Structural organisation of the head-to-tail interface of a bacterial virus

In tailed icosahedral bacteriophages the connection between the 5-fold symmetric environment of the portal vertex in the capsid and the 6-fold symmetric phage tail is formed by a complex interface structure. The current study provides the detailed analysis of the assembly and structural organisation of such an interface within a phage having a long tail. The region of the interface assembled as part of the viral capsid (connector) was purified from DNA-filled capsids of the Bacillus subtilis bacteriophage SPP1. It is composed of oligomers of gp6, the SPP1 portal protein, of gp15, and of gp16. The SPP1 connector structure is formed by a mushroom-like portal protein whose cap faces the interior of the viral capsid in intact virions, an annular structure below the stem of the mushroom, and a second narrower annulus that is in direct contact with the helical tail extremity. The layered arrangement correlates to the stacking of gp6, gp15, and gp16 on top of the tail. The gp16 ring is exposed to the virion outside. During SPP1 morphogenesis, gp6 participates in the procapsid assembly reaction, an early step in the assembly pathway, while gp15 and gp16 bind to the capsid portal vertex after viral chromosome encapsidation. gp16 is processed during or after tail attachment to the connector region. The portal protein gp6 has 12-fold cyclical symmetry in the connector structure, whereas assembly-na?ve gp6 exhibits 13-fold symmetry. We propose that it is the interaction of gp6 with other viral morphogenetic proteins that drives its assembly into the 12-mer state.     

The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.     

The SPP1 connection

Abstract: The connector of the virulent Bacillus subtilis bacteriophage SPP1 (Styloviridae) is a structure localized at the phage head vertex which attaches the tail. It is formed by oligomerization of SPP1 gene product 6 (gp6; portal protein). The purified protein is found in solution essentially as a homo-tredecamer. Its assembly pattern resembles the turbine-like organization found for other portal proteins and has a defined handedness (Dube et al. (1993) EMBO J. 12, 1303–1309). A preliminary reconstruction of the structure shows that gp6 is composed of a lower ring connected by a narrow region to the upper area consisting of 13 lobes radiating from an inner ring. The assembly is organized around a central channel which spans its full height. A functional characterization of gp6 mutants showed that substitutions of defined amino acids by more basic residues lead to packaging of reduced amounts of DNA into the phage head (Tavares et al. (1992) J. Mol. Biol. 225, 81–92). Since SPP1 encapsidates its DNA by a headful mechanism, these mutations ( siz ) affect most probably a function on the headful sensor—signal transduction—headful cut system. Combination of siz alleles has severe effects in packaging. The resulting gp6 versions lead to the encapsidation of shorter DNA molecules at a lower efficiency than single siz mutants. Gene 6 is expressed late during SPP1 infection. Interestingly, the mass of portal protein inside the cell then increases continuously until lysis, reaching a level several fold higher than the amount required to accomplish its role as a structural component of the virion.     

RNA dependence of the bacteriophage phi 29 DNA packaging ATPase.

The activity of the DNA packaging adenosine triphosphatase (ATPase) of the Bacillus subtilis bacteriophage phi 29 is dependent upon prohead RNA. The 174 nucleotide viral-encoded RNA is positioned on the head-tail connector at the portal vertex of the phi 29 precursor shell (prohead). Here, the RNA interacts with the ATP-binding gene 16 product (gp16) to constitute the DNA-packaging ATPase and initiate DNA packaging in vitro. Both the prohead connector (gene 10 product, gp10) and gp16 may utilize an RNA recognition motif characteristic of a number of RNA-associated proteins, and the binding of gp16 by proheads shields the prohead RNA from RNase A. The ATPase activity of gp16 is stimulated fourfold by RNA and tenfold by proheads with RNA. RNA is needed continuously for the gp16/RNA ATPase activity and is essential for the gp16/prohead ATPase activity. The prohead, with its connector, RNA and associated gp16 in an assembly-regulated configuration, hydrolyzes ATP and drives phi 29 DNA translocation.     

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.     

Binding of pRNA to the N-terminal 14 amino acids of connector protein of bacteriophage phi29

