Introduction

SPP1 is a virulent double-stranded DNA (dsDNA) phage that infects the Gram-positive bacterium Bacillus subtilis strain 168. SPP1 belongs to the Siphoviridae family. The virion is composed of an icosahedral, isometric capsid (∼60 nm diameter) and a long, flexible, noncontractile tail.¹ The phage head encloses the mature SPP1 chromosome, which is a linear double-stranded DNA molecule of ∼45.9 kbp size.^2,3 Replication of the SPP1 genome produces DNA concatemers from which single chromosomes are cleaved and encapsidated into preformed procapsids. SPP1 packages its DNA by a headful packaging mechanism, a strategy first described for bacteriophage T4.⁴ As for phages P22 or P1, in case of SPP1 DNA packaging starts by recognition and cleavage of a defined nucleotide sequence termed pac.^5,6 The concatemeric DNA is then translocated unidirectionally to the viral capsid interior. When the level of capsid DNA headfilling reaches a threshold amount (headful) a second endonucleolytic cleavage occurs. This second cleavage, the “headful cut”, defines the size of the packaged DNA. In contrast to pac cleavage the sequence independent headful cut is imprecise. The size of mature SPP1 DNA therefore varies for about 6% around the average chromosome size of 45.9 kb.^7,8 Since the genome of SPP1 encompasses 44,007 bp,⁹ the chromosomes generated by headful cut are terminally redundant (∼4.1%). While the first round of packaging is initiated by a sequence specific cut at pac, the second and subsequent packaging events start from the DNA end generated by the headful cut of the previous packaging event. Therefore this strategy generates a population of phage chromosomes that are partially circularly permutated and terminally redundant (fig. 1). Terminal redundancy of the packaged DNA molecule is essential for circularisation of the viral genome by homologous recombination after its injection in the host cell at the beginning of a new infection cycle.

Figure 1

Processive sequential packaging of the SPP1 genome by the headful mechanism.^, The substrate for packaging is a concatemer of the viral genome generated by replication of the SPP1 DNA shown as a white bar with black circles (pac sequence) and dark grey (more...)

The aim of this article is to review recent progress on the understanding of the molecular mechanisms used by phage SPP1 to package its DNA and to provide an integrated view of our current knowledge of this process. We identify also some of the central questions that are likely to be tackled in the near future. Reference to other virus systems is made only when of direct relevance for the comprehension of SPP1 DNA packaging.

Assembly of the SPP1 Procapsid, the Proteinaceous DNA Container

The procapsid (or prohead) of SPP1 consists of four proteins: the major capsid protein gp13, the scaffold protein gp11, the portal protein gp6 and a minor component gp7¹⁰ (Table 1). Gp13 forms the head shell of the SPP1 procapsid. The protein composition of the SPP1 procapsid suggests that 415 copies of gp13 form a T=7 icosahedral head shell lattice.¹¹ The interior of the structure is filled with 100-180 copies of gp11 that exit the procapsid at the beginning of DNA packaging. A complex of either 12 or 13 copies of the portal protein is present at a unique vertex of the icosahedron termed the portal vertex, the site of DNA entry during encapsidation of the phage chromosome. Furthermore the procapsid contains 2-3 copies of the minor capsid protein gp7.¹¹

Table 1

Properties of the SPP1 proteins that participate in capsid assembly and DNA packaging.

The role of the individual procapsid components for procapsid assembly was defined by the characterization of SPP1 mutants carrying conditional lethal mutations in the corresponding genes.¹⁰ Expression of various combinations of the cloned procapsid genes in the bacterium Escherichia coli allowed establishing the network of protein-protein interactions during procapsid assembly and their role in procapsid shape determination.¹¹ The four procapsid proteins were shown to assemble into procapsids competent for DNA packaging in vitro.^11,12 During assembly of the head shell lattice the scaffold protein controls the polymerisation of gp13 into an icosahedral shell. Incorporation of the portal complex requires interaction with both the capsid and the scaffolding protein. A particular feature of SPP1 is that the portal protein regulates the size of the procapsid. Coproduction of gp11 and gp13 in E. coli in absence of the portal protein or during infections with SPP1 mutants deficient for portal protein production leads to formation of a mixed population of T=7 and smaller procapsids.¹¹ However, in presence of the portal protein formation of small sized procapsids is suppressed. The decision whether a T=7 or a smaller procapsid is assembled is made at an initial stage of procapsid assembly.¹³ This observation indicates that the portal protein together with the coat protein and the scaffold is part of an initiation complex from which subsequently polymerisation of the head shell proceeds. Presence of the portal protein in the procapsid is an essential requirement for DNA packaging.

