Minor Coat Protein Research Articles

Delineating the Site of Interaction on the pIII Protein of Filamentous Bacteriophage fd with the F-pilus of Escherichia coli

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F + cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.

Citrus tristeza virus ultrastructure and associated cytopathology in Citrus sinensis and Citrus aurantifolia

Citrus tristeza virus ultrastructure and associated cytopathology was documented with three isolates and two hosts, sweet orange 'Madam vinous' (Citrus sinensis (L.) Osbeck) and Mexican lime (Citrus aurantifolia (L.) Swingle). Virions were long, flexuous, and disorganized or in swirled, parallel masses. Infection was common in phloem parenchyma and companion cells and less frequent in mature sieve elements. Immunogold labeling confirmed previous findings that the major coat protein encapsidated the length of purified virions, while the minor coat protein encapsidated one terminal. Three types of inclusions were observed: (i) viral arrays that reacted with antibodies against the major (p25) and minor (p27) Citrus tristeza virus coat proteins, (ii) fibrous inclusions that reacted with antibodies against the Citrus tristeza virus p20 gene product but were sparsely labeled with antibodies against either coat protein, and (iii) accumulated cytoplasmic vesicles associated with aggregated, vesiculating mitochondria. The latter resembled Beet yellows virus-like vesicles, which are typical of closterovirus infection, but did not react with any of our antibodies. Cytopathology did not differ between isolates and plant hosts. Most effects were observed in phloem parenchyma cells, including chloroplast degradation, mitochondria vesiculation, and nuclear membrane invagination. Multivesicular bodies and lipid-filled vesicles were abundant in the cytoplasm. Masses of electron-lucent vesicles and electron-dense bodies were present between the cell membrane and cell wall.Key words: immunolocalization, CTV major coat protein, CTV minor coat protein, CTV p20 gene product, inclusions, isolate severity.

A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases.

In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3' end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3-Jun/Fos-Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.

Macromolecular import into Escherichia coli: the TolA C-terminal domain changes conformation when interacting with the colicin A toxin.

Various macromolecules such as bacteriotoxins and phage DNA parasitize some envelope proteins of Escherichia coli to infect the bacteria. A two-step import mechanism involves the primary interaction with an outer membrane receptor or with a pilus followed by the translocation across the outer membrane. However, this second step is poorly understood. It was shown that the TolA, TolQ, and TolR proteins play a critical role in the translocation of group A colicins and filamentous bacteriophage minor coat proteins (g3p). Translocation of these proteins requires the interaction of their N-terminal domain with the C-terminal domain of TolA (TolAIII). In this work, short soluble TolAIII domains were overproduced in the cytoplasm and in the periplasm of E. coli. In TolAIII, the two cysteine residues were found to be reduced in the cytoplasmic form and oxidized in the periplasmic form. The interaction of TolAIII with the N-terminal domain of colicin A (ATh) is observed in the presence and in the absence of the disulfide bridge. The complex formation of TolAIII and ATh was found to be independent of the ionic strength. An NMR study of TolAIII, both free and bound, shows a significant structural change when interacting with ATh, in the presence or absence of the disulfide bridge. In contrast, such a structural modification was not observed when TolAIII interacts with g3p N1. These results suggest that bacteriotoxins and Ff bacteriophages parasitize E. coli using different interactions between TolA and the translocation domain of the colicin and g3p protein, respectively.

