A thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged

Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450A-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Delta-domain (11kDa) and gp5* (31 kDa): gp5* forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5* hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53 degrees, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation.     

Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T=7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro five- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T=7) and small sized (T=4?) procapsids. Predominant formation of T=7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Efficient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure.     

Identification and characterization of the domain structure of bacteriophage P22 coat protein.

The bacteriophage P22 serves as a model for assembly of icosahedral dsDNA viruses. The P22 procapsid, which constitutes the precursor for DNA packaging, is built from 420 copies of a single coat protein with the aid of stoichiometric amounts of scaffolding protein. Upon DNA entry, the procapsid shell expands and matures into a stable virion. It was proposed that expansion is mediated by hinge bending and domain movement. We have used limited proteolysis to map the dynamic stability of the coat protein domain structures. The coat protein monomer is susceptible to proteolytic digestion, but limited proteolysis by small quantities of elastase or chymotrypsin yielded two metastable fragments (domains). The N-terminal domain (residues 1-180) is linked to the C-terminal domain (residues 205-429) by a protease-susceptible loop (residues 180-205). The two domains remain associated after the loop cleavage. Although only a small change of secondary structure results from the loop cleavage, both tertiary interdomain contacts and subunit thermostability are diminished. The intact loop is also required for assembly of the monomeric coat protein into procapsids. Upon assembly, coat protein becomes largely protease-resistant, baring cleavage within the loop region of about half of the subunits. Loop cleavage decreases the stability of the procapsids and facilitates heat-induced shell expansion. Upon expansion, the loop becomes protease-resistant. Our data suggest the loop region becomes more ordered during assembly and maturation and thereby plays an important role in both of these stages.     

Phage P22 procapsids equilibrate with free coat protein subunits

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

Sequential interactions of structural proteins in phage phi 29 procapsid assembly.

The mechanism of viral capsid assembly is an intriguing problem because of its fundamental importance to research on synthetic viral particle vaccines, gene delivery systems, antiviral drugs, chimeric viruses displaying antigens or ligands, and the study of macromolecular interactions. The genes coding for the scaffolding (gp7), capsid (gp8), and portal vertex (gp10) proteins of the procapsid of bacteriophage phi 29 of Bacillus subtilis were expressed in Escherichia coli individually or in combination to study the mechanism of phi 29 procapsid assembly. When expressed alone, gp7 existed as a soluble monomer, gp8 aggregated into inclusion bodies, and gp10 formed the portal vertex. Circular dichroisin spectrum analysis indicated that gp7 is mainly composed of alpha helices. When two of the proteins were coexpressed, gp7 and gp8 assembled into procapsid-like particles with variable sizes and shapes, gp7 and gp10 formed unstable complexes, and gp8 and gp10 did not interact. These results suggested that gp7 served as a bridge for gp8 and gp10. When gp7, gp8, and gp10 were coexpressed, active procapsids were produced. Complementation of extracts containing one or two structural components could not produce active procapsids, indicating that no stable intermediates were formed. A dimeric gp7 concatemer promoted the solubility of gp8 but was inactive in the assembly of procapsid or procapsid-like particles. Mutation at the C terminus of gp7 prevented it from interacting with gp8, indicating that this part of gp7 may be important for interaction with gp8. Coexpression of the portal protein (gp20) of phage T4 with phi 29 gp7 and gp8 revealed the lack of interaction between T4 gp20 and phi 29 gp7 and/or gp8. Perturbing the ratio of the three structural proteins by duplicating one or another gene did not reduce the yield of potentially infectious particles. Changing of the order of gene arrangement in plasmids did not affect the formation of active procapsids significantly. These results indicate that phi 29 procapsid assembly deviated from the single-assembly pathway and that coexistence of all three components with a threshold concentration was required for procapsid assembly. The trimolecular interaction was so rapid that no true intermediates could be isolated. This finding is in accord with the result of capsid assembly obtained by the equilibrium model proposed by A. Zlotnick (J. Mol. Biol. 241:59-67, 1994).     

Assembly and Maturation of the Bacteriophage Lambda Procapsid: gpC Is the Viral Protease

Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single “portal” structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection. Bacteriophage λ has served as an ideal model system to study the development of the large double-stranded DNA viruses. Within this context, the λ procapsid assembly pathway has been reported to be uniquely complex involving protein cross-linking and proteolytic maturation events. In this work, we identify and characterize the protease responsible for λ procapsid maturation and present a structural model for a procapsid-bound protease dimer. The procapsid protease possesses autoproteolytic activity, it is required for degradation of the internal “scaffold” protein required for procapsid self-assembly, and it is responsible for proteolysis of the portal complex. Our data demonstrate that these proteolytic maturation events are not required for procapsid assembly or for DNA packaging into the structure, but that proteolysis is essential to late steps in particle assembly and/or in subsequent infection of a host cell. The data suggest that the λ-like proteases and the herpesvirus-like proteases define two distinct viral protease folds that exhibit little sequence or structural homology but that provide identical functions in virus development. The data further indicate that procapsid assembly and maturation are strongly conserved in the prokaryotic and eukaryotic virus groups.     

