Phage P22 procapsids equilibrate with free coat protein subunits

Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been viewed as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsid assembly reactions achieve equilibrium or are irreversibly trapped. To assemble a procapsid-like particle in vitro, pure coat protein monomers are mixed with scaffolding protein. We show that free subunits can exchange with assembled structures, indicating that assembly is a reversible, equilibrium process. When empty procapsid shells (procapsids with the scaffolding protein stripped out) were diluted so that the concentration was below the dissociation constant ( approximately 5 microM) for coat protein monomers, free monomers were detected. The released monomers were assembly-competent; when NaCl was added to metastable partial capsids that were aged for an extended period, the released coat subunits were able to rapidly re-distribute from the partial capsids and form whole procapsids. Lastly, radioactive monomeric coat subunits were able to exchange with the subunits from empty procapsid shells. The data presented illustrate that coat protein monomers are able to dissociate from procapsids in an active state, that assembly of procapsids is consistent with reactions at equilibrium and that the reaction follows the law of mass action.     

Purification and organization of the gene 1 portal protein required for phage P22 DNA packaging

The gene 1 protein of Salmonella bacteriophage P22 is located at the DNA packaging vertex of the mature particle. The protein is incorporated into the procapsid shell during shell assembly and is required for DNA packaging. The unassembled precursor form of the gene 1 protein has been purified from cells infected with mutants blocked in procapsid assembly. The purified 90,000-dalton protein was dimeric or monomeric; upon storage in the cold it formed 20S cyclic dodecamers. Computer filtering of negatively stained electron micrographs revealed 12 arms and knobs projecting from a central ring, with a 30-A channel at the center. Similar dodecameric rings were released from disrupted procapsid shells. These results indicate that the gene 1 protein is organized as a cyclic dodecamer within the procapsid shell and serves as the portal through which P22 DNA is threaded during DNA packaging. The presence of a 12-fold ring located at a 5-fold portal vertex appears to be a conserved structural theme of the DNA packaging apparatus of double-stranded DNA phages.     

Initiation of P22 procapsid assembly in vivo

The procapsids of all double-stranded DNA phages have a unique portal vertex, which is the locus of DNA packaging and DNA injection. Procapsid assembly is also initiated at this vertex, which is defined by the presence of a cyclic dodecamer of the portal protein. Assembly of the procapsid shell of phage P22 requires the gene 5 coat protein and the gene 8 scaffolding protein. We report here that removal of gene product (gp) 1 portal protein of P22 by mutation does not slow the rate of polymerization of coat and scaffolding subunits into shells, indicating that the portal ring is dispensable for shell initiation. Mutant scaffolding subunits specified by tsU172 copolymerize with coat subunits into procapsids at restrictive temperature, and also correctly autoregulate their synthesis. However, the shell structures formed from the temperature-sensitive scaffolding subunits fail to incorporate the portal ring and the three minor DNA injection proteins. This mutation identifies a domain of the scaffolding protein specifically involved in organization of the portal vertex. The results suggest that it is a complex of the scaffolding protein that initiates procapsid assembly and organizes the portal ring.     

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.     

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.     

Pressure denaturation of the bacteriophage P22 coat protein and its entropic stabilization in icosahedral shells.

The pressure stability of bacteriophage P22 coat protein in both monomeric and polymeric forms under hydrostatic pressure was examined using light scattering, fluorescence emission, polarization, and lifetime methodology. The monomeric protein is very unstable toward pressure and undergoes significant structural changes at pressures as low as 0.5 kbar. These structural changes ultimately lead to denaturation of the subunit. Comparison of the protein denatured by pressure to that in guanidine hydrochloride suggests that pressure results in partial unfolding, perhaps by a domain mechanism. Fluorescence lifetime measurements indicate that at atmospheric pressure the local environments of the tryptophans are remarkably similar, suggesting they may be clustered. In contrast to the monomeric protein subunit, the protein when polymerized into procapsid shells is very stable to applied pressure and does not dissociate with pressure up to 2.5 kbar. However, under applied pressure the procapsid shells are cold-labile, suggesting they are entropically stabilized. The significance of these results in terms of virus assembly are discussed.     

Bacteriophage P22 portal protein is part of the gauge that regulates packing density of intravirion DNA.

