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Phage assembly and the special role of the portal protein
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ScienceDirect Phage assembly and the special role of the portal protein Peter E Prevelige Jr.1 and Juliana R Cortines2 Virus infections are ultimately dependent on a successful viral genome delivery to the host cell. The bacteriophage family Caudovirales evolved specialized machinery that fulfills this function: the portal proteins complex. The complexes are arranged as dodecameric rings and are a structural part of capsids incorporated at a five-fold vertex. They are involved in crucial aspects of viral replication, such as virion assembly, DNA packaging and DNA delivery. This review focuses on the organization and the mechanism through which these portal complexes achieve viral genome delivery and their similarities to other viral portal complexes. Addresses 1 Department of Microbiology, University of Alabama at Birmingham, 35294, United States 2 Departamento de Virologia, Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro, 21941-902, Brazil Corresponding author: Cortines, Juliana R ([email protected])
Current Opinion in Virology 2018, 30:xx–yy This review comes from a themed issue on Virus structure and expression Edited by Kay Gru¨newald and Thomas Krey
https://doi.org/10.1016/j.coviro.2018.09.004 1879-6257/ã 2018 Published by Elsevier B.V.
Introduction Bacteriophages constitute the most abundant biomass on Earth and play significant roles in nearly every aspect of life, from controlling biogeochemical cycles to gene transfer [1,2]. Among the viruses known to date, 96% are dsDNA bacteriophages from Caudovirales, an order that includes the contractile tailed bacteriophages of the Myoviridae family (i.e. P2-like and T4-like viruses) and noncontractile members of Podoviridae and Syphoviridae families (i.e. P22-like, T7-like, phi29-like and l-like viruses) [3]. Viruses from this order have icosahedral or a derivative prolate capsid. This spatial organization demands capsid proteins to be arranged in clusters of 6 protein subunits (hexamers), with 12 clusters of 5 protein subunits (pentamers) placed at the vertices. The generated closed capsule follows the principles of icosahedral symmetry proposed by Caspar and Klug in 1962. www.sciencedirect.com
In each capsid at a single 5-fold vertex lies the portal dodecameric ring, a hub for attachment of tail and closure proteins. Together, they form the tail machines, structures that can be displayed either internally and/or protruding from the capsids which are responsible for actively packaging and delivering the dsDNA viral genome. Thus, portals are also known as the head–tail connectors. Portal rings have also been described in herpesviruses, adenoviruses and giant viruses [4–6], making these specialized genome delivery machines broadly distributed in the virosphere [7]. The assembly of such infectious bacteriophage particles can be divided into four main events: 1) nucleation, 2) formation of an intermediate structure, the prohead or procapsid, filled with copies of internal scaffolding proteins, that drive capsid size-determination 3) DNA packaging, coupled with scaffolding removal, and structural maturation, 4) the attachment of tail proteins [8]. Portal protein complexes are crucial in events 1, 3 and 4, as described below. Nucleation starts with the association of coat protein, scaffolding protein and a single portal protein ring [9– 11,12,13,14,15]. The formation and recruitment of the portal complex seems to rely on the aid of scaffolding protein [16,17]. Interaction between the two gene products was described for several bacteriophages, including phi29 [17,18], T4 [19], l [20] and even for HSV, deemed an honorary bacteriophage based on its similarities with dsDNA phage [21]. Once formed, the dodecameric rings are the assembly initiators, as observed in in vitro models for T4 [22–24], P22 [15], phi29 [18], and HSV [25]. In the last two cases, the yield of capsid assembly is increased in the presence of the portal complex [18,25]. Coupling nucleation of assembly to the presence of the portal complex ensures that nearly all capsids will have one and only one portal complex. In event 2, a complete, immature icosahedral capsid is built. At this point, scaffolding proteins are present inside procapsids, and assembly proceeds to DNA packaging and maturation (event 3), which triggers the release of scaffolding protein, intact (i.e. P22, phi29, HSV) or proteolyzed (i.e. T5 and HK97), from the interior of procapsids [26]. Here too, there is evidence of the impact of the portal protein as in the case of phi29 the presence of portal protein seems to play a role in ensuring the formation of prolate particles [27]. Viral DNA is selectively bound and delivered by viral terminases to and actively translocated through the portal complex [28]. Current Opinion in Virology 2018, 30:1–8
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2 Virus structure and expression
In headful DNA packaging this process appears to be terminated via portal proteins signaling when the dodecamers undergo conformational changes induced by complete genome packaging. Portal complexes work as head– tail connectors and act as sensors of internal pressure, which upon viral DNA translocation can reach 60 atm [12,29–34]. As a result, mature capsids have increased stiffness as probed by atomic force microscopy [35]. Lastly, to ensure capsid closure and viral infectivity, head completion proteins interact with the portal complex. This set of proteins vary among the bacteriophage families: in podoviruses, they are constituted by plug proteins and the adaptors for tail proteins; in siphoviruses and myoviruses portal interacts with proteins necessary for attachment of a pre-formed tail. The final step is the sequential attachment of the individual tail proteins or the pre-formed tails, rendering the progeny infectious and characterizing the completion of assembly [36]. The newly formed viral progeny will now infect hosts via recognition of cell receptors and injection of viral genome, actions controlled by the tail machines.
