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.     

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)     

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-dependent conformational changes in a viral capsid protein. Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.     

Structural changes during the transformation of bacteriophage T4 polyheads: characterization of the initial and final states by freeze-drying and shadowing Fab-fragment-labelled preparations.

The maturation of the head of bacteriophage T4 requires a cleavage of the major capsid protein subunit, P23, and results in a transformation of the unstable prehead shell to the chemically resistant shell of the mature virion. We have studied this transformation by comparing class I and class III polyheads, which have P23 lattices which correspond to the prehead and mature head, respectively. The inner and outer surface topographies of these structures were determined from optically filtered images of freeze-dried and shadowed preparations. Individual antigenic sites were localized on the polyhead surfaces by labelling them with Fab fragments obtained from antisera raised against polyheads and against sheets composed of a fragment of the P23 molecule. We find that the transformation involves a structural change in the surface lattice which eliminates protrusions on the inside surface and produces new protrusions on the outer surface. Changes in antigenicity include at least one site which disappears from the outer surface, the unmasking of a site which appears on the outer surface, and the movement of at least one site from the inside surface to the outside during the transformation. We discuss the mechanism of the transformation in terms of the changes in tertiary and quaternary structure of the subunits required to account for the observed changes in the polyhead structure and antigenicity.     

Chaperonin-mediated folding of bacteriophage T4 major capsid protein. II. Production of gene product 23 deletion mutants

Folding of bacteriophage T4 major capsid protein, gene product 23 (534 a.a.), is aided by two proteins: E. coli GroEL chaperonin and viral gp31 co-chaperonin. In the present work a set of mutants with extensive deletions inside gene 23 using controlled digestion with Bal31 nuclease has been constructed. Proteins with deletions were co-expressed from plasmid vectors with phage gp31 co-chaperonin. Deletions from 8 to 33 a.a. in the N-terminal region of the gp23 molecule covering the protein proteolytic cleavage site during capsid maturation have no influence on the mutants' ability to produce in E. coli cells proteins which form regular structures—polyheads. Deletions in other regions of the polypeptide chain (187-203 and 367-476 a.a.) disturb the correct folding and subsequent assembly of gp23 into polyheads.     

Letter: Capsid structure of bacteriophage lambda

The arrangement of capsomers in the capsid of phage λ has been investigated by electron micrography of negatively stained fragments of empty capsids, polyheads, and intact virions. The proposed structure is a composite T = 7 levo lattice, with hexamer and pentamer clustering of the D protein and trimer clustering of the E protein. Such a lattice requires that the λ capsid contain 420 copies of the D and E proteins, a number compatible with recent chemical determinations.     

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4.

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.     

Proteolysis of the major capsid protein T4 bacteriophage polyheads limited by quaternary structure.

Bacteriophage T4 carrying an amber mutation in gene 22 plus an amber mutation in gene 21 form aberrant, tubular structures termed rough polyheads, instead of complete phage when they infect Escherichia coli B. These rough polyheads consist almost entirely of the major capsid protein in its uncleaved form (gp23). When rough polyheads are treated under mild conditions with any of the five proteases, trypsin, chymotrypsin, thermolysin, pronase, or the protease from Staphylococcus aureus V8, the gp23 is rapidly hydrolyzed at a limited number of peptide bonds. In contrast, cleaved capsid protein (gp23) in mature phage capsids is completely resistant to proteolysis under the same conditions. A major project in this laboratory requires determining the primary structure of gp23, a large protein (Mr = 58,000) quite rich in those amino acids at which cleavages are achieved by conventional means. Recovery of peptides from the complex mixtures resulting from such cleavages proved to be extremely difficult. The limited proteolysis of gp23 in rough polyheads had yielded a set of large, easily purified fragments which are greatly simplifying the task of determining the primary structure of this protein.     

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.     

Specific labeling of protein domains with antibody fragments

Monovalent antibody Fab fragments, prepared from antisera raised against two different types of crystalline arrays made of either intact, or a proteolytic fragment of bacteriophage T4 major capsid protein, gp23*, were employed to stoichiometrically label different gp23* protein domains on the outer surface of a tubular variant (i.e., "polyheads") of bacteriophage T4 capsids. Computer filtrations of both negatively stained and freeze-dried/metal-shadowed specimens permitted approximate mapping of the Fab binding sites within the capsomere of the polyheads.     

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.     

Bacteriophage SPO1 structure and morphogenesis. II. Head structure and DNA size.

The capsid of bacteriophage SPO1 is icosahedral, and the subunit arrangement on the 87-nm-diameter head suggests the triangulation number T = 16. The major capsid protein (45,700 daltons) is cleaved from a 47,700-dalton precursor. Tubular heads (polyheads) are produced by mutations in genes 5 and 8 and contain cores as well as capped ends. The lattice constant of these structures is 13.4 nm; diameter is 109.5 nm. The size of the double-stranded SPO1 DNA (containing 5' hydroxymethyl uracil in place of thymine) was measured by sedimentation analysis and electron microscopy and has a molecular weight of 86 X 10(6) (about 140 kilobase pairs), which is smaller than several previously reported values.     

Co-expression of gene 31 and 23 products of bacteriophage T4

Folding of the major capsid protein of bacteriophage T4 encoded by gene 23 is aided by Escherichia coli GroEL chaperonin and phage co-chaperonin gp31. In the absence of gene product (gp) 31, aggregates of recombinant gp23 accumulate in the cell similar to inclusion bodies. These aggregates can be solubilized with 6 M urea. However, the protein cannot form regular structures in solution. A system of co-expression of gp31 and gp23 under the control of phage T7 promoter in E. coli cells has been constructed. Folding of entire-length gp23 (534 amino acid residues) in this system results in the correctly folded recombinant gp23, which forms long regular structures (polyheads) in the cell.     

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.     

Large-scale,pH-dependent,quaternary structure changes in an RNA virus capsid are reversible in the absence of subunit autoproteolysis

The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus, Nudaurelia capensis omega virus (N omega V), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 A in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-A-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, N omega V provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in N omega V.     

Assembly-associated structural changes of bacteriophage T7 capsids. Detection by use of a protein-specific probe.

To detect changes in capsid structure that occur when a preassembled bacteriophage T7 capsid both packages and cleaves to mature-size longer (concatameric) DNA, the kinetics and thermodynamics are determined here for the binding of the protein-specific probe, 1,1'-bi(4-anilino)naphthalene-5,5'-di-sulfonic acid (bis-ANS), to bacteriophage T7, a T7 DNA deletion (8.4%) mutant, and a DNA-free T7 capsid (metrizamide low density capsid II) known to be a DNA packaging intermediate that has a permeability barrier not present in a related capsid (metrizamide high density capsid II). Initially, some binding to either bacteriophage or metrizamide low density capsid II occurs too rapidly to quantify (phase 1, duration < 10 s). Subsequent binding (phase 2) occurs with first-order kinetics. Only the phase 1 binding occurs for metrizamide high density capsid II. These observations, together with both the kinetics of the quenching by ethidium of bound bis-ANS fluorescence and the nature of bis-ANS-induced protein alterations, are explained by the hypothesis that the phase 2 binding occurs at internal sites. The number of these internal sites increases as the density of the packaged DNA decreases. The accompanying change in structure is potentially the signal for initiating cleavage of a concatemer. Evidence for the following was also obtained: (a) a previously undetected packaging-associated change in the conformation of the major protein of the outer capsid shell and (b) partitioning by a permeability barrier of the interior of the T7 capsid.     

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.