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.     

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.     

Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation

The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.     

Folding and assembly of oligomeric proteins in Escherichia coli.

High levels of expression of oligomeric proteins in heterologous systems are frequently associated with misfolding and accumulation of the polypeptides in inclusion bodies. This reflects aspects of the folding and assembly pathways of oligomeric proteins, which generally proceed from either folding intermediates or native-like metastable species that are not in their final conformation. Methods for optimizing the yield of correctly assembled oligomers are discussed.     

Quantitative analysis of multi-component spherical virus assembly: scaffolding protein contributes to the global stability of phage P22 procapsids

Assembly of the hundreds of subunits required to form an icosahedral virus must proceed with exquisite fidelity, and is a paradigm for the self-organization of complex macromolecular structures. However, the mechanism for capsid assembly is not completely understood for any virus. Here we have investigated the in vitro assembly of phage P22 procapsids using a quantitative model specifically developed to analyze assembly of spherical viruses. Phage P22 procapsids are the product of the co-assembly of 420 molecules of coat protein and approximately 100-300 molecules of scaffolding protein. Scaffolding protein serves as an assembly chaperone and is not part of the final mature capsid, but is essential for proper procapsid assembly. Here we show that scaffolding protein also affects the thermodynamics of assembly, and for the first time this quantitative analysis has been performed on a virus composed of more than one type of protein subunit. Purified coat and scaffolding proteins were mixed in varying ratios in vitro to form procapsids. The reactions were allowed to reach equilibrium and the proportion of the input protein assembled into procapsids or remaining as free subunits was determined by size exclusion chromatography and SDS-PAGE. The results were used to calculate the free energy contributions for individual coat and scaffolding proteins. Each coat protein subunit was found to contribute -7.2(+/-0.1)kcal/mol and each scaffolding protein -6.1(+/-0.2)kcal/mol to the stability of the procapsid. Because each protein interacts with two or more neighbors, the pair-wise energies are even less. The weak protein interactions observed in the assembly of procapsids are likely important in the control of nucleation, since an increase in affinity between coat and scaffolding proteins can lead to kinetic traps caused by the formation of too many nuclei. In addition, we find that adjusting the molar ratio of scaffolding to coat protein can alter the assembly product. When the scaffolding protein concentration is low relative to coat protein, there is a correspondingly low yield of proper procapsids. When the relative concentration is very high, too many nuclei form, leading to kinetically trapped assembly intermediates.     

Kinetics of c2-repressor synthesis in a regulatory defective P22 mutant

Summary Phage P22 defective in gene 24 and harbouring the o^c mutation k5 in O_R exhibits a strongly increased c2-repressor synthesis after infection of non-lysogenic S. typhimurium. The repressor synthesis depends strictly on an intact c1 gene. The kinetics of its synthesis, as monitored by polyacrylamid gel electrophoresis, is the same as with P22 c ⁺, namely a turn off 8–10 min after infection. — After infection of P22-lysogenic bacteria with either P22 24 ^– k5 or P22 24 ^– k5 cl, much lower amounts of repressor are synthesized but again with the same kinetics. These results suggest a cro-like function acting at P_RE and P_RM of P22. The possible reason for the c2 overproduction is discussed.     

Folding and assembly of beta-barrel membrane proteins

Beta-barrel membrane proteins occur in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The membrane-spanning sequences of beta-barrel membrane proteins are less hydrophobic than those of alpha-helical membrane proteins, which is probably the main reason why completely different folding and membrane assembly pathways have evolved for these two classes of membrane proteins. Some beta-barrel membrane proteins can be spontaneously refolded into lipid bilayer model membranes in vitro. They may also have this ability in vivo although lipid and protein chaperones likely assist with their assembly in appropriate target membranes. This review summarizes recent work on the thermodynamic stability and the mechanism of membrane insertion of beta-barrel membrane proteins in lipid model and biological membranes. How lipid compositions affect folding and assembly of beta-barrel membrane proteins is also reviewed. The stability of these proteins in membranes is not as large as previously thought (<10 kcal/mol) and is modulated by elastic forces of the lipid bilayer. Detailed kinetic studies indicate that beta-barrel membrane proteins fold in distinct steps with several intermediates that can be characterized in vitro. Formation of the barrel is synchronized with membrane insertion and all beta-hairpins insert simultaneously in a concerted pathway.     

Identification of additional coat-scaffolding interactions in a bacteriophage P22 mutant defective in maturation

Scaffolding proteins play a critical role in the assembly of certain viruses by directing the formation and maturation of a precursor capsid. Using electron cryomicroscopy difference mapping, we have identified an altered arrangement of a mutant scaffolding within the bacteriophage P22 procapsid. This mutant scaffolding allows us to directly visualize scaffolding density within the P22 procapsid. Based on these observations we propose a model for why the mutant prevents scaffolding release and capsid maturation.     