New approach for generation of neutralizing antibody against human T-cell leukaemia virus type-I (HTLV-I) using phage clones

We have screened a phage peptide library to address whether clones binding to a monoclonal antibody (mAb) could be isolated and if the selected phage particles would be able to elicit an in vivo immune response against the original antigen. A phage peptide library, consisting of seven random amino acids inserted in the minor coat protein (pIII), was screened for specific binding to a rat mAb LAT-27, which is capable of neutralizing human T-cell leukaemia virus type-I (HTLV-I) by binding to its envelope gp46 epitope, (amino acids LPHSNL). Total 37 clones were selected from the library and one clone named 4-2-22 was tested for its immunogenicity in three rabbits. The all rabbit immune sera showed strong binding activity to a gp46 peptide carrying the neutralization sequence, stained gp46-expressing cells and neutralized HTLV-I in vitro as determined by cell fusion inhibition assay. These results show that the selected phage clone was capable of mimicking the epitope recognized by a HTLV-I neutralizing mAb, and it can be used as an immunogen to induce protective immune response against HTLV-I. Thus, the present methodology could be one of the approaches to develop vaccines against infectious agents in a simple and inexpensive way.

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

Translation at higher than an optimal level interferes with coupling at an intercistronic junction.

In pairs of adjacent genes co-transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single-stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild-type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine-Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.

Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage

Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a “secondary” library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.

Selection of phage-display library peptides recognizing ethanol targets on proteins

There is a forthcoming link between chronic alcohol consumption and proteins covalently modified by ethanol metabolites and their antibodies. To identify sensitive probes of protein–ethanol conjugates, we screened for the ethanol-altered protein domains with a phage-display combinatorial peptide library. In principle, recognition of the epitopes by the library peptides occurs through protein–protein interactions. A general screening, M13-based library with 10 9 random sequences of linear heptameric peptides was used. The peptides were displayed in five copies each, as fusion proteins with phage’s minor coat protein III. They were located on one end of the surface of the phage particles. The targets were a model protein, streptavidin, and protein–ethanol conjugates (hydroxyethyl radical- or acetaldehyde-modified bovine serum albumin). They were either immobilized on a surface by direct coating or affinity captured on floating beads. An enriched library of phages with the tightest peptide binders for each target was selected and amplified in a multiple-cycle biopanning in vitro procedure. Binders were characterized by DNA sequencing of the corresponding phages and by counter-screening with positive and negative targets in either an enzyme-linked immunosorbent assay or plaque assay. We obtained the HPQ motif for streptavidin and two unique subsets of peptides that recognized each ethanol target with a selectivity of two orders of magnitude above the carrier protein and controls. The application of biopanning processes, coupled with phage-display peptide libraries on biological fluids and tissues, could provide a systematic mapping of protein–ethanol conjugates and supply a means for early diagnosis and prognosis of chronic alcohol consumption in human beings.

Comparison of phage pIII, pVIII and GST as carrier proteins for peptide immunisation in Balb/c mice

Carrier proteins are important for improving the efficiency of synthetic peptide vaccines. Recently, genetically-based systems such as filamentous phage display or glutathione S-transferase (GST)-fusion proteins have been employed for immunisation. Whilst these carrier systems can facilitate the evaluation of a potential vaccine by reducing the time and cost of production, their relative efficacy and the kinetics of the immune response to each carrier has not been directly compared. In this study, we have displayed the epitopes of the anti-ErbB-2 Mabs N12 (C531–A586, EP531) and N28 (T216–C235, EP216) on phage minor coat protein pIII, major coat protein pVIII and GST. Balb/c H-2 d mice were immunised with the constructs and the sera were tested after the initial, the 3rd, 5th and 6th immunisations for an anti-peptide, an anti-ErbB-2 and an anti-carrier response. The specificity of the antibody response was also mapped using synthetic peptides. It was found that GST was the best of the three carriers, both in terms of the magnitude and the kinetics of the induced anti-peptide and anti-ErbB-2 response. Multiple (five) administrations of the immunogens were necessary to obtain a high titre of antibodies specific for ErbB-2. It was further noted that whilst an anti-EP531 response was induced using all three carriers, EP216 was not immunogenic irrespective of the carrier used. The lack of immunogenicity of EP216 implies it does not contain a H-2 d T cell recognition site. All three carriers provide a useful system for vaccination and consequently facilitate the identification of T-cell epitopes in Balb/c inbred mice.