In vitro assembly of the herpes simplex virus procapsid: formation of small procapsids at reduced scaffolding protein concentration

The herpes simplex virus 1 capsid is formed in the infected cell nucleus by way of a spherical, less robust intermediate called the procapsid. Procapsid assembly requires the capsid shell proteins (VP5, VP19C, and VP23) plus the scaffolding protein, pre-VP22a, a major component of the procapsid that is not present in the mature virion. Pre-VP22a is lost as DNA is packaged and the procapsid is transformed into the mature, icosahedral capsid. We have employed a cell-free assembly system to examine the role of the scaffolding protein in procapsid formation. While other reaction components (VP5, VP19C, and VP23) were held constant, the pre-VP22a concentration was varied, and the resulting procapsids were analyzed by electron microscopy and SDS-polyacrylamide gel electrophoresis. The results demonstrated that while standard-sized (T = 16) procapsids with a measured diameter of approximately 100 nm were formed above a threshold pre-VP22a concentration, at lower concentrations procapsids were smaller. The measured diameter was approximately 78 nm and the predicted triangulation number was 9. No procapsids larger than the standard size or smaller than 78-nm procapsids were observed in appreciable numbers at any pre-VP22a concentration tested. SDS-polyacrylamide gel analyses indicated that small procapsids contained a reduced amount of scaffolding protein compared to the standard 100-nm form. The observations indicate that the scaffolding protein concentration affects the structure of nascent procapsids with a minimum amount required for assembly of procapsids with the standard radius of curvature and scaffolding protein content.     

Control of virus assembly: HK97 "Whiffleball" mutant capsids without pentons

The capsid of Escherichia coli bacteriophage HK97 assembles as a 420 subunit icosahedral shell called Prohead I which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature Head II shell. Prohead I have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. We report here the properties of a mutant of the capsid protein, E219K, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wild-type Prohead I, when the mutant structure is dissociated into capsomers in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into "Whiffleballs", particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer.     

Modulation of the viral ATPase activity by the portal protein correlates with DNA packaging efficiency

DNA packaging in tailed bacteriophages and herpesviruses requires assembly of a complex molecular machine at a specific vertex of a preformed procapsid. As in all these viruses, the DNA translocation motor of bacteriophage SPP1 is composed of the portal protein (gp6) that provides a tunnel for DNA entry into the procapsid and of the viral ATPase (gp1-gp2 complex) that fuels DNA translocation. Here we studied the cross-talk between the components of the motor to control its ATP consumption and DNA encapsidation. We showed that gp6 embedded in the procapsid structure stimulated more than 10-fold the gp2 ATPase activity. This stimulation, which was significantly higher than the one conferred by isolated gp6, depended on the presence of gp1. Mutations in different regions of gp6 abolished or decreased the gp6-induced stimulation of the ATPase. This effect on gp2 activity was observed both in the presence and in the absence of DNA and showed a strict correlation with the efficiency of DNA packaging into procapsids containing the mutant portals. Our results demonstrated that the portal protein has an active control over the viral ATPase activity that correlates with the performance of the DNA packaging motor.     

Direct Interaction of the Bacteriophage SPP1 Packaging ATPase with the Portal Protein

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.     

Assembly-dependent conformational changes in a viral capsid protein. Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.     

Initiation of P22 procapsid assembly in vivo

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

Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.     

Conformational transformations in the protein lattice of phage P22 procapsids.