During assembly, bacterial virus phi29 utilizes a motor to insert genomic DNA into a preformed protein shell called the procapsid. The motor contains one twelve-subunit connector with a 3.6 nm central channel for DNA transportation, six viral-encoded RNA (packaging RNA or pRNA) and a protein, gp16, with unknown stoichiometry. Recent DNA-packaging models proposed that the 5-fold procapsid vertexes and 12-fold connector (or the hexameric pRNA ring) represented a symmetry mismatch enabling production of a force to drive a rotation motor to translocate and compress DNA. There was a discrepancy regarding the location of the foothold for the pRNA. One model [C. Chen and P. Guo (1997) J. Virol., 71, 3864–3871] suggested that the foothold for pRNA was the connector and that the pRNA–connector complex was part of the rotor. However, one other model suggested that the foothold for pRNA was the 5-fold vertex of the capsid protein and that pRNA was the stator. To elucidate the mechanism of phi29 DNA packaging, it is critical to confirm whether pRNA binds to the 5-fold vertex of the capsid protein or to the 12-fold symmetrical connector. Here, we used both purified connector and purified procapsid for binding studies with in vitro transcribed pRNA. Specific binding of pRNA to the connector in the procapsid was found by photoaffinity crosslinking. Removal of the N-terminal 14 amino acids of the gp10 protein by proteolytic cleavage resulted in undetectable binding of pRNA to either the connector or the procapsid, as investigated by agarose gel electrophoresis, SDS–PAGE, sucrose gradient sedimentation and N-terminal peptide sequencing. It is therefore concluded that pRNA bound to the 12-fold symmetrical connector to form a pRNA–connector complex and that the foothold for pRNA is the connector but not the capsid protein.     

Binding-induced stabilization and assembly of the phage P22 tail accessory factor gp4

To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (Tm 34 degrees C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1)12:gp(4)6 assembly intermediate, which is stably populated at 30 degrees C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1)12:gp(4)12 complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.     

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.     

The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop

The connector protein, also known as the portal protein, located at the portal vertex in the Phi29 bacteriophage has been found to play a key role in the genome DNA packaging motor. There is a disordered region, composed of 12 sets of 18-residue loops N229–N246, that has been assumed to serve as a “clamp” to retain the DNA within the pressurized capsid when DNA is fully packaged. However, the process remains undefined about how the clamping of DNA occurs and what signal is used to engage the channel loops to clamp the DNA near the end of DNA packaging. In this study, we use the planar lipid bilayer (PLB) membrane technique to study the connector with its loops cleaved. The channel properties are compared with those of the connector with corresponding wild-type loops at different membrane potentials. On the basis of the hypothesis of the Donnan effects in the flashing Brownian ratchet model, we associate the PLB experimental results with the outcomes from the relevant biochemical experiments on the proheads containing the connectors without the loops, which enables us to provide a clear picture about how the DNA clamping occurs. A mathematical relationship between the Donnan potential and the DNA packaging density is established, demonstrating that they are both in essence the same signal that is received and transmitted by the connector to dictate DNA clamping and the termination of DNA packaging. At the end of the study, the PLB technique is proposed as a viral research tool, and its potential use to study the functions of specific domains in a portal protein of the tailed bacteriophages is highlighted.     

Large Terminase Conformational Change Induced by Connector Binding in Bacteriophage T7

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.     

噬菌体phi29末端酶gp16蛋白C端结构域在溶液中的构象分析

噬菌体phi29是一种双链DNA病毒,它的增殖和成熟过程需要将较大的子代DNA包装入一个空间极其有限的新生病毒衣壳中,这一步骤由其转运马达完成.转运马达具有三个部分:头颈连接器蛋白(the connector)、pRNA和ATP水解酶蛋白(gp16).在结构预测中发现gp16蛋白的C端结构域可能与pRNA或DNA结合,但尚未有相关文献可以证明这种相互作用,因此其结构的解析成为研究其功能的关键.本文利用大肠杆菌SUMO表达系统,对噬菌体phi29 gp16蛋白的C端结构域进行重组构建并诱导表达后,通过Ni-NTA亲和层析纯化,再用分子排阻色谱获得高纯度的目的蛋白质单体,纯度达到95%左右.最后用X射线小角散射技术(small angle X-ray scattering)对其构象进行分析,收集小角散射数据,当目的蛋白的浓度为1.3 g/L、所得到的目的蛋白的三维结构与同源蛋白FtsK的晶体结构高度拟合,且通过CRYSOL软件反推计算的曲线与实验曲线高度重合.通过原核表达、纯化及结构分析,成功获得了该结构域在溶液中的状态信息.这一研究不仅为后续关于该结构域的功能研究奠定基础,也为病毒感染的诊断和治疗提供新思路.     

DNA packaging motor assembly intermediate of bacteriophage phi29

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.     

