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

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

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

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

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

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

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.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

Studies related to the head-maturation pathway of bacteriophages T4 and T2: I. Morphology and kinetics of intracellular particles produced by mutants in the maturation genes

Mutants in the genes governing the maturation of the head of bacteriophage T4 and in gene 24 were studied by electron microscopy of thin sections. We define morphologically: black particles, comprising mature, stable heads and immature, fragile heads, which break down upon lysis; grizzled particles, which apparently are partially filled or partially emptied; empty large particles without DNA or core Which are all the same size as normal heads; empty small particles without DNA and without core which are of the size of the τ particle, which is the prehead of phage T4. The study of single and double mutants of the maturation genes demonstrates that the phenotypes are only different by the proportions of the different particles made except for 17^? where only empty small and empty large particles accumulate. The mutants in gene 24 are epistatic on all other mutants. Mutants in gene 17 are epistatic on the remaining ones. The results are consistent with the hypothesis that the products of several of the maturation genes act on DNA to render it competent for packaging while the others act directly on the particle. By this uncoupling, bypasses and abortive pathways can result.     

Head maturation pathway of bacteriophages T4 and T2. III. Isolation and characterization of particles produced by mutants in gene 17

We have isolated and characterized two types of particles produced in comparable amounts by mutants in gene 17: the empty large particle and the empty small particle. Dimensions, morphology, stability, and protein composition of the empty large particle are very similar to those of the capsids or empty heads of mature phage. The other type of particle (empty small particle) is very similar in dimensions and stability to the prehead, but differs in that it is composed of processed proteins (gp23, gp24, IpIII). Structural analysis has shown that the protein subunits of the empty small particles are arranged in an unexpanded type of lattice (11.2 to 11.3 nm), whereas the empty large particles have an expanded lattice (13 nm). The characterization of the empty small particle as being composed of cleaved proteins, but still unexpanded, shows that the expansion of the T4 head shell is not necessarily linked to the cleavage of the structural proteins.     

Sequential Steps in Human Immunodeficiency Virus Particle Maturation Revealed by Alterations of Individual Gag Polyprotein Cleavage Sites

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.     

Head maturation pathway of bacteriophages T4 and T2. V. Maturable epsilon-particle accumulating an acridine-treated bacteriophage T4-infected cells.

A maturable head-related particle of bacteriophage T4 has been identified and characterized. This epsilon-particle has the same size as the prehead, but its shell is made of the cleaved product of gene 23 (gp23*). It contains internal matter, most likely the processed core proteins, which is lost or modified by experimental manipulations. It accumulates, together with partially filled ("grizzled") heads, in T4 infected cells that are treated with 9-aminoacridine. On sections of "well-preserved" cells the epsilon-particles are not identifiable with certainty; a more or less empty breakdown product of them becomes visible when cytoplasmic leakage is induced. The number of particles per cell is then in agreement with the biochemically and with the number of particles counted in lysates. Morphologically and biochemically, the isolated epsilon-particles closely resemble the empty small particles of 17- -infected cells described in previous papers of this series. Both are composed of gp23* and are still unexpanded, so that they are not yet able to bind the minor head proteins soc and hoc. We discuss the possibility of the epsilon-particle being an intermediate on the normal T4 wild-type head maturation pathway.     

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.     

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

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

Amber mutants in gene 67 of phage T4. Effects on formation and shape determination of the head