Functional organization of portals The effort to determine the high-resolution structure for portal complexes, common to tailed dsDNA phages is motivated by unravelling their mechanism and perhaps by the technical challenge itself. For these reasons, a plethora of structural information is available on portals and tail machines [4,12,19,37–40,41,42,43]. Portal structures are almost identical (Figure 1), even though they have very little (12%) homology in their primary structure [43]. Indeed, protein folds are more conserved than amino acid sequences, a fact which has been used for classification based solely on specific folds, that is, the HK97-fold. This leads to the conclusion that function exerted by portal proteins is highly fold-dependent [44]. All portals whose structures were obtained from assembled capsids are built of 12 subunits to form 0.4 up to 1 MDa portal dodecamers [43]. An interesting observation is that in vitro, portal proteins exist in various oligomeric arrangements (11-mers, 12-mers and 13-mers) [45,46], suggesting portal protein monomers are built to adjust to considerable structural changes. Structurally, this plasticity may be explained by the fact that portal protomers have negative and positive patches that interact via electrostatic forces, functioning as molecular ‘magnets’ [47] and resulting in the formation of different oligomers in ectopically expressed portal proteins [45]. Each monomer (and the dodecamer as well) has up to five distinct regions, from top to bottom: barrel (where present), crown, wing, stem and stalk [19,42,48]. Figure 2 coordinates each of the described regions to their functions in bacteriophages, described in more details below. Current Opinion in Virology 2018, 30:1–8
The crown and wing are the locations that display the greatest variability among distinct portals. This plasticity is directly related to the protein size [49] and linked to obligatory conformational changes for maturation transitions induced by DNA spooling during packaging [39]. The stem and stalk are more strictly arranged across phages: stems are formed by twelve angled helices (tilted 30–50 in relation to the center of the channel, depending on the virus [50]), and the stalk forms the initial channel for DNA packaging [43]. Computational simulations suggest that the central core of phi29 is stiffer than the extremities, possibly necessary to withstand the pressure imposed by DNA packaging [51]. Portal rings may exhibit an additional barrel region that extends from and is flexibly linked [48] to the crown towards the inner side of the virus capsid, as is the case of T4 [19] and P22 [12,48]. The longest (125 amino acids) protrudes inwards the mature-only P22 viruses, with a helical arrangement. Glutamine is the most abundant amino acid in this region (17%), a common residue found in DNA binding proteins [52]. Indeed, the barrel changes conformation upon genome packaging, going from unstructured to helical. The newly packaged genome is found to be tightly arranged around the barrel [12,48,53]. It acts as a pressure sensor which reflects into a transducing signal for downstream conformational changes that take place during genome packaging [12]. In phi29 and T7, the barrel is not present [42]. Coincidentally, the packaging termination signaling does not follow the head-full mechanism, supporting the barrel’s involvement in the process. Below the barrel (where present) or directly facing the inside of the viral capsid lies the wide end of the portal complex, the crown. Genome packaging induces conformational changes in this region, resulting in channel widening and consequently in helical structure gain of the barrel in the mature P22 [12]. The crown is flexibly linked to the wing, the site for intimate interaction with capsid and scaffolding proteins [12,19,54]. It is the most variable structure in size observed for portal due to the portal protein size across bacteriophages (33 kDa in HK97-like to 80 kDa in P22) [43,55]. The flat surface of the wing is predominantly negative to prevent DNA binding to portal [12,19]. The wing is connected to the stem region by a flexible loop, a disordered element detected in portal structures of several phages. The loops from each portal protomer protrudes towards the center of the portal channel, generating a constriction in the ring that plays a crucial role in preventing leakage of the packaging DNA [56]. The viral genome is translocated upwards via the channel formed mainly by the wing and stem regions. Mutations in the stem helix affect DNA packaging: in P22, altering residues located between 105 through 132 results in viral www.sciencedirect.com
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Portal proteins in bacteriophages Prevelige and Cortines 3
Figure 1
T4
P22-IV
P22-MV Phi29
1 nm Current Opinion in Virology
Portal complexes from contractile and non-contractile tailed bacteriophages. Even though amino acid homology is low, the overall structure of different portal monomers and rings are conserved. Representative three-dimensional structures from T4 (contractile tail), P22 and phi29 (noncontractile tails), labeled at the top of each structure row. Portal protein monomers are depicted in the top row. The barrel (blue), crown (green), wing (yellow), stem (red) and stalk (purple) regions are highlighted by the colored dashed lines in the P22 mature virus monomeric structure. Once oligomerized, bacteriophage P22 portal rings go through a drastic conformational rearrangement upon DNA packaging termination/virus maturation. Therefore, two structures are shown: the immature virus’ and the mature virus’ portal monomers and dodecamers (middle and bottom rows, respectively). Middle row: top view of portal rings. A single portal monomer is highlighted in red. Bottom row: side view of protein dodecamers. A single portal monomer is highlighted in red. [PDB IDs: 3JA7 (T4), 5JJ1 (P22 immature virus’ portal), 5JJ3 (P22 mature virus’ portal) and 1FOU (phi 29)]. All structures are drawn to scale. Scale bar: 1 nm. IV: immature virus; MV: mature virus. Reader is encouraged to access [43] for a collection of other homologous portal ring structures.
