An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

ABSTRACT

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages.

IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs.

INTRODUCTION

During viral assembly, double-stranded DNA (dsDNA) viruses, such as the tailed bacteriophages and herpesviruses, package their genomic DNA into a preformed viral shell (prohead) (1, 2). This process is driven by a viral genome-encoded molecular motor that assembles at a unique vertex of the prohead and utilizes the energy from ATP binding and hydrolysis to translocate DNA. During this process, the DNA is compacted to a nearly crystalline density and the motor must overcome the barriers of electrostatic repulsion and entropy that oppose this process. Indeed, the dsDNA viral packaging motors are among the most powerful molecular motors yet characterized (3).

The packaging process involves distinct phases (Fig. 1A), including (i) initiation, which is comprised of assembly events that culminate in a prohead-motor-DNA substrate complex primed for packaging; (ii) translocation, whereby the DNA is driven into the head in an ATP-dependent manner; and (iii) termination, when DNA translocation ceases and the DNA-filled head is stabilized while the transient parts of the motor are readied for disassembly (2). While the mechanics of translocation are repeated over thousands of mechanochemical cycles, it is the singular events of the initiation phase that serve to provide the specificity for packaging. These include genome selection, thereby distinguishing the viral DNA from other DNAs in the cell and establishing the polarity of DNA packaging; DNA cleavage to generate a free DNA end from a concatemeric substrate; and coassembly of the prohead-motor-DNA substrate into a ternary initiation complex.

FIG 1.

DNA packaging in ϕ29. (A) DNA packaging pathway depicting key events that culminate in the production of an infectious virion. Illustrations are derived from various cryo-electron microscopy reconstructions of the ϕ29 prohead-motor complex (15) and virion (43). (B) The packaging motor of ϕ29 showing the dodecameric connector (portal) in green, the pentameric pRNA ring in magenta, and the pentameric ring ATPase gp16 in blue. (Reprinted from reference 42.) (C) Secondary structure of the 174-base pRNA (domains I and II). The 120-base functional form is comprised of domain I only and includes the prohead binding domain in blue (bases 25 to 95) and the A-helices that provide the scaffold for ATPase assembly in red (bases 1 to 24 and 96 to 117). Domain II, of unknown function (bases 131 to 174, which comprise the structured region, and bases 118 to 130, which comprise the single-stranded region), is shown in green. Yellow shading, bases involved in the intermolecular interaction between pRNAs that create the pentameric ring.

In general, the phage packaging motors are comprised of a head-tail connector (portal protein) embedded in a unique vertex of the prohead, a packaging ATPase (the large subunit of the terminase complex [terL] in most dsDNA phages), and a DNA recognition component (the small subunit of the terminase complex [terS] in most phages) (1, 2). The connector is a dodecameric ring with a central channel through which the DNA passes into the head during packaging and out of the head upon ejection. Packaging ATPases are members of a large class of cellular ring ATPases (4) and, when bound to the viral head, provide the power for DNA translocation (1, 2). For phages with concatemeric DNA substrates, the packaging ATPase (terL) also includes an endonuclease domain required to generate the termini needed in packaging. TerS is involved in recognition/binding of the DNA substrate and binds to the terL subunit, thereby recruiting the DNA to be packaged to the force-generating ATPase for encapsidation into the viral head. For at least phage T4, the terS subunit is not essential for DNA translocation in vitro (5).

The Bacillus subtilis phage ϕ29 serves as an excellent model system for mechanistic studies of DNA packaging due to its highly efficient in vitro packaging system and relatively simple composition (6). Extensive genetic, biochemical, structural, and single-molecule studies have generated a nearly complete pseudoatomic structure of the motor, assigned functions to structural elements in the motor components (6), demonstrated a high degree of motor coordination, and elucidated the complete mechanochemical cycle of the motor (7, 8). The ϕ29 motor has the connector (gp10) and ring ATPase (gp16; analogous to terL in other phages) components common to all phage packaging motors (Fig. 1B). Since the packaging substrate for ϕ29 is unit-length DNA, there is no requirement for an endonuclease function in the ϕ29 packaging ATPase (6). Covalently bound to each 5′ end of the ϕ29 DNA is the terminal protein gp3, which is essential for DNA packaging, as well as DNA replication. Relative to the genetic map, packaging initiates at the left terminal protein and proceeds in a left-to-right direction (9); consequently, the right end is the first end ejected and contains the genes needed to successfully complete the ejection process (10, 11). While there is no exact equivalent of terS in ϕ29, the DNA-binding function of terS suggests a function analogous to that of the gp3 terminal proteins (12).