The complex double-stranded DNA bacteriophages assemble DNA-free protein shells (procapsids) that subsequently package DNA. In the case of several double-stranded DNA bacteriophages, including P22, packaging is associated with cutting of DNA from the concatemeric molecule that results from replication. The mature intravirion P22 DNA has both non-unique (circularly permuted) ends and a length that is determined by the procapsid. In all known cases, procapsids consist of an outer coat protein, an interior scaffolding protein that assists in the assembly of the coat protein shell, and a ring of 12 identical portal protein subunits through which the DNA is presumed to enter the procapsid. To investigate the role of the portal protein in cutting permuted DNA from concatemers, we have characterized P22 portal protein mutants. The effects of several single amino acid changes in the P22 portal protein on the length of the DNA packaged, the density to which DNA is condensed within the virion, and the outer radius of the capsid have been determined. The results obtained with one mutant (NT5/1a) indicate no change (+/- 0.5%) in the radius of the capsid, but mature DNA that is 4.7% longer and a packing density that is commensurately higher than those of wild-type P22. Thus, the portal protein is part of the gauge that regulates the length and packaging density of DNA in bacteriophage P22. We argue that these findings make models for DNA packaging less likely in which the packing density is a property solely of the coat protein shell or of the DNA itself.     

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.     

Determinants of bacteriophage P22 polyhead formation: the role of coat protein flexibility in conformational switching

We have investigated determinants of polyhead formation in bacteriophage P22 in order to understand the molecular mechanism by which coat protein assembly goes astray. Polyhead assembly is caused by amino acid substitutions in coat protein at position 170, which is located in the β‐hinge. In vivo scaffolding protein does not correct polyhead assembly by F170A or F170K coat proteins, but does for F170L. All F170 variants bind scaffolding protein more weakly than wild‐type as observed by affinity chromatography with scaffolding protein‐agarose and scaffolding protein shell re‐entry experiments. Electron cryo‐microscopy and three‐dimensional image reconstructions of F170A and F170K empty procapsid shells showed that there is a decreased flexibility of the coat subunits relative to wild‐type. This was confirmed by limited proteolysis and protein sequencing, which showed increased protection of the A‐domain. Our data support the conclusion that the decrease in flexibility of the A‐domain leads to crowding of the subunits at the centre of the pentons, thereby favouring the hexon configuration during assembly. Thus, correct coat protein interactions with scaffolding protein and maintenance of sufficient coat protein flexibility are crucial for proper P22 assembly. The coat protein β‐hinge region is the major determinant for both features.     

In vitro unfolding/refolding of wild type phage P22 scaffolding protein reveals capsid-binding domain.

The scaffolding proteins of double-stranded DNA viruses are required for the polymerization of capsid subunits into properly sized closed shells but are absent from the mature virions. Phage P22 scaffolding subunits are elongated 33-kDa molecules that copolymerize with coat subunits into icosahedral precursor shells and subsequently exit from the precursor shell through channels in the procapsid lattice to participate in further rounds of polymerization and dissociation. Purified scaffolding subunits could be refolded in vitro after denaturation by high temperature or guanidine hydrochloride solutions. The lack of coincidence of fluorescence and circular dichroism signals indicated the presence of at least one partially folded intermediate, suggesting that the protein consisted of multiple domains. Proteolytic fragments containing the C terminus were competent for copolymerization with capsid subunits into procapsid shells in vitro, whereas the N terminus was not needed for this function. Proteolysis of partially denatured scaffolding subunits indicated that it was the capsid-binding C-terminal domain that unfolded at low temperatures and guanidinium concentrations. The minimal stability of the coat-binding domain may reflect its role in the conformational switching needed for icosahedral shell assembly.     

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.     

Regulation of coat protein polymerization by the scaffolding protein of bacteriophage P22.

In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approximately 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly (King and Casjens. 1974. Nature (Lond.). 251:112-119). To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble subunits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.     

Conformational states of the bacteriophage P22 capsid subunit in relation to self-assembly

The formation of closed icosahedral capsids from a single species of coat protein subunit requires that the subunits assume different conformations at different lattice positions. In the double-stranded DNA bacteriophage P22, formation of correctly dimensioned capsids is mediated by interaction between coat protein subunits and scaffolding protein. Raman spectroscopy has been employed to compare the conformations of coat protein subunits which have been polymerized to form capsids in the presence and absence of the of scaffolding protein display a Raman spectrum characterized by a broad amide I band centered at 1665 cm-1 with a discernible shoulder near 1653 cm-1, and a broad amide III profile centered at 1238 cm-1 but asymmetrically skewed to higher frequency. These spectral features indicate that the protein conformation in procapsid shells is rich in beta-sheet secondary structure but contains also a significant distribution of alpha-helix. When biologically active, purified subunits assemble in the absence of scaffolding protein, they form polydisperse multimers lacking the proper dimensions of procapsid closed shells. We designate these multimers as "associated subunits" (AS). The Raman spectrum of associated subunits indicates a narrower distribution of secondary structure. The associated subunits are characterized by a sharper and more intense Raman amide I band at 1666 cm-1, with no prominent amide I shoulder of lower frequency. An analogous narrowing of the Raman amide III profile is also observed for AS particles, with an accompanying shift of the amide III band center to 1235 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational transformations in the protein lattice of phage P22 procapsids.