The role of the fourth procapsid protein in SPP1 assembly, gp7, is poorly understood. Gp7 strongly binds to gp6. This interaction recruits gp7 to the procapsid structure but it is disrupted at a late stage of SPP1 assembly leaving gp7 probably bound to the DNA packed inside the capsid.¹⁴ Gp7 is not essential for the formation of biologically active procapsids but its absence leads to a 5 to 10-fold reduction in the biological activity of phages formed in DNA packaging reactions in vitro.¹¹

Procapsid assembly: question for future research

• How is the portal oligomer recruited to the initiation complex of procapsid assembly?
• What is the molecular nature of the initiation complex in procapsid assembly?
• How is the portal protein embeddedin a nonmatching symmetry enviroment?
• What is the function of gp7 in SPP1 assembly?

Selective Recognition and Cleavage of SPP1 DNA by the Terminase Complex gp1-gp2

Packaging of SPP1 DNA into procapsids is initiated by pac cleavage of the concatemeric DNA precursor (fig. 1). This endonucleolytic cleavage is performed by the terminase enzyme. The terminase of SPP1 is a hetero-oligomer of small (gp1) and large (gp2) subunits. The holoenzyme possesses several activities including recognition, binding and cleavage of the DNA substrate, and ATP-hydrolysis.

Gp1 is an oligomer in solution with a native mass of about 200 kDa. Electron micrographs of negatively stained gp1 molecules and analysis of the native protein by gel filtration suggested that ten molecules of gp1 form a ring-shaped particle.^15,16 Gp1 is composed of several functional domains.¹⁵ Information about this domain organisation of gp1 was gained by comparing the sequence of gp1 with the sequence of the corresponding functional analog of closely related phages (SF6 and ρ15) and functional analysis of chimeras between the SPP1 and SF6 proteins¹⁵ (fig. 2). The two proteins show 71 % identity that is clustered in three discrete regions (I, II and III). Segment I contains a putative NTP binding motif (B-type) and a DNA-binding motif (putative helix-turn-helix motif ).^15,16 Furthermore, a gp1-gp2 interaction region is located in segment I.¹⁷ The region responsible for the interaction between gp1 subunits is located in segment II, the central part of the protein¹⁷ (fig. 2). Even though the segment II additionally contains a NTP hydrolysis motif, purified gp1 shows no ATPase activity in vitro.¹⁵ No function could yet be assigned to segment III.

Figure 2

Comparison between the amino acid sequences of gp1 from the homologous phages SPP1, ρ15, and SF6. The number of amino acids of each gp1 form is shown on the right side. High amino acid sequence homology between the three proteins is found in three (more...)

Gp1 alone binds specifically to SPP1 DNA at the packaging initiation sequence, pac (fig. 3), but is unable to cleave it. The target pac sequence is asymmetric. DNAse I footprints have shown that gp1 binds to two sites within pac that were termed pacL, at the nonencapsidated left end, and pacR, at the encapsidated right end.¹⁶ These two sites flank the site of cleavage pacC (fig. 3). The intrinsically bent sequence pacL and pacR share no sequence homology and form dissimilar complexes with gp1. In case of pacL the pattern of DNaseI cleavage indicates that pacL DNA wraps around the gp1 decamer. Based on the cooperative binding of the two gp1 decamers to pac DNA and based on footprinting data it was suggested that the gp1 decamers bound to pacL and pacR interact and thereby generate a DNA loop of 204 bp length that includes pacC¹⁶ (fig.4). This model is further supported by the finding that purified gp1 promotes cyclization in vitro of a DNA fragment containing pacL.¹⁶ The ensemble of the data showed that binding of gp1 to pac generates an asymmetric nucleoprotein complex.

