Structure and assembly of the capsid of bacteriophage P22.

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.     

Determinants of bacteriophage phi29 head morphology

Bacteriophage phi29 requires scaffolding protein to assemble the 450 x 540 A prolate prohead with T = 3 symmetry end caps. In infections with a temperature-sensitive mutant scaffolding protein, capsids assemble predominantly into 370 A diameter isometric particles with T = 3 symmetry that lack a head-tail connector. However, a few larger, 430 A diameter, particles are also assembled. Cryo-electron microscopy shows that these larger particles are icosahedral with T = 4 symmetry. The prolate prohead, as well as the two isometric capsids with T = 3 and T = 4 symmetry, are composed of similar pentamers and differently skewed hexamers. The skewing of the hexamers in the equatorial region of proheads and in the T = 4 isometric particles reflects their different environments. One of the functions of the scaffolding protein, present in the prohead, may be to stabilize skewed hexamers during assembly.     

Second-Site Suppressors of a Cold-Sensitive Prohead Accessory Protein of Bacteriophage φX174

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.     

Control of the synthesis of phage p22 scaffolding protein is coupled to capsid assembly

The assembly of the precursor shells of bacteriophage P22 entails the co-polymerization of gene 5 coat protein with gene 8 scaffolding protein into double shell structures. During DNA encapsidation, the inner shell of scaffolding molecules dissociates and exits from the prohead. These molecules then recycle, catalyzing the assembly of newly synthesized coat protein to form new proheads (King and Casjens, 1974).Although gene 5 and gene 8 are adjacent on the phage chromosome, we find that the synthesis of the two proteins is differentially regulated. In productively infected cells, scaffolding protein is synthesized at a low rate relative to the coat protein. In contrast, cells that are infected with mutants blocked in DNA packaging and accumulate precursor shells synthesize scaffolding protein at a much higher rate. If a mutation is introduced into the coat protein gene, however, preventing shell assembly, the rate of scaffolding protein synthesis decreases to less than the wild-type rate.The experiments are consistent with models in which either continued synthesis of scaffolding protein depends upon co-polymerization with coat subunits, or soluble scaffolding subunits (but not assembled subunits) depress their own further synthesis. The finding that amber fragments of the scaffolding protein are synthesized at a very low rate is inconsistent with the second model. There is evidence, however, that fragments of the protein may have regulatory activity.The regulatory circuit couples scaffolding protein synthesis to morphogenesis. Gene dosage experiments show that regulation results in the maintenance of coat and scaffolding subunits in the proper ratio for shell assembly.     

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.     

A proposed structure of bacteriophage T4 gene product 22--a major prohead scaffolding core protein

Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted Mr of 29.9 and a pI of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an alpha-helical conformation and form seven peculiar alpha-helices. A model suggesting the spatial organization of gp22 is presented. Three long alpha-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short alpha-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the alpha alpha-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.     

Proteolysis of bacteriophage phi X174 prohead accessory protein gpB by Escherichia coli OmpT protease is not essential for phage maturation in vivo.

To examine whether cleavage of the phi X174 prohead accessory protein, gpB, by the OmpT protease is required for phage development in vivo, a phage mutant lacking the OmpT cleavage site and an Escherichia coli C delta ompT strain were constructed. The results of burst size experiments suggest that neither the cleavage site nor the OmpT protein is required for phi X174 development.     

Structure of phage P22 coat protein aggregates formed in the absence of the scaffolding protein.

Previous studies have shown that the assembly of the precursor shell (prohead) of bacteriophage P22 requires the copolymerization of the gene 5 coat protein with the gene 8 scaffolding protein. Removal of the scaffolding protein by mutation prevents efficient coat protein assembly, but some aberrant particles do form. We have now isolated these structures and characterized them with respect to morphology, protein composition, and small-angle X-ray scattering properties.The aberrant particles fall into three morphological classes, i.e. complex spirals and closed shells of two sizes. Small-angle X-ray scattering studies confirm that the larger particles are hollow shells with the radius of proheads ($\text{r = 260}\text{A}\text{?}$), and not of the mature virus ($\text{r = 285}\text{A}\text{?}$). These structures lack the inner shell of scaffolding protein found in proheads. The small particles have a radius of 195 Å, smaller than proheads, and appear to contain material, not scaffolding protein, within the outer shell.The aberrant particles contain two minor protein species, the gene 9 tail-spike protein, and an unidentified 67,000 molecular weight polypeptide, probably from the host. Neither is found in normal proheads. Removal of gene.9 product by mutation did not affect the formation of the aggregates. Fractionation of the morphological classes of particles revealed that the 67,000 molecular weight band was associated with the closed shells. It may be serving as a pseudo-initiator.Earlier studies had shown that treatment of proheads with sodium dodecyl sulfate in vitro resulted in loss of the scaffolding protein, and expansion of the shell to the mature radius of 285 Å. When the 8^? prohead-sized shells were treated similarly, they also expanded to the mature-sized shell. These results support the idea that there are at least two stable states of the coat protein, one of which, the prohead form, is an obligatory precursor of the mature form.     

Form determination of the heads of bacteriophages

The shape of the DNA-containing heads of many bacteriophages is not only determined by the properties of the protein subunits which build the shell (capsid) but also by the scaffolding core which is a transient structure of the prohead. The form-determining properties of the scaffolding proteins have been characterized by genetic methods based on conditional mutants and site-directed mutagenesis. The mechanism of form determination has been studied by in vitro assembly experiments. The theoretical background is discussed and different models for mechanisms of form determination are considered. Definitive decisions about the validity of a model is still limited by the difficulty of obtaining unambiguous answers on the stoichiometry and the fine structure of the scaffold because of their high instability.     

