A minimum of 60 copies of the capsid protein is necessary to make an icosahedral particle, in which case all the subunits have identical environments. Not all T values are permissible although some disallowed T numbers can occur in practice [ 38 ] as some T numbers cannot be arranged with quasi-symmetric environments. However, for each virus there is usually a dominant capsid organization.
This implies that virus assembly must ensure that the capsid proteins form a unique structure with a specific T number. A different way of increasing capsid size is assembling an elongated or prolate head, by inserting an additional cylindrical section between the two icosahedral end-caps.
Such a structure is described by two triangulation numbers: a T number is used for the terminal caps and a Q number for the cylindrical section. As was pointed out by Moody [ 40 ], elongation of the head in one direction does not affect receptor binding sites in bacteriophages, because only the vertex where the tail is attached to the head is functional for cell binding.
In a prolate head there are two types of five-fold vertices, the ten surrounding the cylindrical section and the two five-folds at the center of the caps. These differences would affect receptor attachment and uncoating in viruses that otherwise use any of the five-fold vertices during infection e. Most dsDNA tailed phages assemble their capsid from multiple copies of one capsid protein. However, bacteriophage T4 encodes two capsid proteins, one forming the pentameric capsomers, consisting of gp24, and the other hexameric capsomers, consisting of gp Although both are present in the wild-type T4 phage, a mutant gp23 can form both hexameric and pentameric capsomers, producing a capsid that contains only one type of capsid protein [ 41 — 43 ].
Many eukaryotic viruses, like herpesvirus, adenovirus, picornaviruses, PBCV-1 and others, code for more than one capsid protein [ 45 ]. Although the presence of several different proteins requires a larger viral genome, it might provide some evolutionary advantages.
For example, the problem of accommodating quasi-equivalent environments in the vicinity of pentameric vertices is mitigated. Additionally, mutations would be easier to accommodate, as they would affect only certain positions in the capsid [ 23 ]. An empty shell, called the prohead or procapsid, is usually a required stable assembly intermediate which is then packaged with DNA. The difference in the structure of proheads and mature heads is discussed in Section 2.
Prohead assembly is not unique to bacteriophages, but also occurs in many other viruses such as adenoviruses, herpesviruses, poxviruses and the giant mimivirus [ 18 — 21 ].
The similarities between tailed phages and herpesviruses are even more striking as they were shown to share the same capsid protein fold [ 24 ], termed a HKlike fold, which was first found in bacteriophage HK97 [ 46 ]. However, some phages have domains in the capsid protein structure that are not part of the HK97 fold. Such domains are structurally distinct in different phages, represented by BIG2-like domain in phi29 [ 47 ], a chitin-binding-like domain in T4 [ 44 ] and an insertion domain in P22 [ 48 — 50 ].
In T4 and phi29 these insertion domains stabilize the capsid, by bridging the neighboring molecules within one capsomer T4 or between neighboring capsomers phi Although the capsid protein of HK97 does not have an insertion domain, the capsid is stabilized through a network of covalent cross-links [ 46 ].
Although complementation showed that T7 capsid without the larger protein is as stable as the wild-type virus, this frameshift is conserved in the related phage T3 [ 51 ].
A capsid protein by itself is not capable of ensuring the correct geometry of the capsid shell, requiring an additional scaffolding protein for prohead assembly [ 8 , 52 , 53 ].
Most phages have a separate scaffolding protein gene, with the exception of HK97 and T5 that have scaffolding domains, or delta-domains, within capsid protein sequences [ 54 — 56 ].
In assembled procapsids, scaffolding proteins form a core inside the prohead, which does not have the icosahedral symmetry of the outer capsid shell. Many phage scaffolding proteins, including those of P22, phi29 and SPP1, form dimers and tetramers [ 57 — 59 ], increasing the local concentration of capsid proteins and therefore acting as an entropic sink and promoting association of coat proteins [ 8 , 53 ].
Without scaffolding most capsid proteins form aberrant structures. In the case of P22, the assembly of aberrant particles proceeds ten-times slower than head assembly in the presence of the scaffolding protein [ 60 ]. A mutation in the scaffolding protein of phi29 leads to the formation of isometric particles instead of prolate [ 61 ].