During the packaging of double-stranded DNA by bacterial viruses, the precursor procapsid loses its internal core of scaffolding protein and undergoes a substantial expansion to form the mature virion. Here we show that upon heating, purified P22 procapsids release their scaffolding protein subunits, and the coat protein lattice expands in the absence of any other cellular or viral components. Following these processes by differential scanning calorimetry revealed four different transitions that correlated with structural transitions in the coat protein shells. Exit of scaffolding protein from the procapsid occurred reversibly and just above physiological temperature. Expansion of the procapsid lattice, which was exothermic, occurred after the release of scaffolding protein. Partial denaturation of coat subunits within the intact shell structure was detected prior to the major endothermic event. This major endotherm occurred above 80 degrees C and represents particle breakage and irreversible coat protein denaturation. The results indicate that the coat subunits are designed to form a metastable precursor lattice, which appears to be separated from the mature lattice by a kinetic barrier.     

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.     

Improved method for selecting RNA-binding activities in vivo.

RNA challenge phages are modified versions of bacteriophage P22 that allow one to select directly for a specific RNA-protein interaction in vivo. The original construction method for generating a bacteriophage that encodes a specific RNA target requires two homologous recombination reactions between plasmids and phages in bacteria. An improved method is described that enables one to readily construct RNA challenge phages through a single homologous recombination reaction in vivo. We have applied the new method to construct a derivative of P22R17, an RNA challenge phage that undergoes lysogenic development in bacterial cells that express the bacteriophage R17/MS2 coat protein.     

Cavity defects in the procapsid of bacteriophage P22 and the mechanism of capsid maturation

Bacteriophage P22 belongs to a family of double-stranded DNA viruses that share common morphogenetic features like DNA packaging into a procapsid precursor and maturation. Maturation involves cooperative expansion of the procapsid shell with concomitant lattice stabilization. The expansion is thought to be mediated by movement of two coat protein domains around a hinge. The metastable conformation of subunit within the procapsid lattice is considered to constitute a late folding intermediate. In order to understand the mechanism of expansion it is necessary to characterize the interactions stabilizing procapsid and mature capsid lattices, respectively. We employ pressure dissociation to compare subunit packing within the procapsid and expanded lattice. Procapsid shells contain larger cavities than the expanded shells, presumably due to polypeptide packing defects. These defects contribute to the metastable nature of the procapsid lattice and are cured during expansion. Improved packing contributes to the increased stability of the expanded shell. Comparison of two temperature-sensitive folding (tsf) mutants of coat protein (T294I and W48Q) with wild-type coat revealed that both mutations markedly destabilized the procapsid shell and yet had little effect on relative stability of the monomeric subunit. Thus, the regions affected by these packing defects constitute subunit interfaces of the procapsid shell. The larger activation volume of pressure dissociation observed for both T294I and W48Q indicates that the decreased stability of these particles is due to increase of cavity defects. These defects in the procapsid lattice are cured upon expansion suggesting that the intersubunit contacts affected by tsf mutations are absent or rearranged in the mature shell. The energetics of the in vitro expansion reaction also suggests that entropic stabilization contributes to the large free energy barrier for expansion.     

RNA recognition site of PP7 coat protein

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.     

Unraveling the Role of the C-terminal Helix Turn Helix of the Coat-binding Domain of Bacteriophage P22 Scaffolding Protein

Many viruses encode scaffolding and coat proteins that co-assemble to form procapsids, which are transient precursor structures leading to progeny virions. In bacteriophage P22, the association of scaffolding and coat proteins is mediated mainly by ionic interactions. The coat protein-binding domain of scaffolding protein is a helix turn helix structure near the C terminus with a high number of charged surface residues. Residues Arg-293 and Lys-296 are particularly important for coat protein binding. The two helices contact each other through hydrophobic side chains. In this study, substitution of the residues of the interface between the helices, and the residues in the β-turn, by aspartic acid was used examine the importance of the conformation of the domain in coat binding. These replacements strongly affected the ability of the scaffolding protein to interact with coat protein. The severity of the defect in the association of scaffolding protein to coat protein was dependent on location, with substitutions at residues in the turn and helix 2 causing the most significant effects. Substituting aspartic acid for hydrophobic interface residues dramatically perturbs the stability of the structure, but similar substitutions in the turn had much less effect on the integrity of this domain, as determined by circular dichroism. We propose that the binding of scaffolding protein to coat protein is dependent on angle of the β-turn and the orientation of the charged surface on helix 2. Surprisingly, formation of the highly complex procapsid structure depends on a relatively simple interaction.     

Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.