Structure of T4 polyheads: II. A pathway of polyhead transformations as a model for T4 capsid maturation

We have studied the aberrant tubular polyheads of bacteriophages T4D and T2L as a model system for capsid maturation. Six different types of polyhead surface lattice morphology, and the corresponding protein compositions are reported and discussed. Using in vitro systems to induce transformations between particular polyhead types, we have deduced that the structural classes represent successive points in a transitional pathway. In the first step, coarse polyheads (analogous to the prohead τ-particle) are proteolytically cleaved by a phagecoded protease, a fragment of the gene 21 product. This cleavage of P23 to P23¹ induces a co-operative lattice transformation in the protein of the surface shell, to a conformation equivalent to that of T2L giant phage capsids. These polyheads (derived either from T4 or T2L lysates) can accept further T4-coded proteins. In doing so, they pass through intermediate structural states, eventually reaching an end point whose unit cell morphology is indistinguishable from that of the giant T4 capsids. At least one protein (called soc (Ishii & Yanagida, 1975)) is bound stoichiometrically to P23¹ in the end-state conformation. The simulation of several aspects of capsid maturation (cleavage of P23 to P23¹, stabilization, and lattice expansion) in the polyhead pathway suggest that it parallels the major events of phage T-even capsid maturation, decoupled from any involvement of DNA packaging.     

Folding and capsomere morphology of the P23 surface shell of bacteriophage T4 polyheads from mutants in five different head genes

Mutants in five different “head formation” genes (20, 22, 24, 40, IPIII)² of bacteriophage T4 produce polyheads. “Coarse” polyheads, which contain uncleaved P23, constitute over 90% of these tubular particles in fresh lysates. Using optical diffraction and filtration, we show that the pseudo-hexagonal net and the capsomere morphology are common to all coarse polyheads, regardless of genetic origin or polyhead diameter. Micropolymorphism is exhibited in each genetic class with respect to the cylindrical folding of the hexagonal net. We find that the frequency distribution of the diameters and pitch angles is significantly different for polyheads made by mutants affecting either of the major prohead core proteins (IPIII and P22). In every case, the foldings differ from the unique folding characteristic of giant phage capsid, suggesting that the assembly error responsible for producing polyheads instead of proheads involves a misdirection in arranging the P23 shell. By analysing the properties common to the various structures which may be formed out of this net (single-layered polyheads, multi-layered polyheads, proheads), we find that the P23 molecules possess form-determining specificity in terms of an intrinsic curvature of the capsomere bonding. These observations are discussed within the context of form determination of the phage prohead (τ-particle) and of its subsequent conservative maturation to the head of the infective wild-type phage.     

Structural changes during the transformation of bacteriophage T4 polyheads: II. Evidence that a conformational change in the major capsid protein precedes the expansion

The shell of the bacteriophage T4 prehead is transformed after the maturation cleavages from a fragile to a highly chemically resistant structure. A “cleaved but anchored” shell, in which the capsid protein has been cleaved but expansion to the mature structure has not yet occurred, is thought to be an intermediate in the transformation. We have compared native, trypsinized, temperature-sensitive mutant, and cleaved but anchored polyheads for differences and similarities in their capsomeres. Our results show that the altered capsomeres of the cleaved but anchored state must be attributed to a conformational change in the subunits, and not simply to the loss of the amino-terminal peptide by proteolysis.     

Isolation and characterization of bacteriophage T4 mutant preheads.

To determine the function of individual gene products in the assembly and maturation of the T4 prehead, we have isolated and characterized aberrant preheads produced by mutations in three of the T4 head genes. Mutants in gene 21, which codes for the T4 maturation proteases, produce rather stable preheads whose morphology and protein composition are consistent with a wild-type prehead blocked in the maturation cleavages. Mutants in gene 24 produce similar structures which are unstable because they have gaps at all of their icosahedral vertices except the membrane attachment site. In addition, greatly elongated "giant preheads" are produced, suggesting that in the absence of P24 at the vertices, the distal cap of the prehead is unstable, allowing abnormal elongation of broth the prehead core and its shell. Vertex completion by P24 is required to allow the maturation cleavages to occur, and 24- preheads can be matured to capsids in vitro by the addition of P24. Preheads produced by a temperature-sensitive mutant in gene 23 are deficient in core proteins. We show that the shell of these preheads has the expanded lattice characteristic of the mature capsid as well as the binding sites for the proteins hoc and soc, even though none of the maturation cleavage takes place. We also show that 21- preheads composed of wild-type P23 can be expanded in vitro without cleavage.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins

Bacteriophage P22 forms an isometric capsid during normal assembly, yet when the coat protein (CP) is altered at a single site, helical structures (polyheads) also form. The structures of three distinct polyheads obtained from F170L and F170A variants were determined by cryo-reconstruction methods. An understanding of the structures of aberrant assemblies such as polyheads helps to explain how amino acid substitutions affect the CP, and these results can now be put into the context of CP pseudo-atomic models. F170L CP forms two types of polyhead and each has the CP organized as hexons (oligomers of six CPs). These hexons have a skewed structure similar to that in procapsids (precursor capsids formed prior to dsDNA packaging), yet their organization differs completely in polyheads and procapsids. F170A CP forms only one type of polyhead, and though this has hexons organized similarly to hexons in F170L polyheads, the hexons are isometric structures like those found in mature virions. The hexon organization in all three polyheads suggests that nucleation of procapsid assembly occurs via a trimer of CP monomers, and this drives formation of a T = 7, isometric particle. These variants also form procapsids, but they mature quite differently: F170A expands spontaneously at room temperature, whereas F170L requires more energy. The P22 CP structure along with scaffolding protein interactions appear to dictate curvature and geometry in assembled structures and residue 170 significantly influences both assembly and maturation.     

Assembly and disassembly of bacteriophage T4 polyheads

The assembly of the product of bacteriophage T4 gene 23 (gp23), the uncleaved form of the main shell protein, has been studied. Assembly and disassembly follow the predictions for entropy-driven processes; assembly is strongly favored by conditions of high salt concentrations and high temperatures, whereas low salt and low temperatures promote disassembly. In the absence of the scaffolding core proteins in vitro, only polyheads, the tubular variant of the prohead, are produced. Kinetic studies show that the rate of polyhead dissociation depends on the concentration of associated protein, not on the number and length of the particles. Comparable to crystal formation, assembly of gp23 occurs above a critical concentration, which is dependent on salt concentration, pH and temperature. These characteristics are common to most self-assembling systems. The oligomeric states of gp23 have been investigated by analytical ultracentrifugation, which indicated the existence, at very low salt concentration and low temperature, of an equilibrium between monomers and higher oligomers, culminating in the hexamer. At pH 9.0 polyheads are completely dissociated into their monomeric gp23 subunits. Our data suggest that the hexamer is a true intermediate of polyhead assembly.     

Studies on the morphopoiesis of the head of phage T-even

Summary The kinetics of the assembly of polyheads produced by infecting Escherichia coli B with T₄ amber mutants in gene 20 was measured and compared with the growth of wild type phage. The rates of production of polyheads and of phages were found to be about the same. The final yields in lysis-inhibited cells were approximately 600 phage equivalents per infected bacterium. The initial appearance of polyheads is delayed 15–20 min compared with wild type phage production, although it is not due to a reduced rate of protein synthesis in mutant-infected cells. In such cells an accumulation of precursor protein for polyhead is thus caused. This pool is about three times larger than the one measured during wild type infection. The delay is extended if the amount of subunits available for polyhead formation is reduced. We conclude that the initiation of polyhead assembly depends upon the subunit concentration. Polyhead assembly continues at the same rate for several minutes when protein synthesis is inhibited with chloramphenicol at different times. The maturable polyhead precursor was estimated by measuring the amount of polyheads assembled after adding the drug, and it was found that 25% of the total protein pool was converted into polyheads. Using a new technique for the observation of single cells with the electron microscope we found that polyheads are arranged in bundles oriented parallel to the long axis of the cell. The average length of polyheads is roughly the same at all times during their formation.     

Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity

The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines. A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5514-5518). In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation. We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)). The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement. Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface. In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy. These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.     