In the absence of scaffolding proteins, capsid proteins of T4 and lambda can assemble into long cylindrical structures [ 62 — 64 ]. Similarly, bacteriophage T5 capsid protein can form open tubular assemblies under certain conditions, even though the scaffolding domain is a part of the capsid protein sequence [ 56 ].
Assembly of bacteriophage T7 into polycapsids occurs in the presence of scaffolding protein, but only when the ratio of scaffolding protein to the capsid protein is 0. The resulting polycapsids consist almost entirely of head protein [ 66 ]. Both the phi29 scaffolding protein and the C-terminal portion of the P22 scaffolding protein have a helix-loop-helix structure [ 58 , 67 ].
Moreover, the sequence alignment suggests that the N-terminal delta domains of HK97 and T5 would have a similar motif [ 58 ]. The binding sites of P22 scaffolding protein on the capsid were determined by electron microscopy, but the arrangement of the scaffolding protein inside the capsid could not be visualized due to icosahedral averaging [ 57 , 68 , 69 ].
The reconstruction of the phi29 prohead assuming no symmetry showed a cage-like scaffolding protein density inside the capsid, organized in several shells with different symmetry [ 58 ]. In contrast to the majority of phages that code for one scaffolding protein, the bacteriophage T4 capsid assembly requires six different scaffolding, or core, proteins [ 70 , 71 ].
Co-expression of these proteins results in formation of tubular polycores which are unique to T4 [ 72 ].
Although it was previously shown that the core of T4 has six-fold symmetry, a later re-examination indicated that polycores could have six-, eight- or ten-fold symmetry [ 73 ]. How exactly scaffolding proteins influence the shape of the head is unknown. The third essential component of head assembly is a portal protein, also called a connector, which is situated on one pentameric vertex of the capsid.
As these names suggest, portal or connector is required for DNA entry and release, as well as for attachment of the neck proteins to the head. Additionally, the phage head assembly is probably initiated from the portal vertex by copolymerization of the scaffolding and capsid proteins [ 3 , 15 , 74 ]. In bacteriophage T4, the ellipsoidal cores are formed in the absence of the capsid protein [ 75 ], whereas in many other phages the scaffolding proteins cannot assemble without the presence of capsid proteins.
Assembly initiation in bacteriophage T4 additionally requires a membrane scaffolding protein, which interacts with the connector protein [ 76 ]. The membrane association of proheads during the assembly was not demonstrated for other phages. The recombinantly expressed portal proteins were shown to assemble into different oligomers, but in all structurally characterized phages the portal protein forms a dodecameric ring [ 10 ].
Therefore, to ensure proper prohead assembly the portal has to co-assemble with the capsid and scaffolding proteins [ 77 , 78 ]. Electron microscopy and X-ray crystallography have shown that the basic morphology of the portal ring is similar in all known tailed phages [ 79 — 82 ]. Some phages, like P22, HK97 and T7, can form prohead-like structures in the absence of portal proteins. However, these virus-like particles cannot package DNA and represent a dead end assembly.
It was noted earlier that aberrant particles often form slower than the viral precursors, indicating that connector and scaffolding affect not only the accuracy but the kinetics of the assembly process [ 60 , 83 , 84 ]. Once the head is assembled, the packaging complex binds and utilizes ATP hydrolysis to translocate the genome into the head [ 5 , 13 , 85 , 86 ]. Because of a symmetry mismatch between the dodecameric portal ring and the five-fold capsid vertex, it was suggested that the portal ring is rotating during packaging [ 87 ].
Later single-molecule spectroscopy of the phi29 connector [ 88 ] and studies on T4 connector in which its motion was restricted by binding to the capsid [ 89 ] showed that connector rotation does not accompany packaging. Many phages produce head-to-tail multimers, or concatemers of DNA, and these are used as a packaging substrate in T4, lambda, and P In that case, the ATPase packages the length of the genome or more in some phages and cuts the concatemer.
This enzyme is also called a large terminase to distinguish it from the small terminase, which does not cut the DNA. The small terminase modulates the activity of the large terminase and is involved in packaging initiation [ 5 , 90 ]. The crystal structure of a small terminase from a Podoviridae phage Sf was solved by X-ray crystallography, suggesting it could form a ring below the large terminase [ 91 ].