Experimental test of connector rotation during DNA packaging into bacteriophage phi29 capsids

The bacteriophage phi29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Interaction of gp16 with pRNA and DNA for genome packaging by the motor of bacterial virus phi29

One striking feature in the assembly of linear double-stranded (ds) DNA viruses is that their genome is translocated into a preformed protein coat via a motor involving two non-structural components with certain characteristics of ATPase. In bacterial virus phi29, these two components include the protein gp16 and a packaging RNA (pRNA). The structure and function of other phi29 motor components have been well elucidated; however, studies on the role of gp16 have been seriously hampered by its hydrophobicity and self-aggregation. Such problems caused by insolubility also occur in the study of other viral DNA-packaging motors. Contradictory data have been published regarding the role and stoichiometry of gp16, which has been reported to bind every motor component, including pRNA, DNA, gp3, DNA-gp3, connector, pRNA-free procapsid, and procapsid/pRNA complex. Such conflicting data from a binding assay could be due to the self-aggregation of gp16. Our recent advance to produce soluble and highly active gp16 has enabled further studies on gp16. It was demonstrated in this report that gp16 bound to DNA non-specifically. gp16 bound to the pRNA-containing procapsid much more strongly than to the pRNA-free procapsid. The domain of pRNA for gp16 interaction was the 5'/3' paired helical region. The C18C19A20 bulge that is essential for DNA packaging was found to be dispensable for gp16 binding. This result confirms the published model that pRNA binds to the procapsid with its central domain and extends its 5'/3' DNA-packaging domain for gp16 binding. It suggests that gp16 serves as a linkage between pRNA and DNA, and as an essential DNA-contacting component during DNA translocation. The data also imply that, with the exception of the C18C19A20 bulge, the main role of the 5'/3' helical double-stranded region of pRNA is not for procapsid binding but for binding to gp16.     

A second symmetry mismatch at the portal vertex of bacteriophage T7: 8-fold symmetry in the procapsid core

Like other bacteriophages, T7 has a singular vertex that is the site of a symmetry mismatch involving the portal/connector protein, a 12-fold ring at the vertex site which is also a 5-fold axis for the icosahedral capsid. In the mature virion, a 6-fold-symmetric tail extends outwards from the connector. T7 also has a cylindrical "core" that assembles on the inner surface of the connector during procapsid formation, is retained in the mature virion, and is required for infectivity. We have investigated the core structure by cryo-electron microscopy and image analysis of procapsids and find that it observes 8-fold symmetry. Stoichiometry data indicate that its major constituent is an octamer of gp15.     

The opening of the SPP1 bacteriophage tail, a prevalent mechanism in Gram-positive-infecting siphophages

The SPP1 siphophage uses its long non-contractile tail and tail tip to recognize and infect the Gram-positive bacterium Bacillus subtilis. The tail-end cap and its attached tip are the critical components for host recognition and opening of the tail tube for genome exit. In the present work, we determined the cryo-electron microscopic (cryo-EM) structure of a complex formed by the cap protein gp19.1 (Dit) and the N terminus of the downstream protein of gp19.1 in the SPP1 genome, gp21(1-552) (Tal). This complex assembles two back-to-back stacked gp19.1 ring hexamers, interacting loosely, and two gp21(1-552) trimers interacting with gp19.1 at both ends of the stack. Remarkably, one gp21(1-552) trimer displays a "closed" conformation, whereas the second is "open" delineating a central channel. The two conformational states dock nicely into the EM map of the SPP1 cap domain, respectively, before and after DNA release. Moreover, the open/closed conformations of gp19.1-gp21(1-552) are consistent with the structures of the corresponding proteins in the siphophage p2 baseplate, where the Tal protein (ORF16) attached to the ring of Dit (ORF15) was also found to adopt these two conformations. Therefore, the present contribution allowed us to revisit the SPP1 tail distal-end architectural organization. Considering the sequence conservation among Dit and the N-terminal region of Tal-like proteins in Gram-positive-infecting Siphoviridae, it also reveals the Tal opening mechanism as a hallmark of siphophages probably involved in the generation of the firing signal initiating the cascade of events that lead to phage DNA release in vivo.     

Sequential action of ATPase, ATP, ADP, Pi and dsDNA in procapsid-free system to enlighten mechanism in viral dsDNA packaging

Many cells and double-stranded DNA (dsDNA) viruses contain an AAA⁺ ATPase that assembles into oligomers, often hexamers, with a central channel. The dsDNA packaging motor of bacteriophage phi29 also contains an ATPase to translocate dsDNA through a dodecameric channel. The motor ATPase has been investigated substantially in the context of the entire procapsid. Here, we report the sequential action between the ATPase and additional motor components. It is suggested that the contact of ATPase to ATP resulted in its conformational change to a higher binding affinity toward dsDNA. It was found that ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action that is speculated to push dsDNA to pass the connector channel. Our results suggest that dsDNA packaging goes through a combined effort of both the gp16 ATPase for pushing and the channel as a one-way valve to control the dsDNA translocation direction. Many packaging models have previously been proposed, and the packaging mechanism has been contingent upon the number of nucleotides packaged per ATP relative to the 10.5 bp per helical turn for B-type dsDNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five or six discrete steps of dsDNA translocation. Combination of the two distinct roles of gp16 and connector renews the perception of previous dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP.     

The high-resolution functional map of bacteriophage SPP1 portal protein

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.