Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages

Group A RNA phages consist of four genes-maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli(group A), Pseudomonas aeruginosa(PP7) RNA phages and Rg-lysis protein from Qbeta phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub-groups A-I and A-II of E. coli RNA phages were clearly dispersed into two clusters.     

A filamentous phage of Vibrio parahaemolyticus O3:K6 isolated in Laos.

A filamentous phage, 'lvpf5,' of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Principles of selective inactivation of the virus genome. IV. The effect of UV-irradiation of phage MS2 on its binding with anti-MS2-immunoglobulins

Ultraviolet (254 nm) irradiation of the bacteriophage MS2 results in the decrease of the number of antigenic determinants exposed on the virion surface. The cross-section of the decrease, as measured by the number of anti-MS2 IgG molecules bound per virion, is 10(-16) mm2 per photon. The decrease of the phage-antibody binding proceeds after irradiation with a rate constant of about 5 x 10(-3) min-1. Since the antigenic determinants of the phage MS2 coat protein does not contain photoreactive amino acid residues, the irradiation-induced decrease of the phage antibody binding is determined, most probably, by the shielding of the antigenic determinants. Such shielding could be caused by rearrangement of coat protein molecules and/or of the capsid induced by photomodification of non-antigenic fragments of coat protein and/or of intraphage RNA.     

A Filamentous Phage of Vibrio parahaemolyticus O3:K6 Isolated in Laos

A filamentous phage, ‘lvpf5’, of Vibrio parahaemolyticus O3:K6 strain LVP5 was isolated and characterized. The host range was not restricted to serotype O3:K6, but 7 of 99 V. parahaemolyticus strains with a variety of serotypes were susceptible to the phage. The phage was inactivated by heating at 80 C for 10 min and by treating with chloroform. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phage exhibited a 3.8 kDa protein. The amino-terminal amino acid sequence of the coat protein was determined as AEGGAADPFEAIDLLGVATL. The phage genome consisted of a single-stranded DNA molecule. The activity of the phages was inhibited by anti-Na2 pili antibody.     

Conformation of the coat protein of filamentous bacteriophage Pf1 determined by neutron diffraction from magnetically oriented gels of specifically deuterated virions

The structure of filamentous bacteriophage Pf1 has been studied using neutron diffraction from magnetically oriented gels of native and valine-deuterated phage. Neutron diffraction intensities were measured to approximately 8 A resolution along the equator and first six layer-lines, and differences due to the deuterated valine residues were apparent. Analysis of equatorial data indicate that one valine residue is located at a radius of about 13 A, three are in the hydrophobic center of the protein coat at an average of about 22 A radius, and one is near the outer surface of the virion at about 28 A radius. Analysis of the three-dimensional data was initiated using the rod model for the alpha-helices of the coat protein derived from earlier X-ray diffraction studies. This model was refined against the neutron diffraction intensities from native phage to obtain a phase set that was used to calculate a difference map between the valine-deuterated and native phage. The difference map exhibits peaks that correspond to the positions of the five valine residues in the coat protein. From the amino acid sequence and the alpha-helical conformation of the coat protein, the five valine residues can be unambiguously assigned to the difference peaks. This assignment indicates that the two alpha-helices of the coat protein are parallel to one another, connected by a short stretch of non-helical peptide. The valine positions also indicate that the helical surface lattice of the phage particle is right-handed.     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Expression of an immunogenic region of HIV by a filamentous bacteriophage vector.