Ligand Binding Regions in the Receptor for Urokinase-type Plasminogen Activator: STRUCTURAL REQUIREMENTS FOR A MULTIDOMAIN BINDING REGION AND RECEPTOR-RECEPTOR INTERACTION

The interaction between urokinase plasminogen activator (uPA) and its cellular receptor (uPAR) is a key event in cell surface-associated plasminogen activation, relevant for cell migration and invasion. In order to define receptor recognition sites for uPA, we have expressed uPAR fragments as fusion products with the minor coat protein on the surface of M13 bacteriophages. Sequence analysis of cDNA fragments encoding uPA-binding peptides indicated the existence of a composite uPA-binding structure including all three uPAR domains. This finding was confirmed by experiments using an overlapping 15-mer peptide array covering the entire uPAR molecule. Four regions within the uPAR sequence were found to directly bind to uPA: two distinct regions containing amino acids 13--20 and amino acids 74--84 of the uPAR domain I, and regions in the putative loop 3 of the domains II and III. All the uPA-binding fragments from the three domains were shown to have an agonistic effect on uPA binding to immobilized uPAR. Furthermore, uPAR-(154--176) increased uPAR-transfected BAF3-cell adhesion on vitronectin in the presence of uPA, whereas uPAR-(247--276) stimulated the cell adhesion both in the absence or presence of uPA. The latter fragment was also able to augment the binding of vitronectin to uPAR in a purified system, thereby mimicking the effect of uPA on this interaction. These results indicate that uPA binding can take place to particular part(s) on several uPAR molecules and that direct uPAR-uPAR contacts may contribute to receptor activation and ligand binding.

Beet necrotic yellow vein virus particles localize to mitochondria during infection.

Fluorescent beet necrotic yellow vein virus (BNYVV) particles were produced by replacing part of the readthrough domain of the minor coat protein P75 with the green fluorescent protein (GFP). The recombinant virus was functional in plants and P75-GFP was incorporated at one end of the rod-shaped virions. Laser scanning confocal microscopy and transmission electron microscopy showed that virus-like particles, almost certainly authentic BNYVV virions, localized to the cytoplasmic surface of mitochondria at early times postinfection but relocated at later times to semiordered clusters in the cytoplasm. This is the first report of specific targeting of plant virus particles to the mitochondria in vivo.

Synthesis of ligand-specific phage-display ScFv against the herbicide picloram by direct cloning from hyperimmunized mouse.

Immunoglobulin genes were directly isolated from the splenocytes of a BALB/C mouse hyperimmunized with the auxinic herbicide picloram conjugated to bovine serum albumin. Variable light and heavy domain DNA were joined to produce single-chain Fv (scFv) DNA, which was cloned into phage vector fd-tet-GIIID to display multiple copies of scFv on the filamentous phage minor coat protein gIIIp. The phage-display scFv library (10(4) clones) was selected against picloram conjugated to ovalbumin. After five rounds of panning, individual clones were analyzed. ScFv with different affinities to picloram (IC(50) values ranging from 20 ppb to 10 ppm) were detected in the final enriched pool. The increased avidity of the phage vector enhanced the selection (i.e., panning) of multiple picloram-specific recombinant antibodies. Stringent selection was required to isolate the clones with the highest affinity. Nucleotide sequence analysis of six isolated clones revealed that all of the V(L) belonged to the V kappa 9A family joined to J kappa 2 segments. All of the V(H) belonged to the V(H)()7183 family and joined to two different J segments (i.e., J(H)()2 or J(H)()4). Different from the immune response to large molecular weight molecules (MW > 10,000 Da), which requires both VDJ segment rearrangement and somatic hypermutations, production of high-affinity antibodies to picloram, a small ligand having a formula weight of 241.5 Da, predominantly requires somatic hypermutations.