Transformation and characterization of mutant human fibroblasts defective in peroxisome assembly.

Human skin fibroblasts deficient in peroxisome biogenesis were transformed by transfecting SV40 ori- DNA with the use of an electroporator, and the biochemical, immunocytochemical, and cytogenetic properties of the transformants were analyzed. Cells (1 x 10(6)) from a patient with Zellweger syndrome and one with neonatal adrenoleukodystrophy were suspended with 2 micrograms of SV40 ori- DNA in PBS; then a high-voltage pulse (2000 V, 30 microseconds) was generated two times. Several colonies expressing large T-antigen were picked up 4 weeks after transfection. Doubling time of the transformants was about half of that and the saturation density was 5 to 10 times greater than that of the parental cells. Biochemical abnormalities including defective lignoceric acid oxidation, dihydroxyacetone phosphate acyltransferase deficiency, and disturbed biosynthesis of peroxisomal beta-oxidation enzymes were preserved in the transformants. Peroxisomes were defective in all colonies, as determined by immunofluorescence staining using anti-catalase IgG. Cell fusion studies confirmed that the transformants belong to the same complementation groups as those of the parental cells. These transformed mutant cell lines are expected to be useful tools for investigating the pathogenesis of inherited diseases related to defects in peroxisome biogenesis.     

E proteins of bacteriophage P22. I. Identification and ejection from wild-type and defective particles.

Of the nine proteins found in the virion of phage P22, four are ejected into the cell after adsorption. The four ejected proteins, termed E proteins, are gp16, gp20, gp26, and gp7. This was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled phage that had been adsorbed to cells and then eluted off the surface with distilled water. Phage particles that lack gp7 (7- particles) or gp20 (20- particles) successfully eject all their E proteins. The 16- particles do not eject gp7. Analysis of phage ghosts showed that they lack gp16, gp20, and gp7, but they have gp26 in close to normal quantities. Our results suggest roles for gp16 and gp26 in DNA and E protein ejection. All four E proteins are possible candidates for roles in helping the phage DNA cross the plasma membrane.     

In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins

A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.     

Changes in the capsid structure and stability of defective particles of bacteriophage R17.

Serological and chemical methods were used to compare the capsid structure and stability of R17 phage and amA31 defective particles. Immunodiffusion analysis demonstrated identity between intact R17 and amA31 capside and between dissociated subunits of both R17 and amA31 and purified coat protein. Radioimmunoassays detected an antibody in R17 antisera that binds to intact R17 but could not be absorbed from R17 antisera with amA31. The R17 antibody remaining in amA31-absorbed sera did not neutralize infectivity of R17 phage. Differences between the surface composition of R17 and amA31 capsids were also detected by iodination. Capsids of R17 bound approximately four times more 125I than amA31, which was accounted for by a decreased 125I labeling of coat protein. Finally, amA31 capsids dissociated under milder conditions of sodium dodecyl sulfate treatment than R17 capsids. The sodium dodecyl sulfate dissociation of both R17 and amA31 capsids resulted in the formation of a transient 38,000-dalton intermediate, which subsequently dissociated to coat protein monomers. Preparations of dissociated R17 capsids also contained assembly protein was also found in preparations of dissociated amA31 capsids.     

Assembly-controlled autogenous modulation of bacteriophage P22 scaffolding protein gene expression.

In the assembly of bacteriophage P22, precursor particles containing two major proteins, the gene 5 coat protein and the gene 8 scaffolding protein, package the DNA molecule. During the encapsidation reaction all of the scaffolding protein molecules are released intact and subsequently participate in further rounds of DNA encapsidation. We have previously shown that even though it lies in the center of the late region of the genetic map, the scaffolding protein gene is not always expressed coordinately with the remainder of the late proteins and that some feature of the phage assembly process affects its expression. We present here in vivo experiments which show that there is an inverse correlation between the amount of unassembled scaffolding protein and the rate of scaffolding protein synthesis and that long amber fragments of the scaffolding protein can turn down the synthesis of intact scaffolding protein in trans. These results support a model for scaffolding protein regulation in which the feature of the assembly process which modulates the rate of scaffolding protein synthesis is the amount of unassembled scaffolding protein itself.     

Conformational stability of adrenodoxin mutant proteins.

Adrenodoxin and the mutants at the positions T54, H56, D76, Y82, and C95, as well as the deletion mutants 4-114 and 4-108, were studied by high-sensitivity scanning microcalorimetry, limited proteolysis, and absorption spectroscopy. The mutants show thermal transition temperatures ranging from 46 to 56 degrees C, enthalpy changes from 250 to 370 kJ/mol, and heat capacity change delta Cp = 7.28 +/- 0.67 kJ/mol/K, except H56R. The amino acid replacement H56R produces substantial local changes in the region around positions 56 and Y82, as indicated by reduced heat capacity change (delta Cp = 4.29 +/- 0.37 kJ/mol/K) and enhanced fluorescence. Deletion mutant 4-108 is apparently more stable than the wild type, as judged by higher specific denaturation enthalpy and resistance toward proteolytic degradation. No simple correlation between conformational stability and functional properties could be found.     