Figure 3

Bacteriophage SPP1 pac site. Gp1 binds to pacL (nonencapsidated DNA end, or left end) and pacR (encapsidated DNA end or right end) promoting 1 bp staggered nicking at the pacC site that is carried out by the gp2 endonuclease activity.^, The predominant (more...)

Figure 4

Model of the terminase-pac nucleoprotein complex before and after pac cleavage.^, The gp1 cyclical decamers bound to pacL and pacR were proposed to position two molecules of gp2 to cut at the positions indicated by the arrows within the pacC sequence. (more...)

The large subunit of the terminase, gp2, is a protein of 422 residues with a predicted molecular mass of 49 kDa. Analysis of gp2 has long been hampered because the protein could not be overproduced and purified in adequate amounts. However, this problem was recently overcome by affinity purification of hexa-histidine gp2 (H6-gp2), a gp2 derivative that fully complements the deficiency of SPP1 conditional lethal mutants in gene 2.^6,17 H6-gp2 interacts with gp1, binds to dsDNA in a sequence independent manner and possesses a modest ATPase activity.⁶ H6-gp2 introduces nicks and breaks in dsDNA with poor substrate specificity. Interestingly, in presence of distamycin H6-gp2 specifically nicks both DNA strands at the bona fide pacC sequence. Distamycin competes with gp1-pac complex formation and, like gp1, introduces local distortions into DNA. It was therefore suggested, that the DNA structure generated by gp1 promotes cleavage at pacC by gp2. Interaction with the two gp1 decamers was proposed to place two molecules of gp2 in close vicinity to the b sites of pacC (fig.3, fig. 4) within the pac-terminase nucleoprotein complex.⁶ Upon cleavage at both b boxes the gp2 molecule bound at the b box proximal to pacL would unbind gp1 and degrade the nonencapsidated left DNA end (fig.4). The gp2 molecule bound to the b box proximal to pacR, however, remains in the packaging complex. Interaction of the gp2 monomer with both gp1 decamers would stimulate the ATPase activity of gp2 thereby energising ATP-driven DNA translocation.⁶ The model is supported by the fact that raising the concentration of gp1 stimulates the ATPase activity of H6-gp2 but exerts a negative effect on the H6-gp2 endonuclease activity in vitro.⁶ The terminase-DNA complex subsequently participates in DNA translocation to the procapsid interior through the portal protein pore.

In the model described above asymmetry of the pac site causes formation of an asymmetric DNA-terminase complex, thereby providing the structural basis for discrimination between the nonencapsidated left end and the encapsidated right end of the DNA concatemer.^6,16 This mechanism ensures the unidirectionality of DNA packaging.

Terminase-DNA interaction: questions for future research

• What is exact mechanism ensuring sequence specific cleavage at pac by gp2?
• How is pac cleavage contolled and how is excessive pac cleavage throughout the DNA concatemer prevented?
• How does the terminase switch between the DNA cleavage and DNA translocation activities?

Assembly of the DNA Packaging Machine, DNA Translocation, and Capsid Expansion

Specific recognition of the viral DNA and cleavage at pac is mediated by the terminase complex in the absence of other SPP1 proteins.^6,18 Productive SPP1 DNA encapsidation, however, requires the presence of procapsid structures containing an active portal protein.^11,19,20 This feature is common to every tailed phage system presently characterised. In analogy to other phage systems, in case of SPP1 the viral DNA-terminase complex is thought to dock at the portal vertex to assemble the machinery that pumps DNA to the procapsid interior.²¹ The interaction activates the ATPase activity of the terminase large subunit gp2 in order to power DNA translocation to the procapsid interior.²¹ The organisation of the DNA-terminase-procapsid ternary complex in SPP1 is not known. DNA packaging in vitro using crude cell extracts showed that neither the presence of the isolated portal protein nor of procapsids lacking the portal structure inhibits DNA packaging in a competitive fashion.¹² The terminase-DNA complex therefore has the capacity to discriminate between the pool of free portal protein, procapsids incompetent for DNA packaging and the portal protein embedded in the procapsid portal vertex. This distinction is essential to avoid nonproductive interactions that would lead to abortive packaging reactions. A possible structural basis for the specificity of the terminase-DNA complex with procapsid-embedded portal could be a structural transition of the portal protein. While isolated gp6 displays 13-fold symmetry, gp6 with a 12-fold symmetry is found in DNA-filled capsids. This change was proposed to occur during procapsid assembly.²²