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

Specific targeting of a DNA-binding protein to the SPP1 procapsid by interaction with the portal oligomer

The icosahedral procapsid of tailed bacteriophages is composed of a large number of identical subunits and of minor proteins found in a few copies. Proteins present in a very low copy number are targeted to the viral procapsid by an unknown mechanism. Bacteriophage SPP1 procapsids and mature virions contain two copies of gp7 on average. Gp7 forms stable complexes with the SPP1 portal protein gp6. Deletion of the gp6 carboxyl-terminus and the mutation Y467-->C localized in the same region prevent gp6-gp7 complex formation. Gp7 binds double-stranded and single-stranded DNA. Gp6 competes for this interaction, and purified gp6-gp7 complexes do not bind DNA. Procapsid structures assembled in the absence of gp6 or carrying the mutant gp6 Y467-->C lack gp7. The gp6-gp7 interaction thus targets gp7 to the procapsid where the portal protein is localized asymmetrically at a single vertex of the icosahedral structure. The interaction between the two proteins is disrupted during viral assembly. Proteins homologous to gp6 and gp7 are coded by contiguous genes in a variety of phage genomes from Gram-positive bacteria, suggesting that the gp6-gp7 complex is widespread in this group of phages. Transient association with the portal protein, an essential component of tailed bacteriophages and herpes viruses, provides a novel strategy to target minor proteins to the virion structure that might be operative in a large number of viruses.     

Stepwise expansion of the bacteriophage ϕ6 procapsid: possible packaging intermediates

The initial assembly product of bacteriophage ?6, the procapsid, undergoes major structural transformation during the sequential packaging of its three segments of single-stranded RNA. The procapsid, a compact icosahedrally symmetric particle with deeply recessed vertices, expands to the spherical mature capsid, increasing the volume available to accommodate the genome by 2.5-fold. It has been proposed that expansion and packaging are linked, with each stage in expansion presenting a binding site for a particular RNA segment. To investigate procapsid transformability, we induced expansion by acidification, heating, and elevated salt concentration. Cryo-electron microscopy reconstructions after all three treatments yielded the same partially expanded particle. Analysis by cryo-electron tomography showed that all vertices of a given capsid were either in a compact or an expanded state, indicating a highly cooperative transition. To benchmark the mature capsid, we analyzed filled (in vivo packaged) capsids. When these particles were induced to release their RNA, they reverted to the same intermediate state as expanded procapsids (intermediate 1) or to a second, further expanded state (intermediate 2). This partial reversibility of expansion suggests that the mature spherical capsid conformation is obtained only when sufficient outward pressure is exerted by packaged RNA. The observation of two intermediates is consistent with the proposed three-step packaging process. The model is further supported by the observation that a mutant capable of packaging the second RNA segment without previously packaging the first segment has enhanced susceptibility for switching spontaneously from the procapsid to the first intermediate state.     

The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution

The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits. The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18A and the maximum diameter is 550A. The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization. The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion. Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar. The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits. The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation. A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.     

The mechanism for producing two symmetries at the head-tail junction of bacteriophages: a hypothesis

Some double-stranded DNA bacteriophages consist of DNA packaged in a proteinaceous capsid. The capsid has a DNA-enclosing outer shell (head) attached to an external projection (tail). At the head-tail junction is a ring of subunits (connector) that has either six or twelve-fold rotational symmetry, and is joined to the head at an axis of the head's five-fold rotational symmetry. The head is made of subunits in either an icosahedral array or an array consisting of two icosahedral hemispheres separated by a cylinder of subunits. During infection of a host, the head with connector is assembled as a procapsid that subsequently packages DNA and joins a tail. The mechanism for producing two symmetries at the head-tail junction has in the past been an unsolved problem. The observation that the connector of bacteriophage T7 does not nucleate assembly of the outer shell of T7's icosahedral procapsid (P. Serwer and R. H. Watson [1982] J. Virol. 42, 595-601) places a constraint on a solution for the above problem. To solve the above problem for icosahedral procapsids, it is proposed here that: (a) assembly of the outer shell of procapsids is nucleated by a six-membered ring of hexameric aggregates of the major outer shell protein, (b) the connector is assembled in the center of this ring, (c) one of the hexamers dissociates from the ring, creating a five-membered ring and forcing the connector to the inside of the outer shell. A related mechanism is proposed for nucleation of the elongated procapsid of bacteriophage T4.     

Maturation dynamics of bacteriophage HK97 capsid

Maturation of the bacteriophage HK97 capsid requires a large conformational change of the virus capsid. Experimental studies have identified several intermediates along this maturation pathway. To gain insights into the molecular mechanisms of capsid maturation, we examined the fluctuation dynamics of the procapsid and mature capsid using a residue-level computational approach. The most cooperative motions of the procapsid are found to be consistent with the observed change in configuration that takes place during maturation. A few dominant modes of motion are sufficient to describe the anisotropic expansion that accompanies maturation. Based upon these modes, maturation is proposed to occur via an overall expansion and reconfiguration of the capsid initiated by puckering of the pentamers, followed by flattening and crosslinking of the hexameric subunits, and finally crosslinking of the pentameric subunits. The highly mobile E loops are stabilized by anchoring to highly stable residues belonging to neighboring subunits.