genome over packaging of 2.6–4 kb and mutations in this region halts packaging in SPP1 [57,58,59]. The central channel aperture is on average 30 A˚, just enough to allow passage of hydrated DNA molecules and maintain tight contact. There is a noticeable difference in diameter for immature and mature P22; in procapsids, the channel is restricted to only 25 A˚ and upon maturation, conformational changes induce an enlargement to 40 A˚, possibly alleviating space constraints for www.sciencedirect.com
DNA delivery ease [12]. Representatives of portal proteins from all three bacteriophage families present a predominant negative charged channel [19], while in podoviruses conserved positive amino acids are hypothesized to aid in DNA binding [60]. In phi29, four lysines are arranged in a way that their side chains interact electrostatically to DNA phosphate groups at any given moment during packaging [38]. The protein:DNA interaction may be responsible for a decrease in translocation velocity in phi29 as compared to T4 [19]. These residues Current Opinion in Virology 2018, 30:1–8
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Figure 2
Barrel Head-full sensor
Crown Head-full sensor Interaction with coat protein Interaction with scaffolding protein
Wing DNA interacttion/translocation DNA channel
Stem DNA channel
Stalk Interaction with adaptor proteins Interaction with Large terminase Initiation of DNA packaging Current Opinion in Virology
Functional organization of portal complexes. Bacteriophage P22 was chosen as model since it displays all 5 regions from portal complexes involved in DNA packaging. Each of the functional/structural region of portal dodecamer is color-coded as follows: blue, barrel; green, crown; yellow, wing; red, stem; and purple, stalk. The main functions they correlate to during packaging is described in colored boxes to the right of the structure (PDB ID: 5JJ1). The functional regions were colored based on Ref. [19].
are part of a stem-wing connector loop, observed as unstructured in immature virions. Finally, placed at the bottom of the portal complex is the stalk, site of interaction for adaptor proteins and for the translocation motor that powers DNA packaging [28,43,48]. In T4 portal, positive amino acids line the bottom of stalk region and are most likely involved in DNA capture and translocation initiation [19]. The stalk region assumes several conformations, depending on the portal oligomeric state, capsid maturation state, tail proteins and DNA presence. This conformational variability is important for concerted packaging termination and tail attachment [12]. Based on the recently published structure of P22 portal protein (PDB IDs: 5JJ1 and 5JJ3) [12], the vast majority Current Opinion in Virology 2018, 30:1–8
of the protein is organized either as a-helices or coils, in detriment of beta-folds [41%, 50%, 9%; values are approximate as coordinates are not available for residues 1–3 (immature conformation), 1–7 (mature conformation) and 463–491]. Helical folds are predominant in proteins involved in intimate interaction with other biomolecules [61], such as DNA. As mentioned previously, portal is the initiator of both capsid and tail machine assembly, being thus the most important docking site for protein interaction in bacteriophages.