The working hypothesis for DNA encapsidation is that chemical energy generated by the ATPase activity of gp2^6,21 is converted to mechanical energy by the packaging apparatus powering DNA translocation against a steep concentration gradient. DNA movements to enter and exit the viral capsid are believed to occur through the portal protein central channel (fig. 5). Single amino acid changes in gp6 were shown to block or reduce the efficiency of DNA translocation revealing the central role of the portal structure in this process.^19,20 The most recent models for DNA translocation are built on the seminal concept proposed by Roger Hendrix that symmetry mismatches between components of the translocation machine provide the structural basis for a DNA rotary pumping action carried out by the portal protein.²³ A model that exploits the symmetry mismatch between the 10-fold helical symmetry of DNA and the symmetry of the portal protein was proposed.²⁴ The model was originally developed for a 13-fold symmetric portal protein but it applies also to a protein composed of 12 subunits. The portal protein was proposed to interact with the DNA phosphate backbone leading to the pumping step of 2 base-pairs through the portal channel. The step would consume 1 ATP molecule hydrolysed by the terminase. The portal protein rotates relative to the DNA and to the capsid in order to bring a new subunit in contact to the DNA molecule to exert the following pumping step. This strategy ensures a constant positioning of the portal protein subunits relative to the DNA and leads to translation of the double-helix to the capsid interior without significant rotations relative to the capsid.²⁴ Determination of the portal protein structure from bacteriophage Φ29 provided enough detail to propose a more elaborated molecular mechanism for DNA translocation that exploits also the symmetry interactions between the portal protein and DNA²⁵ (Grimes and Anderson, this volume).

Figure 5

Structure of the SPP1 portal protein at 18 Å resolution. Stereo view of wild type gp6 oligomer cut-open along a vertical plane. A B-form DNA structure is modelled in the central channel to illustrate the hypothesis that gp6 encircles the double-helix (more...)

DNA packaging leads to a drastic conformational change of the SPP1 major capsid protein. The roundish procapsid with an outer diameter (d_o) of about 55 nm expands to a larger capsid structure (d_o ∼ 66 nm) with a hexagonal outline.¹¹ It is not known if the trigger for expansion is a signal initiated at the portal vertex during DNA encapsidation or if it is due to the increasing pressure generated by the packed DNA on the capsid lattice inner surface (cf., refs. 20,26).

The DNA packaging machine and DNA translocation: question for future research

• What is the structural organization of the DNA translocating machine?
• HOw is the chemical energy generated by the terminase ATP hydrolysis transduced to mechanical pumping of DNA?
• Does the pumping of DNA require rotation of the portal protein?
• How is DNA organized inside the capsid and what stabilizes its tight packaing?

Termination of DNA Packaging: The Headful Cleavage

Encapsidation of the SPP1 chromosome is terminated by endonucleolytic cleavage of the viral DNA concatemer. The site of this sequence independent cleavage is determined by the mass of DNA encapsidated. The mechanism requires a sensor that measures the level of DNA headfilling and an effector headful nuclease. Mutations siz (for sizing) that affect the mass of DNA packaged were found exclusively in the portal protein coding gene³ (fig.1), a feature also reported for bacteriophage P22.²⁷ The siz mutations lead to an undersizing of the mature viral chromosome that correlates with a reduced packing density of DNA inside the viral capsid.³ The capsid size does not change in these mutants. The topology of the portal oligomer in the capsid structure is ideally suited for a sensor activity. The molecule wider region is exposed to the capsid interior while its stem faces the capsid outside where the terminase is believed to bind. The central channel of gp6 is delimited by a tentacles fringe in the wider area of the molecule (fig. 5). The fringe is flexible and a significant difference on its structure was found when the wild type portal protein and a SizA sensor mutant portal protein were compared.²⁸ The pressure generated on this flexible sensor domain by the increasing amount of packaged DNA was proposed to be the trigger for headful cleavage.