Bacteriophage phi29 does not have a small terminase, but it utilizes a unique viral encoded structural RNA, called p-RNA [ 92 , 93 ]. Phages that utilize concatemeric DNA package more than one length of the genome, or a headful.
The determination of when the head is full is not well understood. There are known mutations in T4, P22 and SPP1 that demonstrate the interplay between the terminase and portal protein ring [ 74 , 94 — 97 ].
Therefore, conformational changes in the connector are thought to provide the signal for the terminase to cut the DNA and dissociate from the head [ 5 , 74 , 82 ]. Single molecule studies of the phi29 packaging motor [ 98 , 99 ] and the crystal structure of T4 terminase [ ], as well as the structure of the dsRNA packaging enzyme from phage phi12 [ ], have formed the basis for several packaging mechanisms [ 13 , — ].
The packaged genome inside the phage head is wound into a spool-like structure such that several layers of dsDNA are visible in electron micrographs of individual virions as well as in cryo-EM image reconstructions of phage heads [ 10 , ]. There are several proposed models for the formation of the spool structure [ 5 , 12 , , ]. The DNA end that is packaged first is likely associated with the inside surface of the capsid, whereas the end that is packaged last is probably the first to be ejected [ 5 ].
During or before packaging the scaffolding proteins either exit from the capsid P22, phi29 or are proteolytically cleaved by phage encoded protease HK97, T4. The recycled P22 scaffolding protein can be reused four more times in the assembly process [ ]. Lambda scaffolding protein can also exit from the capsid without being cleaved, as was shown for the phage with a genetically inactivated protease [ ].
The release of scaffolding proteins could be induced by their interaction with DNA, possibly through a leucine-zipper motif, identified in the scaffolding protein of phi29 [ 58 ]. The exit of the scaffolding proteins and the packaging of DNA initiate maturation, involving a large structural transition of the prohead and resulting in a bigger, angular and more stable head with a thinner shell [ 3 , 6 , 8 , 9 , 14 , 40 , 64 ].
In contrast to mature heads, proheads can dissociate into subunits at low concentration [ ], which could allow proofreading and correction of misassembled intermediates.
Additionally, the thick shell of the prohead might make it easier to control the curvature during assembly [ 40 ]. In some phages, like HK97 and T4, cleavage of the capsid and scaffolding proteins precedes the expansion, but the cleaved-unexpanded intermediates are short-lived. The viral protease of T4 cleaves about 3, peptide bonds per virion [ 70 ]. Cleavages of capsid protein can affect the thermodynamic stability of the capsid through changes of quaternary interactions [ 23 ] and could also influence the kinetic stability of capsid protein as shown for self-cleaving enzymes [ , ].
In bacteriophage lambda, in addition to the capsid protein cleavage, the connector protein is also cleaved [ ]. Although this cleavage is not essential for assembly, it might play a role during DNA ejection [ 23 ].
In vitro treatment of proheads with denaturants can also trigger maturation, probably through unfolding of the domains that are cleaved by protease in vivo. Maturation intermediates of HK97 proheads and T4 polyheads have been trapped in vitro [ — ]. Head expansion is probably initiated at one end by the portal protein [ ] and is then propagated through the prohead.
Such a wave was captured in a giant capsid of phage T4, for which several different maturation states were observed along the axis of the head [ ]. In phage P22 skewed capsomers have central holes, through which the scaffolding proteins could exit [ 49 , ]. Although detailed information about the head expansion during maturation is derived mostly from the work on HK97, it is likely to be applicable to other tailed phages, all of which have the same capsid protein fold.
Comparison of the prohead and the mature head crystal structures of HK97 showed that the capsid proteins are probably trapped in a distorted form.
During maturation of HK97, the cross-linking reaction drives the procapsid into a metastable state, where the capsid protein refolds into its lower energy conformation after the delta-domain has been cleaved [ 14 ]. Although the cross-linking is unique for HK97, the binding of capsid stabilization proteins, represented by gp soc in T4 and gpD in lambda, could act similarly during maturation and promote the head expansion [ 14 , — ]. Both gp soc and gpD can only attach to matured capsids, but have different effects on capsid stability.
In the absence of gpD, lambda capsids cannot package the full genome [ ], whereas gp soc is only required in the extremes of pH and temperature [ ]. In the course of head assembly many phages incorporate minor or pilot proteins into the head.