The genome packaging revolution For the bacteriophage DNA to be packaged, portal proteins must transiently work in synchrony with a viral terminase hetero-ring shaped complex (frequently named TerS and TerL, for terminase small and large subunit). The small terminase subunit recognizes genomic DNA www.sciencedirect.com
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Portal proteins in bacteriophages Prevelige and Cortines 5
based on the presence of a packaging signal and delivers the viral concantemeric DNA to the TerL pentameric ring, which is docked on the portal complex. The terminase and portal proteins coordinate their action to package the DNA in an ATP-dependent fashion and subsequently cleave the DNA when the head is full [28]. The packaged genome size is determined by the recognition of one unit-length genome via sequence recognition, or by the headful mechanism. An exception to this general rule is seen in phi29 which signals packaging termination by gp3-DNA site recognition [28,62]. Another exclusive feature of phi29 is the presence of a catalytic prohead RNA (pRNA) that is also arranged as a ring in close contact with TerL and plays a homologous role as the TerS [28]. In order to be successful, the main goal of the portal:terminase complex is to overcome DNA bending, repulsion and consequent entropic penalties relative to the package of near-crystalline DNA [63]. The stoichiometry of interaction between portal ring: TerL seem to follow a constant proportion among many bacteriophages: 12:5 protomers, respectively. TerL appears as monomers in solution in the absence of portal protein, suggesting the large terminase may need to dock onto the portal stalk region to oligomerize [28,64]. This unexpected mismatch may be circumvented by the fact that projections of the stalk region fit a quasi-5-fold symmetry as shown by recent cryo-electronmicroscopy derived P22 procapsid structure [12]. TerS oligomers may range from 8-mers to 12-mers [43]. The first attempt to elucidate the portal-dependent genome packaging was to describe the protein complex as a ball-race machine, powered by an outer stator, in which portal rotation would result in DNA packaging [65]. However, when T4 portal complex was cross-linked to the capsid, DNA packaging occurred successfully [66]. In addition, using sophisticated single particle imagining of Cy3 labeled phi29 portal protein, no evidence for rotation of portal was detected [67,68]. Also in consonance with these hypotheses, molecular dynamics simulation predicts that the DNA molecule rotates and bends in order to be packaged by the concerted work of the portal and terminase complex [63]. The three-dimensional organization of portal protomers around the central translocation channel displays a tilted stem helix that supports DNA revolution by providing transient salt bridges between the viral genome and each portal subunit [69]. Ensuring this is a one-way path, the channel’s loops are a physical barrier to prevent leakage of the genome [56]. DNA packaging results in significant conformational changes of the portal complex. First, the wing domain becomes wider, generating a downstream signaling for packaging to terminate. The transition of immature to www.sciencedirect.com
mature portal protein releases portal:TerL interaction caused by the symmetry rearrangement of the stalk region, going from a quasi-5-fold to a 6-fold. Also related to a conformation change during transition is the remarkable folding of the helical barrel in P22, not observable in the immature capsid. The formation of a straight, folded barrel may be crucial for delivering the genome in the next infection [12]. Indeed, cryogenic electron microscopy reconstruction data of genome-extruded mature P22 virus showed that the biggest structural change was observed at C-terminal end of the barrel region, where it is found to be spread inside the capsids [41]. Taken together, in the case of headful packaging, structural and biochemical results suggest that the portal complex would be responsible for: first, genome sizing [57]; second, sensing internal capsid pressure induced by full-length genome [12]; third, signaling for packaging termination due to high pressure-induced conformational change in the wing and stalk regions [12,39,57] and fourth, maintaining the viral DNA inside the mature virion once packaging is complete via a one-way check-valve mechanism [63,69–71].
Conclusions Protein fold conservation is a common feature in all kingdoms of life since. This is also true for viruses: structural data for various bacteriophages show a striking fold conservation mostly independent of aminoacid sequences, reflected by the widely disseminated HK97-folds in capsid proteins and the portal protein dodecameric ring shaped assemblies present in the Caudovirales members. Folds deeply relates to function, that is, portal proteins hold striking similarities to cellular DNA translocating proteins such as sliding clamps and TrwB [38,49]. All portal proteins studied so far are extremely amenable to conformational changes induced during virus assembly, maturation, hetero–protein interactions and genome delivery which are dependent on protein–protein and protein–DNA interactions [72]. This ‘promiscuous’ behavior can be used in the advantage of applying portals as basis for the production of nanomaterials. These oligomers are well-ordered self-assembling complexes that can be functionalized to attend specific application requirements. To this end, SPP1 [73], phi29 [74] and the termophilic bacteriophage G20c (member of the Syphoviridae family) [75] portal proteins have been utilized membrane-embedded channels for peptide, DNA and ion translocation, respectively. These systems may have applications in detecting protein oligomeric states, as biomarker detectors, and for drug and gene delivery [73,74,75]. Deepening our knowledge of these viral complexes will aid their applications in life sciences. Current Opinion in Virology 2018, 30:1–8
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6 Virus structure and expression
Funding The authors did not receive any specific grant from funding agencies in the public, commercial, or non-forprofit sectors.
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Declarations of interest None.
Acknowledgements We apologize to all authors we were unable to cite, but that certainly honored the field with their work. To Dr. Kristin Parent for sharing information needed for the conclusion of this review.
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through membrane-adapted phi29 motor protein nanopores. Nat Nanotechnol 2009, 4:765-772. 75. Cressiot B, Greive SJ, Si W, Pascoa TC, Mojtabavi M, Chechik M, Jenkins HT, Lu X, Zhang K, Aksimentiev A et al.: Porphyrinassisted docking of a thermophage portal protein into lipid bilayers: nanopore engineering and characterization. ACS Nano 2017, 11:11931-11945. Application of portal complex as a membrane-embedded pore.
