Characterization of immune response to synthetic peptides derived from the hemagglutinin of measles virus

We describe here four synthetic peptides derived from the hemagglutinin of measles virus. The peptides were predicted by a computer program combining hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus hemagglutinin. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using immunostimulating complex as adjuvant. Anti-peptide antisera raised in rabbits against the peptide conjugates reacted well with the homologous peptides and with measles virus antigen as tested with plate ELISA. None of these sera had either neutralizing or hemagglutination inhibiting antibody or reacted with measles hemagglutinin protein in Western blot and reacted weakly in immunofluorescence. Human sera positive for measles virus antibody reacted with the synthesized peptides indicating that the selected locations function as partial antigenic sites.     

Predict7, a program for protein structure prediction

We describe a program for protein sequence analysis which runs in IBM PC computers. Protein sequences are loaded from files in Mount-Conrad and Lipman-Pearson format. Seven features are analyzed: hydrophilicity, hydropathy, surface probability, side chain flexibility, antigenicity, secondary structure and N-glycosylation sites. Numeric results can be shown, printed or stored in files exportable to other programs. Graphics of up to four predictions can be displayed on the screen, printed out or plotted, with several definable options. This program has been designed to be fast, user-friendly and to be shared with the scientific community.     

Fitness Costs of Mutations at the HIV-1 Capsid Hexamerization Interface

The recently available x-ray crystal structure of HIV-1 capsid hexamers has provided insight into the molecular interactions crucial for the virus’s mature capsid formation. Amino acid changes at these interaction points are likely to have a strong impact on capsid functionality and, hence, viral infectivity and replication fitness. To test this hypothesis, we introduced the most frequently observed single amino acid substitution at 30 sites: 12 at the capsid hexamerization interface and 18 at non-interface sites. Mutations at the interface sites were more likely to be lethal (Fisher’s exact test p = 0.027) and had greater negative impact on viral replication fitness (Wilcoxon rank sum test p = 0.040). Among the interface mutations studied, those located in the cluster of hydrophobic contacts at NTD-NTD interface and those that disrupted NTD-CTD inter-domain helix capping hydrogen bonds were the most detrimental, indicating that these interactions are particularly important for maintaining capsid structure and/or function. These functionally constrained sites provide potential targets for novel HIV drug development and vaccine immunogen design.     

Phosphorylation of the duck hepatitis B virus capsid protein associated with conformational changes in the C terminus.

The capsid protein of duck hepatitis B virus (DHBV) is phosphorylated at multiple sites during viral infection. A cluster of sites is located near the C terminus of the 262-amino-acid protein. We have used site-directed mutagenesis to show that three serines and one threonine serve as phosphate acceptor amino acids in the C terminus. An additional six potential phosphate acceptor sites in this region were apparently not utilized. Each serine or threonine that served as a phosphate acceptor was adjacent to a downstream proline, while all six serines that were not acceptors for phosphate residues lacked adjacent downstream prolines. Mutation of the downstream proline to glycine at each site had the same effect as mutating the serine itself, suggesting an SP or TP motif as an essential feature for capsid protein phosphorylation. Phosphorylation at these four sites resulted in complex shifts in electrophoretic mobility in sodium dodecyl sulfate gels of the capsid protein or of a C-terminal peptide containing the phosphorylated sites, suggesting that specific conformations of the C terminus are associated with different combinations of phosphorylated serines. We speculate that distinct functions of the C terminus may be associated with different phosphorylated domains on the intact capsid.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Genetic incorporation of a herpes simplex virus type 1 thymidine kinase and firefly luciferase fusion into the adenovirus protein IX for functional display on the virion

An advantage of the adenoviral vector is its molecular flexibility, which allows for vector tropism modifications for the purpose of cell targeting. In addition to targeting ligands, the capacity to incorporate heterologous peptides has allowed capsid incorporation of other functionalities. We have defined the minor capsid protein IX (pIX) as a locus capable of presenting incorporated ligands on the virion surface. Thus, we sought to exploit the possibility of incorporating functional proteins at pIX. In our current study, we sought to expand the potential utility of our capsid labeling strategy by developing simultaneous imaging capacity for dedicated small animal positron emission tomography and bioluminescence imaging on a single adenoviral vector. Therefore, we constructed an adenovirus that incorporates a fusion protein of herpes simplex virus type 1 thymidine kinase and firefly luciferase (Luc) (TK-Luc) into adenovirus capsid pIX. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional TK-Luc as a component of their capsid surface. Most importantly, Ad-pIX-TK-Luc retained dual enzymatic functions in vitro and in vivo. This dual-modality approach will allow dynamic or real-time imaging analysis of adenovirus-based interventions with maximized analytic flexibility and enhanced resolution potential.     