Molecular rearrangements involved in the capsid shell maturation of bacteriophage T7

Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A- and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Initial cos cleavage of bacteriophage lambda concatemers requires proheads and gpFI in vivo

The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.     

Composition and mass of the bacteriophage phi29 prohead and virion

The protein composition of the Bacillus subtilis bacteriophage phi29 prohead and virion was determined by combustion of gel bands of (3)H-labeled proteins. Copy numbers of individual proteins were calculated relative to the 12 copies of the head-tail connector protein. The mean numbers of copies of the major capsid protein in the prohead and virion were 241 and 218, respectively, approaching the 235 copies determined previously by cryoelectron microscopy. The mean numbers of copies of the dimeric head fiber on the prohead and virion were 24 and 31, respectively, demonstrating partial occupancy of the 55 fiber binding sites. Measured copies of neck and tail proteins in the virion included 11 of the lower collar, 58 of the appendage, and 9 of the tail; if the true copies of these proteins are 12, 60, and 9, respectively, the entire neck and tail of phi29 has quasi-sixfold symmetry. The mass of the fiberless prohead with pRNA was about 14.2 MDa, and the mass of the prohead determined by scanning transmission electron microscopy was consistent with the biochemical data. The mass of the fiberless virion containing the 12.8-MDa DNA genome was about 30.4 MDa. A full complement of dimeric fibers on the prohead or virion would increase the mass of the particle by about 3.2 MDa. The data complement studies relating the structure of phi29 components to dynamic functions in morphogenesis and infection.     

INVESTIGATION OF SECONDARY STRUCTURES AND MACROMOLECULAR INTERACTIONS IN BACTERIOPHAGE P22 BY LASER RAMAN SPECTROSCOPY

Laser Raman spectra of the DNA bacteriophage P22 and of its precursor particles and related structures have been obtained using 514.5-nm excitation. The spectra show that P22 DNA exists in the B form both inside of the phage head and after extraction from the phage. The major coat protein (gp5) contains a secondary structure composed of 18% α-helix, 20% β-sheet and 62% irregular conformations. The scaffolding protein (gp8) in the phage prohead is substantially richer than gp5 in α-helical content. Among the amino acid residues which give prominent Raman lines, the spectra show that tryptophans are exposed to solvent and most tyrosines are hydrogen bonded to positive donor groups. The above features of phage DNA and protein structures are nearly invariant to changes in temperature up to 80°C, indicating a remarkable thermal stability of the phage head and its encapsulated DNA.     

Second-Site Suppressors of a Cold-Sensitive External Scaffolding Protein of Bacteriophage φX174

This report describes the isolation and characterization of second-site suppressors of a cold-sensitive (cs) external scaffolding protein, gpD, of bacteriophage X174. Seven genetically distinct suppressors were isolated. Six of them are located in gene F which encodes the major coat protein of the virus. The seventh is located in gene J which encodes the DNA-binding protein. A subset of the suppressors are trans-acting. These second-site suppressors do not exhibit allele specificity; they are able to suppress defects associated with a csD protein for which they were not selected. The initial characterization of the second-site suppressors and their locations within the major coat protein suggest that the mechanism of suppression may involve both structural and stoichiometric phenomena.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.     

Maturation of phage T7 involves structural modification of both shell and inner core components

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.     

Proteolysis of bacteriophage phi X174 prohead protein gpB by a protease located in the Escherichia coli outer membrane.

The gene B protein (gpB) of bacteriophage phi X174 is required for prohead assembly and is removed from prohead during phage maturation. Protease activity was observed in isolated prohead which specifically cleaved gpB. Cleavage of gpB produced two fragments that had apparent molecular weights of 12,300 and 3,700 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the fragments confirmed that they resulted from the cleavage of gpB and identified the cleavage site as an Arg-Arg at amino acids 76 to 77 of the 120-amino-acid protein. gpB-specific protease activity was observed in both phi X174-infected and uninfected Escherichia coli extracts. This protease activity was localized to the outer-membrane fraction of uninfected cells. Protease activities present in the outer membrane and in isolated prohead produced identical fragments and had the same protease inhibition profile. The gpB-specific activity in uninfected cells was induced by growth at 42 degrees C and was inhibited by the protease inhibitors, 1,10-phenanthroline, EDTA, and N-ethylmaleimide.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway.

Three classes of particles have been identified in restrictive phi 29 suppressor-sensitive (sus) mutant infections of Bacillus subtilis, including DNA-containing heads or phage, prohead, and empty heads. Pulse-chase labeling experiments indicate that the prohead, the first particle assembled in 14-infected cells, is converted to DNA-filled heads and phi 29. In addition to the proteins Hd, P10, and F found in mature phi 29, the prohead contains a "core" protein P7 that exits as the prohead matures and appears to recycle during subsequent rounds of prohead assembly. Prohead-like structures accumulate in UV-irradiated cells and are present in restrictive infections with sus mutants of cistrons 9 and 16. Empty heads are observed only when infection results in the formation of DNA-containing particles; this and other evidence indicates that the empty heads are probably not true intermediates. Phage phi 29 assembly apparently occurs by a single pathway in which neck and tail components interact to stabilize the completed DNA-containing head.