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ScienceDirect Phage assembly and the special role of the portal protein Peter E Prevelige Jr.1 and Juliana R Cortines2 Virus infections are ultimately dependent on a successful viral genome delivery to the host cell. The bacteriophage family Caudovirales evolved specialized machinery that fulfills this function: the portal proteins complex. The complexes are arranged as dodecameric rings and are a structural part of capsids incorporated at a five-fold vertex. They are involved in crucial aspects of viral replication, such as virion assembly, DNA packaging and DNA delivery. This review focuses on the organization and the mechanism through which these portal complexes achieve viral genome delivery and their similarities to other viral portal complexes. Addresses 1 Department of Microbiology, University of Alabama at Birmingham, 35294, United States 2 Departamento de Virologia, Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro, 21941-902, Brazil Corresponding author: Cortines, Juliana R ([email protected])
Current Opinion in Virology 2018, 30:xx–yy This review comes from a themed issue on Virus structure and expression Edited by Kay Gru¨newald and Thomas Krey
https://doi.org/10.1016/j.coviro.2018.09.004 1879-6257/ã 2018 Published by Elsevier B.V.
Introduction Bacteriophages constitute the most abundant biomass on Earth and play significant roles in nearly every aspect of life, from controlling biogeochemical cycles to gene transfer [1,2]. Among the viruses known to date, 96% are dsDNA bacteriophages from Caudovirales, an order that includes the contractile tailed bacteriophages of the Myoviridae family (i.e. P2-like and T4-like viruses) and noncontractile members of Podoviridae and Syphoviridae families (i.e. P22-like, T7-like, phi29-like and l-like viruses) [3]. Viruses from this order have icosahedral or a derivative prolate capsid. This spatial organization demands capsid proteins to be arranged in clusters of 6 protein subunits (hexamers), with 12 clusters of 5 protein subunits (pentamers) placed at the vertices. The generated closed capsule follows the principles of icosahedral symmetry proposed by Caspar and Klug in 1962. www.sciencedirect.com
In each capsid at a single 5-fold vertex lies the portal dodecameric ring, a hub for attachment of tail and closure proteins. Together, they form the tail machines, structures that can be displayed either internally and/or protruding from the capsids which are responsible for actively packaging and delivering the dsDNA viral genome. Thus, portals are also known as the head–tail connectors. Portal rings have also been described in herpesviruses, adenoviruses and giant viruses [4–6], making these specialized genome delivery machines broadly distributed in the virosphere [7]. The assembly of such infectious bacteriophage particles can be divided into four main events: 1) nucleation, 2) formation of an intermediate structure, the prohead or procapsid, filled with copies of internal scaffolding proteins, that drive capsid size-determination 3) DNA packaging, coupled with scaffolding removal, and structural maturation, 4) the attachment of tail proteins [8]. Portal protein complexes are crucial in events 1, 3 and 4, as described below. Nucleation starts with the association of coat protein, scaffolding protein and a single portal protein ring [9– 11,12,13,14,15]. The formation and recruitment of the portal complex seems to rely on the aid of scaffolding protein [16,17]. Interaction between the two gene products was described for several bacteriophages, including phi29 [17,18], T4 [19], l [20] and even for HSV, deemed an honorary bacteriophage based on its similarities with dsDNA phage [21]. Once formed, the dodecameric rings are the assembly initiators, as observed in in vitro models for T4 [22–24], P22 [15], phi29 [18], and HSV [25]. In the last two cases, the yield of capsid assembly is increased in the presence of the portal complex [18,25]. Coupling nucleation of assembly to the presence of the portal complex ensures that nearly all capsids will have one and only one portal complex. In event 2, a complete, immature icosahedral capsid is built. At this point, scaffolding proteins are present inside procapsids, and assembly proceeds to DNA packaging and maturation (event 3), which triggers the release of scaffolding protein, intact (i.e. P22, phi29, HSV) or proteolyzed (i.e. T5 and HK97), from the interior of procapsids [26]. Here too, there is evidence of the impact of the portal protein as in the case of phi29 the presence of portal protein seems to play a role in ensuring the formation of prolate particles [27]. Viral DNA is selectively bound and delivered by viral terminases to and actively translocated through the portal complex [28]. Current Opinion in Virology 2018, 30:1–8
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2 Virus structure and expression
In headful DNA packaging this process appears to be terminated via portal proteins signaling when the dodecamers undergo conformational changes induced by complete genome packaging. Portal complexes work as head– tail connectors and act as sensors of internal pressure, which upon viral DNA translocation can reach 60 atm [12,29–34]. As a result, mature capsids have increased stiffness as probed by atomic force microscopy [35]. Lastly, to ensure capsid closure and viral infectivity, head completion proteins interact with the portal complex. This set of proteins vary among the bacteriophage families: in podoviruses, they are constituted by plug proteins and the adaptors for tail proteins; in siphoviruses and myoviruses portal interacts with proteins necessary for attachment of a pre-formed tail. The final step is the sequential attachment of the individual tail proteins or the pre-formed tails, rendering the progeny infectious and characterizing the completion of assembly [36]. The newly formed viral progeny will now infect hosts via recognition of cell receptors and injection of viral genome, actions controlled by the tail machines.