These are usually present in low copy numbers less than 12 subunits and are nonessential for the formation of the structure, but crucial for infectivity of the virion. There are three minor proteins in P22, which modulate DNA ejection [ 52 , ] and were thought to localize in the shaft above the connector [ 82 ]. However, the shaft density was later reassigned to be part of the connector [ 50 ]. A more elaborate shaft structure, called the inner core, is present in the capsid of bacteriophage T7 [ 65 , , ] and consists of a ring of proteins with twelve-fold symmetry immediately above the connector, followed by an eight-fold and a four-fold symmetric protein rings.
A difference in the core structure before and after DNA packaging is perhaps important for the release of terminase after packaging. A similar signal could be propagated by a conformational change of the connector [ 82 , ]. In Siphoviridae and Myoviridae phages there are two types of neck protein, each making a ring below the portal. The neck proteins that form a ring closest to the connector have similar structure in Siphoviridae phages SPP1 and HK97, but have a different fold in lambda.
However, the neck proteins forming the second ring have similar folds in SPP1 and lambda [ — ]. Additionally, a Myoviridae prophage was identified that has structurally similar neck proteins to those of Siphoviridae [ ].
A related structure of the tail-binding platform among Sipho - and Myoviridae phages indicates a common tail binding mechanism and suggests an evolutionary relationship, as well as a possibility that there once existed a phage which could attach two different tail structures. Naturally disordered proteins that participate in protein-protein interactions are abundant in cells and by becoming folded can influence the sequence of assembly [ ].
Additionally, it has been shown that a small disordered protein, which does not have a hydrophobic core, can donate a large surface area to the binding interface. On the other hand, an ordered protein has to be much larger to donate an equivalent surface area to complex.
The presence of unstructured proteins inside the cell was suggested to prevent overcrowding [ ], whereas for the virus it could play a role in keeping the genome size small. After the completion of the head assembly, the tail proteins of Podoviridae phages are sequentially attached to the capsid [ , ]. However, for Sipho- and Myoviridae phages, there is a separate tail assembly branch to the assembly pathway, allowing the preformed tail to bind the head via the neck proteins [ 15 ].
In the case of T4, this is followed by an attachment of the preassembled fibers [ 71 , ]. Although the tail and the head assemblies occur independently, within each pathway protein association follows a strict order, implying that the third component does not bind until the first two proteins form a complex. Likewise, if an assembly component is absent, the assembly is stalled, and all the proteins that would be added after the missing component should have assembled will remain free in solution.
Such a sequential assembly was shown, for example, for the distal part of the T4 contractile tail, called the baseplate [ — ]. In such processes the monomers are added to a growing complex and are not wasted on incomplete intermediates [ 15 ]. The sequential attachment of proteins can be controlled by different mechanisms. One of the mechanisms, very common in protein assemblies, is called conformational switching. Such a process occurs when a protein structure changes upon attachment to an initiator complex and often involves refolding of some part of the structure, for example a loop-to-helix.
Conformational switching is observed in viral capsids with T numbers higher than one, when a capsid protein has to adopt several quasi-equivalent conformations [ — ]. The sequence of assembly can also be controlled though formation of composite binding surfaces, created by more than one protein.
In such a situation the portion of the surface donated by a single protein is insufficient for stable attachment of an assembly component [ 15 , ]. The tail assembly in Siphoviridae and Myoviridae phages starts from the initiator complex, which forms the absorption device of the phage at the distal end of the tail.
The size of this complex ranges from six proteins in Siphoviridae phage lambda or eight in Myoviridae phage Mu [ , ] to about in the baseplate of Myoviridae phage T4 [ 71 , ]. In addition to priming the tail assembly, baseplate complexes undergo structural changes during infection that involve large motions of the component proteins, as was shown for baseteriophage T4 [ ] and Lactococcal Siphoviridae phage p2 [ ].
During tail assembly, baseplate initiates polymerization of the cylindrical section of the tail, which contributes to the majority of the tail mass. In Siphoviridae the cylinder of the tail is composed of multiple copies of the tail tube [ , ], whereas in Myoviridae phages the tail tube is covered by an outer contractile sheath [ , , ].