Vectors derived from the Escherichia coli filamentous phage, fd-tet, expressing parts of the human immunodeficiency virus (HIV) gag genes were constructed and analyzed. The immunoreactive domain of HIV Gag antigens was produced in the form of a fusion protein, with a phage minor coat protein, called protein III, playing an important role in phage infectivity. A micropanning procedure, utilizing the strong affinity of biotinylated antibody to streptavidin, was applied for the selection of clones. A simple preparation procedure consisting of polyethyleneglycol precipitation of the recombinant phage from the E. coli supernatant allowed us to detect HIV antigens by enzyme-linked immunosorbent assay (ELISA). Cloned FUSE-gag, as isolated using anti-Gag RL4.72.1 monoclonal antibody (mAb), contained a nucleotide sequence coding for 91 amino acids of HIV Gag p24. It specifically reacted with the mAb in the ELISA. Construction of the mAb-selectable phages permitted localization of epitopes for mAb. Infectivity of the phage clone was specifically neutralized by the anti-HIV mAb. Immunoelectroblotting analysis of recombinant phages revealed the presence of an about 65-kDa band reacting with anti-HIV mAb. This Mr corresponded to the size of the fused form of the FUSE 1 protein III. Human sera from HIV-infected and uninfected individuals reacted with recombinant protein III, as well as the original form of protein III.     

Phages from landscape libraries as substitute antibodies

In 'landscape' phage, as in traditional phage-display constructs, foreign peptides or proteins are fused to coat proteins on the surface of a filamentous phage particle. Unlike conventional constructs, however, each virion displays thousands of copies of the peptide in a repeating pattern, subtending a major fraction of the viral surface. The phage body serves as an interacting scaffold to constrain the peptide into a particular conformation, creating a defined organic surface structure ('landscape') that varies from one phage clone to the next. By testing landscape libraries with three representative antigens (streptavidin from the bacterium Streptomyces avidinii, avidin from chicken egg white and beta-galactosidase from Escherichia coli) we have shown that landscape phages may be used as a new type of substitute antibodies-filaments that can bind protein and glycoprotein antigens with nanomolar affinities and high specificity. In many ways these substitute antibodies are more convenient than their natural immunoglobulin counterparts.     

Transmembrane region of wild-type and mutant M13 coat proteins. Conformational role of beta-branched residues.

Although transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, beta-sheet promoters (Val, Ile, Thr) often account for approximately 40% of TM residue composition. We are examining the conformational role(s) of these residues, using as a model system the major coat protein of the filamentous bacteriophage M13. This 50-residue protein, which is located at the Escherichia coli host membrane during phage reproduction, contains a prototypic 19-residue hydrophobic midregion (residues 21-39: YIGYAWAMVVVIVGATIGI). Using "Eckstein" site-directed mutagenesis, we have generated several viable M13 coat protein mutants with beta-branched amino acid substitutions within their TM region. Mutant coat proteins, including Ile32----Val (I32V) and Ala27----Thr (A27T), were obtained in milligram quantities by growing M13 mutant phages in liter preparations, confirming that these coat proteins are capable of assuming their normal biological function(s) in phage reproduction. Circular dichroism spectroscopy performed in the membrane-mimetic medium of deoxycholate micelles indicated comparable alpha-helical contents of mutants I32V and A27T to wild-type protein. 13C nuclear magnetic resonance experiments with mutant A27T demonstrated that the combination of additional beta-branched content and introduction of an -OH substituent induced chemical shift and temperature-dependent changes and influenced the local protein environment at sites up to 12 residues remote from the mutation site. In contrast, mutant I32V (of which a salient feature is a mid-TM pentavaline segment) behaved very similarly to wild-type coat. These findings are interpreted in terms of the range of TM secondary structure and stability which can be accommodated by viable M13 coat protein mutants.     

Antibody-selectable filamentous fd phage vectors: affinity purification of target genes

Foreign DNA fragments can be inserted into a minor coat protein gene of filamentous phage, creating a fusion protein that is incorporated into the virion; we call these particles "fusion phage". The foreign amino acids are displayed on the surface, allowing fusion phage bearing antigenic determinants from a target gene to be purified in infectious form by affinity to antibody directed against the gene product. Here we introduce fusion-phage vectors that accept foreign DNA inserts with little effect on phage function; and describe affinity purification of virions bearing a target determinant from a 10(8)-fold excess of phage not bearing the determinant, using minute amounts of antibody. These "antibody-selectable" vectors are a promising alternative to conventional expression systems for using antibodies to clone genes, though the ability to isolate rare clones from actual libraries remains to be demonstrated.     

Chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly.

The major (gene VIII) coat protein of bacteriophage fd was radiolabelled by treating the virus with methyl[3H]acetimidate without causing any loss of infectivity. Complete amidination of lysine-8 in the amino acid sequence of the protein was achieved but little or no modification of the lysine residues near the C terminus was observed. This supports the assumption that the coat protein is oriented in the viral filament with its N terminus on the outside and its C-terminal region abutting the DNA. Escherichia coli was co-infected with radiolabelled bacteriophage and with unlabelled miniphage, a shorter defective form of phage fd. Radiolabel was detected in the progeny miniphage, proving that individual coat protein subunits can be recycled and assembled onto progeny miniphage DNA. About 35% of the coat protein subunits of phage particles infecting E. coli were recycled in 1 h. These facts support a model of the assembly and disassembly of the virion at the bacterial membrane in which the end of the particle containing the minor adsorption (gene III) protein, which is presumably the first to disassemble during infection, is the last to assemble during morphogenesis.     

Use of bioinformatics to predict a function for the GS element in Mycobacterium avium subspecies paratuberculosis

Filamentous bacteriophages are nonlytic, male-specific bacteriophages which infect Escherichia coli carrying an F-episome. The molecular mechanism of infection remains elusive, including the role of the major coat protein pVIII. In order to evaluate the contributions of major coat protein pVIII in the process of infection, two phage display libraries were generated. One library consisted of random amino acids at positions 2, 4, 5, 8, 11 and 12 of the N-terminus of major coat protein pVIII. The second library was generated by randomizing these positions as well as position 1. All these residues were previously shown to be exposed at the surface of the virions by being accessible to ligands. The infectivity of randomly selected mutant phages was analyzed. The present results demonstrate that phages modified at these positions can be correctly assembled and secreted into the exoplasm, although the efficiency was slightly lower than that of wild-type phage. Their infectivity varied greatly, and a general structural pattern underlying infectivity did not emerge. However, residual differences were observed between infectious and defective phage; in general, uncharged polar amino acids present at positions 5 and 11 of the N-terminus of pVIII reduced phage infectivity, whereas polar residues at position 8 facilitated infection. The first position of pVIII is remarkably critical for infection; when this alanine was substituted with other residues, most of the phages lost their infectivity. These results shed new light on the true complexity of random peptide pVIII phage display libraries.     

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

The basic structure of filamentous phage and its use in the display of combinatorial peptide libraries

Combinatorial peptide libraries have been playing a major role in the search for new drugs, ligands, enzyme substrates, and other specifically interacting molecules. The principal features of these libraries require a versatile repertoire, an easily identifiable tag for each of the library members, a simple method of synthesis, and a compability with the biochemical milieu. Two types of combinatorial libraries are in use: synthetic libraries and biological (mainly phage display) ones. An advantage of the biological libraries is due to the ability of each of the library members to replicate itself and to the fact that they carry their own coding sequences. The uniqueness of filamentous phage is that of its five virion proteins, three can tolerate the insertion of foreign peptides, each in a distinctive manner. The major coat protein, pVIII, is capable of displaying hundreds of peptide copies over the phage virion, pIII can display either one or five copies, and pVI, as opposed to the first two, displays its peptides such that the carboxy terminus is oriented outward. A major drawback of filamentous phage is its size. The length of an intact phage particle is 930 nm and it contains an ssDNA of 6400 bp. 2800 copies of the major coat protein form a “fish scale” cover over most of the virion DNA, whereas five copies of pIII, which has been the major protein used for library display, and five copies of pVI are located at one end of the filamentous virion. There is no doubt that in order to improve the quality of filamentous phage libraries, the size of phage should be drastically reduced. Comprehensive research on the phage life cycle and its structure will lead us to the construction of miniature phage and to other methods that will enable an in vivo expanding of the library repertoire as well as to binding-induced specific clone-proliferation.     

Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII.

Filamentous phage assemble at the membrane of infected cells. The phage filament is released from the membrane at the end of assembly, after four to five copies of the minor proteins, pIII and pVI, have been added to the end of the virion. In the absence of pIII or pVI, phage filaments are not released, but remain associated with the cells. The C-terminal portion of pIII, termed the "C" domain, is required for the release of stable virions.With the use of pIII C-terminal fragments of increasing size, termination of assembly can be divided into various steps. An 83-residue fragment leads to the incorporation of pVI into the assembling phage, but does not release it from the membrane. A slightly longer fragment (93 residues) is sufficient to release the particle into the culture supernatant. However, these released particles are unstable in the detergent, sarkosyl, which does not disrupt wild-type phage. A fragment of >121 residues is needed for the particle to become detergent resistant. Thus, the C-domain can be divided into two subdomains: C2, sufficient for release, and C1, required for virion stability.A model for termination of phage assembly is proposed in which pIII and pVI dock to the membrane-associated filament and form a pre- termination complex. Then, a conformational change involving the C2 domain of pIII disrupts the hydrophobic interactions with the inner membrane, releasing the phage from the cells. The pIII-mediated release of phage from the membranes points to one possible mechanism for excision of membrane-anchored protein complexes from lipid bilayers.     

Molecular genetics of bacteriophage P22 scaffolding protein's functional domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.     

Effects of virion and salt concentrations on the Raman signatures of filamentous phages fd, Pf1, Pf3, and PH75

Filamentous phages consist of a single-stranded DNA genome encapsidated by several thousand copies of a small alpha-helical coat protein subunit plus several copies of four minor proteins at the filament ends. The filamentous phages are important as cloning vectors, vehicles for peptide display, and substrates for macromolecular alignment. Effective use of a filamentous phage in such applications requires an understanding of experimental factors that may influence the propensity of viral filaments to laterally aggregate in solution. Because the Raman spectrum of a filamentous phage is strongly dependent on the relative orientation of the virion with respect to the polarization direction of the electromagnetic radiation employed to excite the spectrum, we have applied Raman spectroscopy to investigate lateral aggregation of phages fd, Pf1, Pf3, and PH75 in solution. The results show that lateral aggregation of the virions and anisotropic orientation of the aggregates are both disfavored by high concentrations of salt (>200 mM NaCl) in solutions containing a relatively low virion concentration (<10 mg/mL). Conversely, the formation of lateral aggregates and their anisotropic orientation are strongly favored by a low salt concentration (<0.1 mM NaCl), irrespective of the virion concentration over a wide range. The use of Raman polarization effects to distinguish isotropic and anisotropic solutions of filamentous phages is consistent with previously reported Raman analyses of virion structures in both solutions and fibers. The Raman data are supported by electron micrographs of negatively stained specimens of phage fd, which permit an independent assessment of salt effects on lateral aggregation. The present results also identify new Raman bands that serve as potential markers of subunit side-chain orientations in filamentous virus assemblies.     

Automated sequencing of insoluble peptides using detergent. Bacteriophage fl coat protein.

Peptides which are highly nonpolar and insoluble under moderate conditions of pH and ionic strength cannot be subjected to automated sequence analysis. We report a method for solubilization of one such peptide, bacteriophage fl coat protein, by chemical modification in the presence of sodium dodecyl sulfate. Following this treatment the 50-residue peptide was degraded stepwise in an automated sequenator using a single cleavage Quadrol program with high repetitive yield through residue 47. We also report a modified program using detergent incorporated into dimethylallylamine buffer which permitted sequencing with high repetitive yields for at least the first 18 residues of the unmodified and otherwise highly insoluble coat protein. The presence of detergent caused no observable difficulties in detection of residues by gas chromatography, thin layer chromatography, or amino acid analysis.     

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.