Outer surface protein of bacteriophage lambda

The bacteriophage λ capsid is composed of a main shell protein (pE) and an outer surface protein (pD). The outer surface protein was purified from sources of free protein and assembled protein. The amino acid composition, C- and N-terminals, iso-electric point, molecular weight, and state of aggregation were determined. In vitro the outer surface protein binds specifically to structures composed of λ main shell protein in the expanded configuration i.e. to enlarged preheads, pD-deficient bacteriophage particles, and polyheads.We discuss the binding of pD to the shell surface as a “pseudo-crystallisation process”, its clustering on the surface as trimers and its role as stabiliser of the filled head.     

The maturation-dependent conformational change of phage T4 capsid involves the translocation of specific epitopes between the inner and the outer capsid surfaces

After polymerization of the phage T4 prohead is complete, its capsid expands by approximately 16%, is greatly stabilized, and acquires the capacity to bind accessory proteins. These effects are manifestations of a large-scale, irreversible, conformational change undergone by the major capsid protein, gp23 (521 residues) which is cleaved to gp23* (residues 66-521) during this maturation process. In order to explore its structural basis, we have performed immunoelectron microscopy with antibodies raised against synthetic peptides that correspond to precisely defined segments of the amino acid sequence of gp23. These antibodies were used to label purified polyheads (tubular polymorphic variants of the normal icosahedral capsid), in experiments designed to impose constraints on the possible foldings of the gp23/gp23* polypeptide chains in their successive conformational states. Peptide 1 (residues 48-57), part of the gp23-delta domain that is excised when gp23 is converted to gp23*, resides on the inner surface of the precursor surface lattice, but--if not proteolyzed--is found on the outer surface of the mature surface lattice. Peptide 2 (residues 65-73), immediately distal to the cleavage site, is located on the inside of the precursor surface lattice, and remains there subsequent to expansion. Peptide 3 (residues 139-146) is translocated in the opposite direction from peptide 1, i.e., from the outer to the inner surface upon expansion; moreover, expansion greatly increases the polyheads' affinity for these antibodies. Peptide 5 (residues 301-308) is located on the inside in both the precursor and the mature states. Taking into account data from other sources, these observations imply that the conformational change that underlies capsid expansion involves a radical reorganization of the proteins' structure, in which at least three distinct epitopes, situated in widely differing parts of the polypeptide chain, are translocated from one side to the other. Moreover, the amino-terminal portion of gp23/gp23*, around the cleavage site, is particularly affected.     

The maturation-dependent conformational change of the major capsid protein of bacteriophage T4 involves a substantial change in secondary structure

We have investigated the conformational basis of the expansion transformation that occurs upon maturation of the bacteriophage T4 prohead, by using laser Raman spectroscopy to determine the secondary structure of the major capsid protein in both the precursor and the mature states of the surface lattice. This transformation involves major changes in the physical, chemical, and immunological properties of the capsid and is preceded in vivo by processing of its major protein, gp23 (56 kDa), to gp23* (49 kDa), by proteolysis of its N-terminal gp23-delta domain. The respective secondary structures of gp23 in the unexpanded state, and of gp23* in the expanded state, were determined from the laser Raman spectra of polyheads, tubular polymorphic variants of the capsid. Similar measurements were also made on uncleaved polyheads that had been expanded in vitro and, for reference, on thermally denatured polyheads. We find that, with or without cleavage of gp23, expansion is accompanied by substantial changes in secondary structure, involving a major reduction in alpha-helix content and an increase in beta-sheet. The beta-sheet contents of gp23* or gp23 in the expanded state of the surface lattice, and even of gp23 in the unexpanded state, are sufficient for a domain with the "jellyroll" fold of antiparallel beta-sheets, previously detected in the capsid proteins of other icosahedral viruses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation between structural transformation and cleavage of the major head protein of T4 bacteriophage

We have studied the maturation of T4 polyheads, the aberrant tubular structures related to the capsid of T4 bacteriophage. Conditions have been found under which more than 95% of the major head protein (P23) undergoes the same cleavage that occurs during development of the normal capsid. The concomitant structural changes in the polyheads have been followed using electron microscope image filtering techniques. As a result of the cleavage, a radical transformation of the hexagonal lattice occurs, Involving a 10–15% expansion in the lattice dimensions. However, a metastable intermediate state similar to the uncleaved structure has been observed immediately after cleavage of the protein subunits. Some kind of additional physical stimulus seems to be required to trigger the major structural change, which appears to be highly cooperative.     