Functional organization of portals The effort to determine the high-resolution structure for portal complexes, common to tailed dsDNA phages is motivated by unravelling their mechanism and perhaps by the technical challenge itself. For these reasons, a plethora of structural information is available on portals and tail machines [4,12,19,37–40,41,42,43]. Portal structures are almost identical (Figure 1), even though they have very little (12%) homology in their primary structure [43]. Indeed, protein folds are more conserved than amino acid sequences, a fact which has been used for classification based solely on specific folds, that is, the HK97-fold. This leads to the conclusion that function exerted by portal proteins is highly fold-dependent [44]. All portals whose structures were obtained from assembled capsids are built of 12 subunits to form 0.4 up to 1 MDa portal dodecamers [43]. An interesting observation is that in vitro, portal proteins exist in various oligomeric arrangements (11-mers, 12-mers and 13-mers) [45,46], suggesting portal protein monomers are built to adjust to considerable structural changes. Structurally, this plasticity may be explained by the fact that portal protomers have negative and positive patches that interact via electrostatic forces, functioning as molecular ‘magnets’ [47] and resulting in the formation of different oligomers in ectopically expressed portal proteins [45]. Each monomer (and the dodecamer as well) has up to five distinct regions, from top to bottom: barrel (where present), crown, wing, stem and stalk [19,42,48]. Figure 2 coordinates each of the described regions to their functions in bacteriophages, described in more details below. Current Opinion in Virology 2018, 30:1–8
The crown and wing are the locations that display the greatest variability among distinct portals. This plasticity is directly related to the protein size [49] and linked to obligatory conformational changes for maturation transitions induced by DNA spooling during packaging [39]. The stem and stalk are more strictly arranged across phages: stems are formed by twelve angled helices (tilted 30–50 in relation to the center of the channel, depending on the virus [50]), and the stalk forms the initial channel for DNA packaging [43]. Computational simulations suggest that the central core of phi29 is stiffer than the extremities, possibly necessary to withstand the pressure imposed by DNA packaging [51]. Portal rings may exhibit an additional barrel region that extends from and is flexibly linked [48] to the crown towards the inner side of the virus capsid, as is the case of T4 [19] and P22 [12,48]. The longest (125 amino acids) protrudes inwards the mature-only P22 viruses, with a helical arrangement. Glutamine is the most abundant amino acid in this region (17%), a common residue found in DNA binding proteins [52]. Indeed, the barrel changes conformation upon genome packaging, going from unstructured to helical. The newly packaged genome is found to be tightly arranged around the barrel [12,48,53]. It acts as a pressure sensor which reflects into a transducing signal for downstream conformational changes that take place during genome packaging [12]. In phi29 and T7, the barrel is not present [42]. Coincidentally, the packaging termination signaling does not follow the head-full mechanism, supporting the barrel’s involvement in the process. Below the barrel (where present) or directly facing the inside of the viral capsid lies the wide end of the portal complex, the crown. Genome packaging induces conformational changes in this region, resulting in channel widening and consequently in helical structure gain of the barrel in the mature P22 [12]. The crown is flexibly linked to the wing, the site for intimate interaction with capsid and scaffolding proteins [12,19,54]. It is the most variable structure in size observed for portal due to the portal protein size across bacteriophages (33 kDa in HK97-like to 80 kDa in P22) [43,55]. The flat surface of the wing is predominantly negative to prevent DNA binding to portal [12,19]. The wing is connected to the stem region by a flexible loop, a disordered element detected in portal structures of several phages. The loops from each portal protomer protrudes towards the center of the portal channel, generating a constriction in the ring that plays a crucial role in preventing leakage of the packaging DNA [56]. The viral genome is translocated upwards via the channel formed mainly by the wing and stem regions. Mutations in the stem helix affect DNA packaging: in P22, altering residues located between 105 through 132 results in viral www.sciencedirect.com
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Portal proteins in bacteriophages Prevelige and Cortines 3
Figure 1
T4
P22-IV
P22-MV Phi29
1 nm Current Opinion in Virology
Portal complexes from contractile and non-contractile tailed bacteriophages. Even though amino acid homology is low, the overall structure of different portal monomers and rings are conserved. Representative three-dimensional structures from T4 (contractile tail), P22 and phi29 (noncontractile tails), labeled at the top of each structure row. Portal protein monomers are depicted in the top row. The barrel (blue), crown (green), wing (yellow), stem (red) and stalk (purple) regions are highlighted by the colored dashed lines in the P22 mature virus monomeric structure. Once oligomerized, bacteriophage P22 portal rings go through a drastic conformational rearrangement upon DNA packaging termination/virus maturation. Therefore, two structures are shown: the immature virus’ and the mature virus’ portal monomers and dodecamers (middle and bottom rows, respectively). Middle row: top view of portal rings. A single portal monomer is highlighted in red. Bottom row: side view of protein dodecamers. A single portal monomer is highlighted in red. [PDB IDs: 3JA7 (T4), 5JJ1 (P22 immature virus’ portal), 5JJ3 (P22 mature virus’ portal) and 1FOU (phi 29)]. All structures are drawn to scale. Scale bar: 1 nm. IV: immature virus; MV: mature virus. Reader is encouraged to access [43] for a collection of other homologous portal ring structures.