ϕ29 is distinct from the other dsDNA phages in that a viral genome-encoded RNA (prohead RNA [pRNA]) is also an essential component of the packaging machine (13). pRNA assembles into a pentameric ring on the prohead via intermolecular base pairing between complementary loops of adjacent RNAs (14, 15). The pRNA ring is positioned between the connector and ATPase components, with projecting spokes serving as the scaffold for assembly of the ring form of the packaging ATPase gp16 (Fig. 1B) (15). Since these phage motors carry out the same fundamental tasks, yet the sizes of the ϕ29 protein motor components are only ∼60% of the sizes of the components found in the other phages, it is likely that the essential function(s) provided by pRNA is carried out by subdomains of the larger motor proteins in the other dsDNA phages (1).

pRNA is a 174-base transcript encoded by the extreme left end of the genome (bases 320 to 147 of 19,285 bp) (Fig. 1C) (16). It is a component of the prohead, making critical interactions with both the connector (17, 18) and the head shell (14, 19, 20). Early purification of pRNA on proheads yielded a 120-base form (13), but upon inclusion of an RNase inhibitor during purification, pRNA was found to actually be 174 bases (16). Phylogenetic analysis and nuclease digestion studies showed that the pRNA is comprised of two domains (domains I and II); the 120-base form of pRNA is comprised only of domain I (Fig. 1C) (21). In previous studies, proheads containing either the 120-base or the 174-base form of pRNA were assayed for DNA packaging and particle maturation in vitro. No differences in biological activity were observed. Thus, no function could be ascribed to the 54 residues of domain II (16). Given the similar in vitro biological activity of these two forms of pRNA, most of the mutational studies and mapping analyses of pRNA in the field have utilized the 120-base form. However, the conservation of domain II in all the pRNA-containing relative phages of ϕ29 (21) suggests that it may have a role in viral assembly.

Here, we report a function for domain II of pRNA. Packaging studies using DNA restriction digests to interrogate DNA end selection show that domain II plays a role in the initiation phase of packaging, providing specificity and stringency to the process. When domain II is present in the packaging motor, the selective left-end orientation of DNA packaging is maintained; in its absence, selectivity is compromised, as the motor packages both left- and right-end restriction fragments. The selectivity in packaging polarity imparted by domain II extended to genomic DNA from a relative of ϕ29; however, in packaging competition assays between these two DNAs, domain II also provided discrimination between ϕ29 and related phage genomes. Additionally, domain II provided stringency to packaging initiation, as motors lacking this domain packaged several restriction fragments into a prohead, indicating multiple initiation events. The presence of domain II restricted initiation to the packaging of just one DNA fragment. Investigation of copy number using engineered heteromeric rings containing both 174-base and 120-base pRNA suggests that the presence of just one or two copies of domain II is sufficient to impart both the specificity and stringency in initiation to the motor. The DNA recognition function for packaging in dsDNA phages is usually the role of the small terminase subunit; here, in ϕ29, the same function is attributed to domain II of pRNA.

MATERIALS AND METHODS

Production of packaging components. (i) Production of pRNA.

pRNA was produced by in vitro transcription from the plasmid pRT72, which contains the ϕ29 pRNA gene (22), using a T7 Quick high-yield RNA synthesis kit (New England BioLabs). For 120-base pRNA production, the plasmid DNA was cut with DdeI, which cleaves at a site engineered into the single-stranded region connecting the two domains. For 174-base pRNA production, the DdeI site was changed back to the wild-type sequence (121GUAC124 to CCUU) and the plasmid DNA was cut with DraI. The resulting RNAs were purified by denaturing urea-PAGE as previously described (23).

(ii) Production of proheads.

Proheads in which the pRNA is assembled to the head in vivo were isolated from an infection with a sus8.5(900)-sus16(300)-sus14(1241) mutant (defective in the nonessential head fibers, the packaging ATPase, and the holin, respectively) of Bacillus subtilis 12A (nonpermissive host) and purified by sucrose gradient centrifugation as previously described (23).