Two amber mutations in gene 67 of bacteriophage T4 were constructed by oligonucleotide-directed mutagenesis and the resulting mutated genes were recombined back into the phage genome and their phenotype was studied. The 67amK1 mutation is close to the amino terminus of the gene, and phage carrying this mutation are unable to form plaques on suppressor-negative hosts. A second mutation, 67amK2, which lies in the middle of the gene, three codons N-terminal to a proteolytic cleavage site, produces a small number of viable phage particles. In suppressor-negative hosts, both mutants produce polyheads and proheads. 67amK1 assembles only few proheads that have a disorganized core structure, as judged from thin sections of infected cells. The proheads and the mature phages of both mutants are mainly isometric rather than having the usual prolate shape. Depending on the 67 mutant and the host, between 20% and 73% of the particles that are produced are isometric, and 1 to 10% are two-tailed biprolate particles. 67amK2 phages grown on a supD suppressor strain that inserts serine in place of the wild-type leucine do not contain gp67* derived from gene product 67 (gp67) by proteolytic cleavage. This demonstrates the importance of the correct amino acid at this position in the protein. Other abnormalities in these 67amK2 phages are the presence of uncleaved scaffolding core proteins (IPIII and gp68), indicating a structural alteration in the prohead scaffold, resulting in only partial cleavage. In wild-type phages these proteins are found in the head only in the cleaved form. With double-mutants of 67 with mutations in the major shell protein gp23 no naked scaffolding cores were found, confirming the necessity of gp67 for the assembly or persistence of a "normal" core.     

Localization of minor protein components of the head of bacteriophage T4.

The bacteriophage T4 capsid contains a number of minor proteins that are required for head assembly but whose detailed function and position in the head are unknown. We have found that by systematically varying the conditions of extraction, some of these minor proteins can be removed while the main capsid structure is left substantially intact. Electron microscopic examination of the residual capsids showed that the extraction of the product of gene 20 is correlated with the loss of a plug that distinguishes one vertex position (presumably the tail attachment site) from the others. Extraction of the product of gene 24 is correlated with the loss of the other 11 (nonproximal) vertexes of the capsid. We further show that antibody to P24 binds specifically to the nonproximal vertexes of both T4 preheads and T4 phages. On the basis of our findings, we suggest that P20 is located at or near the tail attachment site of the capsid, whereas P24 forms the 11 nonproximal vertexes of preheads and P24 forms the nonproximal vertexes of the mature head.     

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.     

A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition

Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.     

Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging

The multiplication of bacteriophage T7 is blocked in Escherichia coli M. The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E. coli K-12 recipient by means of conjugation. We determined at which precise step T7 maturation is blocked. Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected. Instead of infective phages, only preheads were produced. These, however, were maturable in vitro. The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms. The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed. Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging. As intermediates in the packaging process, we found some prehead-DNA complexes. We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form. This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked. Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.     

Cryo-EM structure of a bacteriophage T4 gp24 bypass mutant: the evolution of pentameric vertex proteins in icosahedral viruses

Many large viral capsids require special pentameric proteins at their fivefold vertices. Nevertheless, deletion of the special vertex protein gene product 24 (gp24) in bacteriophage T4 can be compensated by mutations in the homologous major capsid protein gp23. The structure of such a mutant virus, determined by cryo-electron microscopy to 26 angstroms, shows that the gp24 pentamers are replaced by mutant major capsid protein (gp23) pentamers at the vertices, thus re-creating a viral capsid prior to the evolution of specialized major capsid proteins and vertex proteins. The mutant gp23* pentamer is structurally similar to the wild-type gp24* pentamer but the insertion domain is slightly more distant from the gp23* pentamer center. There are additional SOC molecules around the gp23* pentamers in the mutant virus that were not present around the gp24* pentamers in the wild-type virus.     

Cleavage-site mutagenesis alters post-translation processing of Pro-CCK in AtT-20 cells