Purification and properties of the bacteriophage T4 gene 31 protein required for prehead assembly.

A low molecular weight (approximately 16,000), early protein is characterized as the product of the essential T4 head assembly gene 31. This gene is known to be required to allow formation of any ordered head structure from the major T4 capsid protein, P23 (Laemmli, U.K., Beguin, F., and Gujer-Kellenberger, G. (1970) J. Mol. Biol. 47, 69-85). In wild type infection P31 synthesis ceases at late times; in contrast, P31 is overproduced in certain early or regulatory T4 mutant infections, particularly gene 55 mutant infections. P31 was purified preparatively from Escherichia coli infected with the latter mutant, but could only be obtained for the most part in modified form, possibly due to unusual sensitivity to a proteolytic activity. P31 is not cleaved in vivo during normal head assembly, nor does it become a part of the mature head or any ordered prehead structure as determined by an immunological assay using antiserum prepared against the purified protein. However P31 does appear to become a part of the unordered P23 aggregates (lumps) which accumulate when ordered P23 assembly is prevented. We cound find no evidence for P31 association with T4 DNA or the host membrane. Our experiments favor the hypothesis that P31 directly affects the aggregation state and solubility properties of P23.     

Conformational changes of a viral capsid protein. Thermodynamic rationale for proteolytic regulation of bacteriophage T4 capsid expansion, co-operativity, and super-stabilization by soc binding.

We have used differential scanning calorimetry in conjunction with cryo-electron microscopy to investigate the conformational transitions undergone by the maturing capsid of phage T4. Its precursor shell is composed primarily of gp23 (521 residues): cleavage of gp23 to gp23* (residues 66 to 521) facilitates a concerted conformational change in which the particle expands substantially, and is greatly stabilized. We have now characterized the intermediate states of capsid maturation; namely, the cleaved/unexpanded, state, which denatures at tm = 60 degrees C, and the uncleaved/expanded state, for which tm = 70 degrees C. When compared with the precursor uncleaved/unexpanded state (tm = 65 degrees C), and the mature cleaved/expanded state (tm = 83 degrees C, if complete cleavage precedes expansion), it follows that expansion of the cleaved precursor (delta tm approximately +23 degrees C) is the major stabilizing event in capsid maturation. These observations also suggest an advantage conferred by capsid protein cleavage (some other phage capsids expand without cleavage): if the gp23-delta domains (residues 1 to 65) are not removed by proteolysis, they impede formation of the stablest possible bonding arrangement when expansion occurs, most likely by becoming trapped at the interface between neighboring subunits or capsomers. Icosahedral capsids denature at essentially the same temperatures as tubular polymorphic variants (polyheads) for the same state of the surface lattice. However, the thermal transitions of capsids are considerably sharper, i.e. more co-operative, than those of polyheads, which we attribute to capsids being closed, not open-ended. In both cases, binding of the accessory protein soc around the threefold sites on the outer surface of the expanded surface lattice results in a substantial further stabilization (delta tm = +5 degrees C). The interfaces between capsomers appear to be relatively weak points that are reinforced by clamp-like binding of soc. These results imply that the "triplex" proteins of other viruses (their structural counterparts of soc) are likely also to be involved in capsid stabilization. Cryo-electron microscopy was used to make conclusive interpretations of endotherms in terms of denaturation events. These data also revealed that the cleaved/unexpanded capsid has an angular polyhedral morphology and has a pronounced relief on its outer surface. Moreover, it is 14% smaller in linear dimensions than the cleaved/expanded capsid, and its shell is commensurately thicker.     