genome over packaging of 2.6–4 kb and mutations in this region halts packaging in SPP1 [57,58,59]. The central channel aperture is on average 30 A˚, just enough to allow passage of hydrated DNA molecules and maintain tight contact. There is a noticeable difference in diameter for immature and mature P22; in procapsids, the channel is restricted to only 25 A˚ and upon maturation, conformational changes induce an enlargement to 40 A˚, possibly alleviating space constraints for www.sciencedirect.com
DNA delivery ease [12]. Representatives of portal proteins from all three bacteriophage families present a predominant negative charged channel [19], while in podoviruses conserved positive amino acids are hypothesized to aid in DNA binding [60]. In phi29, four lysines are arranged in a way that their side chains interact electrostatically to DNA phosphate groups at any given moment during packaging [38]. The protein:DNA interaction may be responsible for a decrease in translocation velocity in phi29 as compared to T4 [19]. These residues Current Opinion in Virology 2018, 30:1–8
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Figure 2
Barrel Head-full sensor
Crown Head-full sensor Interaction with coat protein Interaction with scaffolding protein
Wing DNA interacttion/translocation DNA channel
Stem DNA channel
Stalk Interaction with adaptor proteins Interaction with Large terminase Initiation of DNA packaging Current Opinion in Virology
Functional organization of portal complexes. Bacteriophage P22 was chosen as model since it displays all 5 regions from portal complexes involved in DNA packaging. Each of the functional/structural region of portal dodecamer is color-coded as follows: blue, barrel; green, crown; yellow, wing; red, stem; and purple, stalk. The main functions they correlate to during packaging is described in colored boxes to the right of the structure (PDB ID: 5JJ1). The functional regions were colored based on Ref. [19].
are part of a stem-wing connector loop, observed as unstructured in immature virions. Finally, placed at the bottom of the portal complex is the stalk, site of interaction for adaptor proteins and for the translocation motor that powers DNA packaging [28,43,48]. In T4 portal, positive amino acids line the bottom of stalk region and are most likely involved in DNA capture and translocation initiation [19]. The stalk region assumes several conformations, depending on the portal oligomeric state, capsid maturation state, tail proteins and DNA presence. This conformational variability is important for concerted packaging termination and tail attachment [12]. Based on the recently published structure of P22 portal protein (PDB IDs: 5JJ1 and 5JJ3) [12], the vast majority Current Opinion in Virology 2018, 30:1–8
of the protein is organized either as a-helices or coils, in detriment of beta-folds [41%, 50%, 9%; values are approximate as coordinates are not available for residues 1–3 (immature conformation), 1–7 (mature conformation) and 463–491]. Helical folds are predominant in proteins involved in intimate interaction with other biomolecules [61], such as DNA. As mentioned previously, portal is the initiator of both capsid and tail machine assembly, being thus the most important docking site for protein interaction in bacteriophages.
The genome packaging revolution For the bacteriophage DNA to be packaged, portal proteins must transiently work in synchrony with a viral terminase hetero-ring shaped complex (frequently named TerS and TerL, for terminase small and large subunit). The small terminase subunit recognizes genomic DNA www.sciencedirect.com
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Portal proteins in bacteriophages Prevelige and Cortines 5
based on the presence of a packaging signal and delivers the viral concantemeric DNA to the TerL pentameric ring, which is docked on the portal complex. The terminase and portal proteins coordinate their action to package the DNA in an ATP-dependent fashion and subsequently cleave the DNA when the head is full [28]. The packaged genome size is determined by the recognition of one unit-length genome via sequence recognition, or by the headful mechanism. An exception to this general rule is seen in phi29 which signals packaging termination by gp3-DNA site recognition [28,62]. Another exclusive feature of phi29 is the presence of a catalytic prohead RNA (pRNA) that is also arranged as a ring in close contact with TerL and plays a homologous role as the TerS [28]. In order to be successful, the main goal of the portal:terminase complex is to overcome DNA bending, repulsion and consequent entropic penalties relative to the package of near-crystalline DNA [63]. The stoichiometry of interaction between portal ring: TerL seem to follow a constant proportion among many bacteriophages: 12:5 protomers, respectively. TerL appears as monomers in solution in the absence of portal protein, suggesting the large terminase may need to dock onto the portal stalk region to oligomerize [28,64]. This unexpected mismatch may be circumvented by the fact that projections of the stalk region fit a quasi-5-fold symmetry as shown by recent cryo-electronmicroscopy derived P22 procapsid structure [12]. TerS oligomers may range from 8-mers to 12-mers [43]. The first attempt to elucidate the portal-dependent genome packaging was to describe the protein complex as a ball-race machine, powered by an outer stator, in which portal rotation would result in DNA packaging [65]. However, when