Characterization of the cis-Acting Elements Controlling Subgenomic mRNAs of Citrus tristeza virus: Production of Positive- and Negative-Stranded 3′-Terminal and Positive-Stranded 5′-Terminal RNAs

Citrus tristeza virus (CTV), a member of the Closteroviridae, has an ∼20-kb positive-sense RNA genome with two 5′ ORFs translated from the genomic RNA and 10 3′ genes expressed via nine or ten 3′-terminal subgenomic (sg) RNAs. The expression of the 3′ genes appears to have properties intermediate between the smaller viruses of the “alphavirus supergroup” and the larger viruses of the Coronaviridae. The sgRNAs are contiguous with the genome, without a common 5′ leader, and are associated with large amounts of complementary sgRNAs. Production of the different sgRNAs is regulated temporally and quantitatively, with the highly expressed genes having noncoding regions (NCR) 5′ of the ORFs. The cis-acting elements that control the highly expressed major coat protein (CP) gene and the intermediately expressed minor coat protein (CPm) gene were mapped and compared. Mutational analysis showed that the CP sgRNA controller element mapped within nts −47 to −5 upstream of the transcription start site, entirely within the NCR, while the CPm control region mapped within a 57 nt sequence within the upstream ORF. Although both regions were predicted to fold into two stem-loop structures, mutagenesis suggested that primary structure might be more important than the secondary structure. Because each controller element produced large amounts of 3′-terminal positive- and negative-stranded sgRNAs, we could not differentiate whether the cis-acting element functioned as a promoter or terminator, or both. Reversal of the control element unexpectedly produced large amounts of a negative-stranded sgRNA apparently by termination of negative-stranded genomic RNA synthesis. Further examination of controller elements in their native orientation showed normal production of abundant amounts of positive-stranded sgRNAs extending to near the 5′-terminus, corresponding to termination at each controller element. Thus, each controller element produced three sgRNAs, a 5′-terminal positive strand and both positive- and negative-stranded 3′-terminal RNAs. Therefore, theoretically CTV could produce 30–33 species of RNAs in infected cells.

Coronary artery ectasia (angiographic and clinical findings of our patients)

For the last 70 years, long before the virus nature of the tristeza disease was recognized, Citrus tristeza virus (CTV), genus Closterovirus, family Closteroviridae, was considered as a serious threat to citrus production. The costs of CTV infections varied geographically and temporally, especially since growers worldwide have recognized the grave consequences of CTV epidemics. A variety of control measures including replacing sensitive rootstocks, prevention of spread with propagation material, and wide scale use of cross-protection for handling stem pitting-related problems are now commonly used in attempts to control the range of CTV diseases.Despite these efforts, the virus which apparently originated in Asia continued its spread and its now present throughout most citrus growing areas of the world, with only a few of the Mediterranean basin countries, and some Western USA areas still free of massive infections. However, even these areas are now under a serious threat due to the recent spread of the most efficient aphid vector to nearby areas. CTV is naturally transmitted in a semipersistent mode by several aphid species. The brown citrus aphid (Toxoptera citricida) is the most efficient vector because of its great abundance on citrus trees and its high efficacy of vectoring highly pathogenic isolates. The symptoms of CTV depend on the nature of the invading isolate and the sensitivity of the citrus host. The most dramatic symptom referred to as ‘quick decline’ results from rapid death of trees grafted onto sour orange rootstocks. Other CTV-related diseases such as ‘stem pitting’ of grapefruit or sweet orange trees are not related to a specific rootstock and do not result in death, but are often associated with stunting and poor productivity. Symptomless CTV isolates are used to provide protection (‘cross-protection’) against stem pitting, but not against the quick decline.The virions of CTV are long flexuous particles (2000 × 12 nm) with an unusual architecture that results from two different coat proteins, the major coat protein (CP), and minor coat protein (CPm) encapsidating approximately 97% and 3%, respectively, of the helically structured particles. Two additional viral proteins, p61 and p64 (a HSP70 homolog), are required for efficient assembly. The 19.3 kbp single-stranded RNA genome is the largest among the plant viruses. Genomic analyses showed a division into 12 open reading frames (ORFs), the organization of which is conserved among isolates. The ORF1a encodes a 349 kDa polyprotein containing two papain-like protease domains plus methyltransferase-like and helicase-like domains. Translation of the polyprotein is thought occasionally to continue through the polymerase-like domain (ORF1b) by a =1 frameshift. Ten 3′ ORFs are expressed by 3′ co-terminal subgenomic messenger RNAs. Infected cells contain a myriad of other subgenomic RNAs. In addition, most CTV isolates contain one or more defective RNAs of different sizes and genomic rearrangements. Among the CTV translation products, at least three function as RNA-silencing repressors, which are correlated with unusual difficulty in conferring pathogen-derived resistance to transgenic citrus plants.

Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes

Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of insertion of this small protein (32 amino acids) into membranes is still unknown. Here we show that the protein is able to insert in monolayers. The limiting surface pressure of 35 mN/m for 1,2-dioleoyl- sn-glycero-3-phosphocholine and 1,2-dioleoyl- sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo. By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside. The orientation after insertion of gene 9 protein is in agreement with the ‘positive inside rule’.

Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition. By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained. The secondary structure of the gene 9 protein predominantly consists of α-helical (67%) and turn (33%) structures. The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly. Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the α-helical domain is mainly transmembrane. The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane. Potential structural consequences for both models are discussed.

Phage display of peptide / major histocompatibility class I complexes.

Major histocompatibility complex class I (MHC-I) molecules sample peptides from the intracellular environment and present them to cytotoxic T cells (CTL). To establish a selection system, and, thereby, enable a library approach to identify the specificities involved (that of the MHC-I for peptides and subsequently that ot the T cell receptor for peptide-MHC-I complex), we have fused a single chain peptide-MHC-I complex to the phage minor coat protein, gpIII, and displayed it on filamentous phage. Expression of peptide-MHC-I complexes was shown with relevant conformation-specific monoclonal antibodies and, more importantly, with a unique "T cell receptor-like" (i. e. peptide-specific, MHC-I-restricted) antibody. Thus, properly assembled and folded peptide-MHC-I complexes can be displayed on filamentous phage. Despite the successful display, interaction with T cells could not be demonstrated.

Size of the ligand complex between the N-terminal domain of the gene III coat protein and the non-infectious phage strongly influences the usefulness of in vitro selective infective phage technology

The selective infective phage (SIP) technology allows a rapid positive selection of interacting pairs of biological molecules that restore to non-infectious phages their ability to infect the bacterial host. After a successful infection, the phage is amplified and the DNA encoding the interacting ligand is isolated from the phage genome and sequenced. In our studies we have evaluated the usefulness of SIP for the identification and cloning of proteins interacting with a biotinylated target binding to a newly designed adapter molecule consisting of streptavidin fused to the C-terminus of the extracellular domain of the phage minor coat protein III. The new adapter was expressed in Escherichia coli and refolded from inclusion bodies. The two different domains joined within the chimaera were found to be biologically functional. We also demonstrated that non-covalent interactions between a non-infectious phage displaying a short peptide, which specifically binds the streptavidin, and the adapter molecule restore phage infectivity. To evaluate the potential of SIP as a general and generic tool for the screening of cDNA libraries that encode the ligands displayed at the surface of the phage and binding to biotinylated targets, we have increased both the size of the displayed ligand on the phage and the size of the biotinylated target bound to the streptavidin domain of the adapter molecule. In our model systems we show that the size of either the ligand or the target is a limiting factor for the technology.

Closterovirus Encoded HSP70 Homolog and p61 in Addition to Both Coat Proteins Function in Efficient Virion Assembly

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution of the key amino acid residues in the ATPase domain of HSP70h, Asp7 to Lys or Glu180 to Arg, reduced assembly, suggesting that the chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.