Folding and stability of membrane transport proteins in vitro

Transmembrane transporters are responsible for maintaining a correct internal cellular environment. The inherent flexibility of transporters together with their hydrophobic environment means that they are challenging to study in vitro, but recently significant progress been made. This review will focus on in vitro stability and folding studies of transmembrane alpha helical transporters, including reversible folding systems and thermal denaturation. The successful re-assembly of a small number of ATP binding cassette transporters is also described as this is a significant step forward in terms of understanding the folding and assembly of these more complex, multi-subunit proteins. The studies on transporters discussed here represent substantial advances for membrane protein studies as well as for research into protein folding. The work demonstrates that large flexible hydrophobic proteins are within reach of in vitro folding studies, thus holding promise for furthering knowledge on the structure, function and biogenesis of ubiquitous membrane transporter families. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

A minor capsid protein P30 is essential for bacteriophage PRD1 capsid assembly.

Bacteriophage PRD1 is a double-stranded DNA virus infecting Gram-negative hosts. It has a membrane component located in the interior of the isometric capsid. In addition to the major capsid protein P3, the capsid contains a 9 kDa protein P30. Protein P30 is proposed to be located between the adjacent facets of the icosahedral capsid and is required for stable capsid assembly. In its absence, an empty phage-specific membrane vesicle is formed. The major protein component of this vesicle is a phage-encoded assembly factor, protein P10, that is not present in the final structure.     

Bacteriophage-specific DNA-binding proteins in P22-lysogenic and in P22-infected Salmonella typhimurium.

Crude extracts of Salmonella typhimurium lysogenic for phages P22 or L contain proteins that specifically retain phage DNA on nitrocellulose filters. Three DNA-binding activities were found after infection with P22. One is P22 specific, accounts for the largest proportion of DNA-binding proteins, and corresponds most likely to the c2 repressor. An early transient binding activity measured with both P22 and L DNA was found to be directly related to the expression of genes c1 and c3. A third, late binding activity for P22 and L DNA is related to phage production.     

Poliovirus capsid proteins derived from P1 precursors with glutamine-valine cleavage sites have defects in assembly and RNA encapsidation.

Assembly of poliovirus virions requires proteolytic cleavage of the P1 capsid precursor polyprotein between two separate glutamine-glycine (QG) amino acid pairs by the viral protease 3CD. In this study, we have investigated the effects on P1 polyprotein processing and subsequent assembly of processed capsid proteins caused by substitution of the glycine residue at the individual QG cleavage sites with valine (QG-->QV). P1 cDNAs encoding the valine substitutions were created by site-directed mutagenesis and were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses which expressed the mutant P1 precursors. The recombinant vaccinia virus-expressed mutant P1 polyproteins were analyzed for proteolytic processing defects in cells coinfected with a recombinant vaccinia virus (VVP3) that expresses the poliovirus 3CD protease and for processing and assembly defects by using a trans complementation system in which P1-expressing recombinant vaccinia viruses provide capsid precursor to a defective poliovirus genome that does not express functional capsid proteins (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The QV-substituted precursors were proteolytically processed at the altered sites both in cells coinfected with VVP3 and in cells coinfected with defective poliovirus, although the kinetics of cleavage at the altered sites were slower than those of cleavage at the wild-type QG site in the precursor. Completely processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor containing a valine at the amino terminus of VP3 (VP3-G001V) were unstable and failed to assemble stable subviral structures in cells coinfected with defective poliovirus. In contrast, capsid proteins derived from the P1 precursor with a valine substitution at the amino terminus of VP1 (VP1-G001V) assembled empty capsid particles but were deficient in assembling RNA-containing virions. The assembly characteristics of the VP1-G001V mutant were compared with those of a previously described VP3-VP1 cleavage site mutant (K. Kirkegaard and B. Nelsen, J. Virol. 64:185-194, 1990) which contained a deletion of the first four amino-terminal residues of VP1 (VP1-delta 1-4) and which was reconstructed for our studies into the recombinant vaccinia virus system. Complete proteolytic processing of the VP1-delta 1-4 precursor also occurred more slowly than complete cleavage of the wild-type precursor, and formation of virions was delayed; however, capsid proteins derived from the VP1-G001V mutant assembled RNA-containing virions less efficiently than those derived from the VP1-delta 1-4 precursor.(ABSTRACT TRUNCATED AT 400 WORDS)