Combination of siz mutations in the portal protein leads to packaging of viral chromosomes significantly smaller than the SPP1 mature chromosome of single siz mutants.²⁹ Additionally the efficiency of packaging is reduced in a number of these multiple gp6 mutants suggesting a cross-talk between some step in DNA encapsidation (e.g., DNA translocation) and the headful trigger/cleavage.^20,29 The data accumulated is compatible with a headful mechanism in which DNA translocation stops and renders the DNA molecule accessible to headful endonuclease attack. Stopping of DNA translocation might be caused by a conformational change of the headful sensor domain that physically blocks the DNA in the portal channel and/or by the incapacity of the DNA translocation machine to exert force for further DNA pumping to the capsid interior. The latter case implies a direct role of the portal protein in the mechanical DNA translocation action as discussed in the previous section. Arrest of DNA translocation would trigger headful cleavage carried out most likely by the terminase large subunit gp2. The process might be associated to a reduction in the gp1/gp2 ratio, a condition shown to de-repress the gp2 endonuclease activity and to inhibit its ATPase activity.^6,21 The studies described provide the first molecular clues to understand the mechanism of headful cleavage.

The headful packaging mechanism: questions for future research

• How does the portal protein measure the level of DNA headfilling inside the viral capsid?
• What is the signal that triggers headful cleavage and how is it transduced to the headful nuclease?
• How is the activity of the headful nuclease (terminase?) regulated?
• Are headful cleavage and binding of the head completion proteins coupled (see also next section)?

Stabilization of Packaged DNA and Control of DNA Release: Assembly and Structure of the SPP1 Connector

DNA is packaged at a high density inside the viral capsid of tailed bacteriophages. A direct correlation between the amount of encapsidated DNA and the sensitivity to chelating agents was demonstrated for a variety of phages including SPP1.^3,30 Divalent cations thus play a central role to stabilise the DNA-filled capsid most probably by neutralizing the negative charges of closely packed DNA phosphate backbones. They might also relieve repulsion between DNA and the capsid lattice interior in case the latter is negatively charged as shown for phage HK97.²⁶ The DNA packing generates an internal pressure that can lead to its spontaneous release from the phage capsid.^8,31-34

After termination of DNA packaging the portal pore needs to be rapidly closed to prevent leakage of the viral chromosome—a process that must be reversed when the virus infects a host cell. In SPP1 this role is achieved by the head completion proteins gp15 and gp16 that bind sequentially to the portal vertex forming two rings of subunits stacked below the portal protein.^22,35 We call the complete structure connector (fig. 6). A central channel that crosses the connector appears closed at the level of the gp16 ring.^22,35 In phages whose capsid was disrupted the connector-tail complex was shown to protect a short DNA fragment of 187 to 288 Å, a size that fits well the height of the connector. This DNA fragment is the extremity of the viral chromosome that is packaged last and is first to exit the virion when ejection is triggered.⁸ The connector is a dynamic structure that binds the viral chromosome, serves as an interface for attachment of the helical tail, and controls DNA release from the virus. When the phage adsorption apparatus interacts with its bacterial receptor a signal is communicated along the tail structure to the connector region to trigger opening of the gp16 valve. One SPP1 chromosome extremity is fixed to this valve ready for polarised ejection from the viral capsid.

Figure 6

Organisation of the SPP1 head-to-tail interface.^, The protein components of the connector structure are identified.