Functional requirements of the yellow fever virus capsid protein

Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.     

Structural polymorphism of the major capsid protein of a double-stranded RNA virus: an amphipathic alpha helix as a molecular switch

The infectious bursal disease virus T=13 viral particle is composed of two major proteins, VP2 and VP3. Here, we show that the molecular basis of the conformational flexibility of the major capsid protein precursor, pVP2, is an amphipatic alpha helix formed by the sequence GFKDIIRAIR. VP2 containing this alpha helix is able to assemble into the T=13 capsid only when expressed as a chimeric protein with an N-terminal His tag. An amphiphilic alpha helix, which acts as a conformational switch, is thus responsible for the inherent structural polymorphism of VP2. The His tag mimics the VP3 C-terminal region closely and acts as a molecular triggering factor. Using cryo-electron microscopy difference imaging, both polypeptide elements were detected on the capsid inner surface. We propose that electrostatic interactions between these two morphogenic elements are transmitted to VP2 to acquire the competent conformations for capsid assembly.     

轮状病毒LLR株VP7抗原表位的预测与验证

在GenBank中检索A组轮状病毒不同血清型的VP7基因信息,在氨基酸水平上与G10血清型LLR株的VP7序列进行序列对比,分析其血清型特异的氨基酸保守序列位点。结合蛋白质二级结构预测理论方案,设计合成3条具有轮状病毒G10血清型特异性氨基酸序列的多肽。通过检测合成肽对轮状病毒免疫血清与LLR抗原的结合抑制,证实三条多肽均具备了LLR表位属性。     

Fluorescent T7 display phages obtained by translational frameshift

Lytic phages form a powerful platform for the display of large cDNA libraries and offer the possibility to screen for interactions with almost any substrate. To visualize these interactions directly by fluorescence microscopy, we constructed fluorescent T7 phages by exploiting the flexibility of phages to incorporate modified versions of its capsid protein. By applying translational frameshift sequences, helper plasmids were constructed that expressed a fixed ratio of both wild-type capsid protein (gp10) and capsid protein fused to enhanced yellow fluorescent protein (EYFP). The frameshift sequences were inserted between the 3′ end of the capsid gene and the sequence encoding EYFP. Fluorescent fusion proteins are only formed when the ribosome makes a −1 shift in reading frame during translation. Using standard fluorescence microscopy, we could sensitively monitor the enrichment of specific binders in a cDNA library displayed on fluorescent T7 phages. The perspectives of fluorescent display phages in the fast emerging field of single molecule detection and sorting technologies are discussed.     

Binding of spermidine and putrescine to energized liver mitochondria.

The binding of spermidine and putrescine to mitochondrial membranes was studied by applying a thermodynamic model of ligand-receptor interactions developed both for equilibrium and far-from-equilibrium binding processes (V. Di Noto, L. Dalla Via, A. Toninello, and M. Vidali Macromol. Theory Simul. 5, 165-181, 1996). Results demonstrate the presence of two monocoordinated binding sites (S1 and S2) for spermidine and one monocoordinated binding site (S2) for putrescine, all exhibiting high capacity and low affinity. It is proposed that differences in the polyamines' flexibility and hydrophilicity perhaps contributes to the observed variations in their interactions with the two sites. A comparison of the binding parameters of these polyamines with those of spermine reveals differences in the specific function of the S1 and S2 sites, identified in studies of spermine binding (L. Dalla Via, V. Di Noto, D. Siliprandi, and A. Toninello Biochim. Biophys. Acta 1284, 247-252, 1996).     

An antiviral compound that blocks structural transitions of poliovirus prevents receptor binding at low temperatures

Drugs such as WIN51711 that inhibit picornavirus replication are thought to block poliovirus infectivity by binding to the capsid and preventing structural transitions required for uncoating. We examined the activity of WIN51711 at temperatures where capsid flexibility is thought to be decreased. Below 37 degrees C, WIN51711 inhibits the binding of wild-type poliovirus to cells but does not affect the binding of a poliovirus mutant which is believed to undergo structural transitions more readily. These results suggest that the poliovirus capsid must undergo structural changes to bind to its cellular receptor.