T4 portal complex was cross-linked to the capsid, DNA packaging occurred successfully [66]. In addition, using sophisticated single particle imagining of Cy3 labeled phi29 portal protein, no evidence for rotation of portal was detected [67,68]. Also in consonance with these hypotheses, molecular dynamics simulation predicts that the DNA molecule rotates and bends in order to be packaged by the concerted work of the portal and terminase complex [63]. The three-dimensional organization of portal protomers around the central translocation channel displays a tilted stem helix that supports DNA revolution by providing transient salt bridges between the viral genome and each portal subunit [69]. Ensuring this is a one-way path, the channel’s loops are a physical barrier to prevent leakage of the genome [56]. DNA packaging results in significant conformational changes of the portal complex. First, the wing domain becomes wider, generating a downstream signaling for packaging to terminate. The transition of immature to www.sciencedirect.com
mature portal protein releases portal:TerL interaction caused by the symmetry rearrangement of the stalk region, going from a quasi-5-fold to a 6-fold. Also related to a conformation change during transition is the remarkable folding of the helical barrel in P22, not observable in the immature capsid. The formation of a straight, folded barrel may be crucial for delivering the genome in the next infection [12]. Indeed, cryogenic electron microscopy reconstruction data of genome-extruded mature P22 virus showed that the biggest structural change was observed at C-terminal end of the barrel region, where it is found to be spread inside the capsids [41]. Taken together, in the case of headful packaging, structural and biochemical results suggest that the portal complex would be responsible for: first, genome sizing [57]; second, sensing internal capsid pressure induced by full-length genome [12]; third, signaling for packaging termination due to high pressure-induced conformational change in the wing and stalk regions [12,39,57] and fourth, maintaining the viral DNA inside the mature virion once packaging is complete via a one-way check-valve mechanism [63,69–71].
Conclusions Protein fold conservation is a common feature in all kingdoms of life since. This is also true for viruses: structural data for various bacteriophages show a striking fold conservation mostly independent of aminoacid sequences, reflected by the widely disseminated HK97-folds in capsid proteins and the portal protein dodecameric ring shaped assemblies present in the Caudovirales members. Folds deeply relates to function, that is, portal proteins hold striking similarities to cellular DNA translocating proteins such as sliding clamps and TrwB [38,49]. All portal proteins studied so far are extremely amenable to conformational changes induced during virus assembly, maturation, hetero–protein interactions and genome delivery which are dependent on protein–protein and protein–DNA interactions [72]. This ‘promiscuous’ behavior can be used in the advantage of applying portals as basis for the production of nanomaterials. These oligomers are well-ordered self-assembling complexes that can be functionalized to attend specific application requirements. To this end, SPP1 [73], phi29 [74] and the termophilic bacteriophage G20c (member of the Syphoviridae family) [75] portal proteins have been utilized membrane-embedded channels for peptide, DNA and ion translocation, respectively. These systems may have applications in detecting protein oligomeric states, as biomarker detectors, and for drug and gene delivery [73,74,75]. Deepening our knowledge of these viral complexes will aid their applications in life sciences. Current Opinion in Virology 2018, 30:1–8
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6 Virus structure and expression
Funding The authors did not receive any specific grant from funding agencies in the public, commercial, or non-forprofit sectors.
15. Motwani T, Lokareddy RK, Dunbar CA, Cortines JR, Jarrold MF, Cingolani G, Teschke CM: A viral scaffolding protein triggers portal ring oligomerization and incorporation during procapsid assembly. Sci Adv 2017, 3:e1700423. Authors show for the first time the incorporation of P22 portal ringin vitro. 16. Weigele PR, Sampson L, Winn-Stapley D, Casjens SR: Molecular genetics of bacteriophage P22 scaffolding protein’s functional domains. J Mol Biol 2005, 348:831-844.
Declarations of interest None.
Acknowledgements We apologize to all authors we were unable to cite, but that certainly honored the field with their work. To Dr. Kristin Parent for sharing information needed for the conclusion of this review.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:
17. Fu C, Uetrecht C, Kang S, Morais MC, Heck AJR, Walter MR, Prevelige PE: A docking model based on mass spectrometric and biochemical data describes phage packaging motor incorporation. Mol Cell Proteom 2010, 8:1764-1773. 18. Fu C, Prevelige PE: In vitro incorporation of the phage Phi29 connector complex. Virology 2009, 394:149-153. 19. Sun L, Zhang X, Gao S, Rao PA, Padilla-Sanchez V, Chen Z, Sun S, Xiang Y, Subramaniam S, Rao VB et al.: Cryo-EM structure of the bacteriophage T4 portal protein assembly at near-atomic resolution. Nat Commun 2015, 6:7548. 20. Rajagopala SV, Casjens S, Uetz P: The protein interaction map of bacteriophage lambda. BMC Microbiol 2011, 11:213.
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