Processivity of DNA Packaging Events during SPP1 Infection

After termination of the first packaging cycle initiated at pac (initiation cycle) a second packaging event is initiated at the nonencapsidated concatemer extremity created by the headful cleavage and additional cycles of encapsidation follow (processive headfuls) (fig. 1). Processivity of SPP1 DNA packaging is high reaching an average of 5-6 headfuls per concatemer. Some of the packaging series can even yield 12 headfuls or more.⁸ The main factor that limits the series of encapsidation cycles during normal infection is host lysis.⁸ Processive DNA packaging requires the generation of concatemers as long as 500 kbp within the time frame of SPP1 infection. It is possible that after the first cut at pac the packaging apparatus follows closely the replication machinery in a dynamic process that would limit the physical length of the concatemer and ensure that newly generated pac sites would be encapsidated before terminase attack.⁸ Long packaging series do indeed absolutely require that the terminase leaves the pac sequences along the substrate concatemer being encapsidated intact. Even if only ∼25% of all the pac sequences were cut randomly along the concatemer¹⁸ the size of the substrate DNA would be considerably limited. Cleavage at pac sequences of concatemers where the initiation packaging cycle occurred must thus be avoided. The observation that DNA encapsidated in vitro when extracts of infected cells are mixed originates exclusively from the terminase donor extract suggests that this extract does not provide active free terminase to bind SPP1 DNA present in the extract lacking terminase.¹² The terminase-DNA complex is therefore the form competent to participate in the DNA packaging reaction in vitro while free terminase might be significantly unstable. Since the number of terminase small subunit decamers (gp1), responsible for recognition of pac, is auto-regulated³⁶ a limited number of terminases are likely to be loaded to the DNA substrate at early stages of the virus infection cycle. The terminase then remains stably associated to the same concatemeric molecule and processive DNA packaging predominates during the rest of the infection cycle. This strategy would ensure specificity of viral DNA encapsidation (terminase binding at pac) and furthermore maintain the integrity of the SPP1 DNA concatemer by avoiding excessive cleavage at pac sequences and thus ensure occurrence of long processive packaging series along a concatemer.⁸ Similar observations were reported for bacteriophage P22.³⁷

SPP1-Mediated Transduction: Packaging of Host DNA

Bacteriophage SPP1 mediates generalised transduction.³⁸ This process involves the transfer of nonviral genetic material between a donor cell and a receptor cell mediated by a virus. SPP1 transducing particles are indistinguishable from SPP1 virions with the exception that they contain a host DNA molecule with a size similar to that of the mature SPP1 chromosome. ³⁹ In absence of homology between the host and SPP1 DNAs the frequency of transduction is very low: 10^-7 – 10^-8 for host chromosome genes³⁸ and 10^-4 – 10^-5 for plasmid DNA.⁴⁰ One SPP1 particle can transduce approximately 1 % of the B. subtilis chromosome.³⁸ Plasmid DNA, which is usually smaller than a phage chromosome unit-length, is encapsidated as a linear concatemer containing multiple copies of the plasmid in a head-to-tail arrangement (fig. 1). SPP1 infection, as in the case of other phages, was shown to change the mode of plasmid replication to generate plasmid concatemers that are the substrate for DNA packaging.^41,42 Generalised transduction is attributed to a low frequency error of the terminase to recognise and initiate processive headful packaging at pac-related sequences in the host DNA or, in some cases, at DNA free ends.

The efficiency of transduction is significantly increased in presence of homology (>47 base pairs) between the host DNA molecule and the virus chromosome.^43,44 This facilitated transduction is attributed to homologous recombination between the two replicons yielding chimeric phage-host DNA molecules. After initiating encapsidation at a pac sequence of the phage genome the highly processive phage packaging machinery would then access host DNA through the region of recombination and package headfuls of it. Presence of pac in the host cell DNA leads to packaging initiation at this sequence and to unidirectional encapsidation of host DNA.⁴⁵

Concluding Remarks

The phenomenology of bacteriophage SPP1 DNA packaging is well known and some of the molecular mechanisms involved are among the best understood in tailed phages systems. These include the terminase recognition and cleavage of its target sequence pac, the structure and function of the portal protein, the mechanism of headful sensor, and connector assembly. Other aspects of the DNA packaging process were not yet studied in detail like the terminase-procapsid interaction, the properties and assembly of the DNA translocating complex, or the mechanism of DNA translocation. A complete picture of the DNA packaging process requires knowledge of all these molecular mechanisms and their integration in the dynamics of the infected cell. An interdisciplinary approach combining genetics, biochemistry, structural biology and bacterial cell biology, on one side, and comparative analysis of the strategies used in different virus systems, on the other side, will undoubtedly be necessary to characterise the unifying mechanisms optimised by tailed phages and herpesviruses to encapsidate their viral genome.

Acknowledgments

We thank T.A. Trautner for critically reading the review and for his long-lasting engagement and support to SPP1 research. We thank E.V. Orlova for kindly providing (fig. 5).

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