Cholecystokinin (CCK) is expressed in the central and peripheral nervous systems and functions as a neurotransmitter and neuroendocrine hormone. The in vivo forms of CCK include CCK-83, -58, -39, -33, -22, -12, and -8. Tissues in the periphery produce the larger forms of CCK, such as CCK-58, whereas the brain primarily produces CCK-8. The different biologically active forms of CCK observed in vivo may result from cell-specific differences in endoproteolytic cleavage during post-translational processing. Evidence suggests that cleavages of pro-CCK occur in a specific sequential order. To further delineate the progression of cleavages during pro-CCK maturation, mutagenesis was used to disrupt putative mono- and dibasic cleavage sites. AtT-20 cells transfected with wild-type rat prepro-CCK secret CCK-22 and -8. Mutagenesis of the cleavage sites of pro-CCK had profound effects on the products that were produced. Substitution of basic cleavage sites with nonbasic amino acids inhibits cleavage and leads to the secretion of pathway intermediates such as CCK-83, -33, and -12. These results suggest that CCK-58 is cleaved to both CCK-33 and -22. Furthermore, CCK-8 and -12 are likely derived from cleavage of CCK-33 but not CCK-22. Alanine substitution at the same site completely blocked production of amidated products, whereas serine substitution did not. The cleavages observed at nonbasic residues in this study may represent the activity of enzymes other than PC1 and carboxypeptidase E, such as the enzyme SKI-1. A model for the progression of pro-CCK processing in AtT-20 cells is proposed. The findings in this study further supports the hypothesis that pro-CCK undergoes parallel pathways of proteolytic cleavages.     

Recovery of Recombinant Crimean Congo Hemorrhagic Fever Virus Reveals a Function for Non-structural Glycoproteins Cleavage by Furin

Crimean Congo hemorrhagic fever virus (CCHFV) is a negative-strand RNA virus of the family Bunyaviridae (genus: Nairovirus). In humans, CCHFV causes fever, hemorrhage, severe thrombocytopenia, and high fatality. A major impediment in precisely determining the basis of CCHFV’s high pathogenicity has been the lack of methodology to produce recombinant CCHFV. We developed a reverse genetics system based on transfecting plasmids into BSR-T7/5 and Huh7 cells. In our system, bacteriophage T7 RNA polymerase produced complementary RNA copies of the viral S, M, and L segments that were encapsidated with the support, in trans, of CCHFV nucleoprotein and L polymerase. The system was optimized to systematically recover high yields of infectious CCHFV. Additionally, we tested the ability of the system to produce specifically designed CCHFV mutants. The M segment encodes a polyprotein that is processed by host proprotein convertases (PCs), including the site-1 protease (S1P) and furin-like PCs. S1P and furin cleavages are necessary for producing the non-structural glycoprotein GP38, while S1P cleavage yields structural Gn. We studied the role of furin cleavage by rescuing a recombinant CCHFV encoding a virus glycoprotein precursor lacking a functional furin cleavage motif (RSKR mutated to ASKA). The ASKA mutation blocked glycoprotein precursor’s maturation to GP38, and Gn precursor’s maturation to Gn was slightly diminished. Furin cleavage was not essential for replication, as blocking furin cleavage resulted only in transient reduction of CCHFV titers, suggesting that either GP38 and/or decreased Gn maturation accounted for the reduced virion production. Our data demonstrate that nairoviruses can be produced by reverse genetics, and the utility of our system uncovered a function for furin cleavage. This viral rescue system could be further used to study the CCHFV replication cycle and facilitate the development of efficacious vaccines to counter this biological and public health threat.     

Functional empty capsid precursors produced by lambda mutant defective for late lambda DNA replication.

This report described lambda phage morphogenesis in a mutant system in which the normal pathways for late phage DNA (concatemer) synthesis are blocked and early (monomeric circular) DNA replication products accumulate. As shown earlier (Dawson et al., 1975) under these conditions, late proteins are synthesized and assembled into headlike structures. These structures that accumulate in the mutant are empty, suggesting the monomeric circular DNA molecules cannot be encapsulated. The present results show that crude extracts of induced lysogens of the mutant contain the complementation activities of preheads (the empty precursors to DNA-filled heads), tails, and DNA terminigenerating protein(s). Sucrose gradients of these crude extracts yield fractions containing prehead activity in relative amounts expected from the concentration of late proteins and empty structures. Furthermore, the proteins present in these fractions coelectrophorese with the known capsid proteins of preheads, and empty structures that look like preheads are observed in electron microscope examination of samples from the fractions. Based on our biological, biochemical, and electron microscope analyses, we conclude that the empty structures that accumulate in the induced lysogen of the mutant are normal preheads, which could become filled phage heads if DNA of the appropriate structure (i.e., "late DNA") were available.