Assembly of bacteriophage T4 head-related strucutres. II. In vitro assembly of prehead-like structures

A protein mixture which is derived from bacteriophage T4 preheads formed in vivo contains all the important prehead proteins: i.e. protein P23, which forms the icosahedral prehead shell; the core proteins P22 and internal protein III; and two quantitatively minor proteins, P24 and P20. Conditions are described under which these proteins assemble in vitro into structures that (1) resemble preheads when visualized by electron microscopy, (2) contain all prehead proteins, and (3) have a similar length and diameter as preheads formed in vivo. It is concluded that prehead-like structures can be assembled in vitro, and that the mechanism that determines the length and diameter of the T4 prehead is active in our in vitro system. Evidence is presented that the core proteins play an important role in specifying the prehead diameter. The result of assembly experiments after partial fractionation of the protein mixture by gel filtration suggests that P20 plays a key role in the assembly of prehead-like structures in vitro, whereas P24 is not required. A possible mechanism by which P20 governs tha assembly of P23 and the core proteins is discussed.     

Possible formation and development of spirochaete attachment sites found on the surface of symbiotic polymastigote flagellates of the termite Reticulitermes flavipes.

We propose that spirochaete attachment sites arise from peripheral protrusions which appear on the surface of polymastigote flagellates. These protrusions develop into bracket-shaped structures which then form the mature attachment site. Next the site becomes detached from the surface of the cell; this latter process may be facilitated by the fusion of vesicles located in the region immediately beneath the spirochaete attachment site. This theory could explain the variability in the number and distribution of attachment sites on the surface of the flagellates.     

Probable localization of the bacteriophage T4 prehead proteinase zymogen in the center of the prehead core.

During the assembly of the bacteriophage T4 prehead, a T4-coded protease zymogen (P21) is built into the structure. At a certain stage in head formation, the protease precursor is activated and specifically cleaves most of the prehead proteins. In this paper we show that a correlation existed between the presence of proteinaceous material in the center of the prehead core, observed by electron microscopy, and the availability of P21 during prehead assembly. In the absence of P21, the core enclosed a hold of about 35 nm long and 20 nm wide. We found the same for (i) in vitro-assembled, negatively stained prehead-like structures and (ii) in vivo-formed preheads in thin sections of T4-infected cells. We concluded that P21 was localized in the center of the prehead core.     

Head morphologies in bacteriophage T4 head and internal protein mutant infections.

Escherichia coli infected with phage T4 mutants defective in synthesis of the three major internal proteins found in the phage head, IPI-, IPII-, IPIII-, or IP degrees (lacking all three) were examined in the electron microscope for head formation. Infection with IPI- or IPII- does not appear to induce increased aberrant head formation, whereas IPII- or IP degrees infections result in production of polyheads and viable phage. Multiple mutants of the early head formation genes 20, 21, 22, 23, 24, 31, 40 and IP degrees were constructed. Combination with IP degrees increases polyhead formation when head formation is not blocked at a more defective stage but results in a qualitative shift to lump formation in association with gene 22 mutants. Thin-sectioning studies show morphologically similar cores in amber 21 and 21am IP degrees tau particles. These morphological observations, genetic evidence for interaction between ts mutants in gene 22 and the IP mutants, and analysis of the protein composition of tau particles further support the idea that p22 and the internal proteins form an unstable assembly core necessary for an early stage of head formation (M. K. Showe and L. W. Black, 1973).     

Isolation and reassembly of bacteriophage T4 core proteins

The products of genes 22, 67 and 68, and the internal proteins IPI, IPII and IPIII, as components of the scaffolding core of the bacteriophage T4 prohead, have been isolated and purified by hydroxylapatite column chromatography. Under conditions promoting reassembly in vitro, the proteins associated into elongated particles of practically constant width but variable length that we have called polycores. Preliminary optical diffraction experiments indicate that polycores may have an ordered structure, possibly helical, as has been suggested for the polyhead core. The coassembly of core proteins and the purified shell protein gp23 results in the formation of core-containing polyheads. Occasionally, prolate core-like particles have been observed but their reproducible formation has not been attained. Attempts to investigate the role of the minor prohead component gp20 in core assembly have been made through the cloning of the corresponding gene in an expression vector and subsequent purification of the protein.