Initiation of the tail tube polymerization in Sipho - and Myoviridae phages probably occurs via conformational switching. Without the initiator complex the lambda tail tube protein, gpV, cannot form a tubular structure and exists as a monomer. One of the structural proteins of the bacterial complex, the type VI secretion system, is structurally homologous to lambda gpV. In contrast to gpV, the secretion system homolog does not require an initiator to form the tube.
Structural comparison of these homologous tube proteins suggested that a loop-to-helix transition is required to initiate polymerization of gpV [ ]. Similarly to lambda gpV, the tail tube protein of Myoviridae phage T4 does not assemble into tubes without baseplates [ ]. Nevertheless, disassembled tubes of T4 can repolymerize without baseplates [ ], probably because of an irreversible conformational switching that occurred during the initial binding of the tube protein to the baseplate.
In Myoviridae phages after the tail tube is assembled, the tail sheath wraps around it. Siphoviridae phage SPP1 has two tail proteins in the ratio , forming a tail tube, with one of them arising from a translational frame shift [ ]. The larger tail protein is predicted to have an additional immunoglobulin-like domain. Although some immunoglobulin-like folds, such as BIG2 domain of phi29, have a function, the roles of others remain unknown. Such domains may have been acquired by phages from hosts [ ].
The cylinder of the Sipho and Myoviridae tail is almost exclusively six-fold symmetric. The two exceptions are the three-fold symmetric tail of Siphoviridae bacteriophages phiCbK [ ] and T5 [ 55 ].
The six-fold symmetry of the tail might be functionally advantageous due to the interaction of the phages with the oligosaccharides which form hexagonal arrays on the outer surface of some bacterial strains [ 40 ]. After polymerization of the cylindrical part of the tail, binding of terminator proteins completes the tail assembly [ — ].
Terminator proteins, in turn, interact with the neck proteins attached to the head and mediate the association of the tail with the head. The tail-terminator protein of Siphoviridae phage lambda is structurally similar to a Myoviridae prophage protein, providing further evidence of the evolutionary relationship of these tails [ ]. Moreover, structural and functional comparison of neck, tail tube and tail completion proteins suggest that these proteins evolved from a single ancestral gene [ ]. The length of Sipho- and Myoviridae tails is determined by a tape-measure or ruler protein, also found in cellular complexes such as an injectesome also called a type III secretion system and the hook of flagellum [ 26 ].
J Gen Virol. Basic characterization of a lipid-containing bacteriophage specific for plasmids of the P, N, and W compatibility groups. Can J Microbiol. Structural proteins of a lipid-containing bacteriophage which replicates in Escherichia coli: phage PR4.
Isolation and characterization of conditional lethal mutants of Escherichia coli defective in transcription termination factor rho. The molecular weight of bacteriophage phi 6 and its nucleocapsid. Suppression of polarity in the gal operon by ultraviolet irradiation. J Bacteriol. Suppressor mutation in Pseudomonas aeruginosa. Lipid-containing bacteriophage PR4: structure and life cycle.
Assembly of bacteriophage PRD1: particle formation with wild-type and mutant viruses. Isolation of nonsense suppressor mutants in Pseudomonas. Manole V ,. Karhu NJ. Affiliations 1 author 1. Share this article Share with email Share with twitter Share with linkedin Share with facebook. Abstract PRD1 is a tailless icosahedrally symmetric virus containing an internal lipid membrane beneath the protein capsid.
Its linear dsDNA genome and covalently attached terminal proteins are delivered into the cell where replication occurs via a protein-primed mechanism.
Extensive studies have been carried out to decipher the roles of the 37 viral proteins in PRD1 assembly, their association in virus particles and lately, especially the functioning of the unique packaging machinery that translocates the genome into the procapsid. These issues will be addressed in this chapter especially in the context of the structure of PRD1. We will also discuss the major challenges still to be addressed in PRD1 assembly. Full text links Read article at publisher's site DOI : Smart citations by scite.
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Electron cryo-microscopy of bacteriophage PR reveals the elusive vertex complex and the capsid architecture. Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses. Similar Articles To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.
Purified membrane-containing procapsids of bacteriophage PRD1 package the viral genome. The major and minor capsid proteins, P3 and P5, occur as soluble multimers before they appear in the empty particles. Nonsense mutants of PRD1 that involve structural proteins of the virion other than P3 form particles that are missing only the defective protein.
Those mutants that are unable to form P3 do not form particles.
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