(iii) Production of reconstituted RNA-free proheads with pRNA.

RNA-free particles were produced by RNase A treatment of purified phage proheads, followed by repurification of the RNA-free particles by ultracentrifugation as described previously (23). Particles were reconstituted with the 120-base or the 174-base pRNA by incubating particles with pRNA at a ratio of 1:10 in 0.5× TMS buffer (25 mM Tris, pH 7.8, 5 mM MgCl₂, 50 mM NaCl) for 10 min prior to packaging as previously described (23).

(iv) Production of DNA packaging substrate.

ϕ29 DNA-gp3, [³H]DNA-gp3, or M2 DNA was extracted from phage with guanidinium chloride and then purified on CsCl density gradients in 0.5× TE buffer (25 mM Tris, pH 7.8, 5 mM EDTA) as previously described (24). Peak fractions were pooled. DNA-gp3 (25 to 100 μl) was dialyzed against 50 ml water or 10 mM Tris, pH 7.6, to remove the CsCl prior to use in packaging reactions or restriction digests.

For restriction digestion of DNA, 2 U enzyme was used per μg DNA and cut in the buffer supplied by the manufacturer at 37°C for 2 h.

(v) Production of gp16.

To facilitate mutagenesis studies of the gp16 ring ATPase for other work, gene 16 was cloned into the SUMOpro plasmid (LifeSensors) for expression in Escherichia coli. A SacI site was introduced downstream of the C-terminal glycine-glycine codons of the SUMO protein moiety in the SUMOpro plasmid to make pSUMO-SacI-XhoI. A SacI-gp16-XhoI PCR product was amplified from the pSAC-gp16 plasmid (25) and ligated into the pSUMO-SacI-XhoI plasmid. PCR was used to remove the 6-nucleotide SacI site, changing the first 3 nucleotides to a serine codon to improve SUMO protease digestion and the second 3 nucleotides to the native aspartate codon corresponding to the second residue in gp16. The plasmid was transformed into competent Rosetta(DE3) cells (Novagen) and selected for by growth in LB supplemented with kanamycin (50 μg/ml) and chloramphenicol (34 μg/ml). The sequence of the SUMO-gp16 fusion construct was verified by DNA sequencing.

(vi) Purification of gp16.

Rosetta cells containing the plasmid carrying SUMO-gp16 were grown overnight in LB medium containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol at 37°C. On the next morning, the culture was diluted 1/100 in LB medium containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol and grown at 37°C to an optical density at 600 nm of 0.5. Isopropyl-β-d-thiogalactopyranoside was added to 0.3 mM, and the culture was incubated at 37°C for 5 min and then shifted to 18°C for protein expression. After overnight induction, the cells were collected by centrifugation, concentrated 15-fold in buffer containing 50 mM Tris-HCl, pH 8, 500 mM NaCl, 5% glycerol (vol/vol), and 1 mM tris(2-carboxyethyl)phosphine (TCEP), and lysed by passage through a French press. MgCl₂ was added to 2.5 mM, DNase was added to 5 μg/ml, and the sample was incubated at room temperature for 15 min. The lysate was clarified by spinning in an SS34 rotor at 10,000 rpm for 40 min at 4°C. The supernatant was incubated with Talon resin (Clontech) that had been equilibrated in wash buffer (50 mM Tris-HCl, pH 8, 400 mM NaCl, 5% glycerol [vol/vol], 1 mM TCEP) at 4°C for 60 min. The resin with SUMO-gp16 bound was then washed with 10 volumes wash buffer. The SUMO-gp16 fusion protein was eluted in 5 volumes elution buffer (50 mM Tris-HCl, pH 8, 400 mM NaCl, 5% glycerol [vol/vol], 100 mM imidazole [pH 8], 1 mM TCEP), and 0.5-ml fractions were collected. The fractions were analyzed by SDS-PAGE to identify peak fractions. To cleave the SUMO tag and recover native gp16 protein, 100 μl of the SUMO-gp16 fusion protein was mixed with 200 μl wash buffer and 2.5 units of SUMO protease (Life Technologies), and the mixture was incubated at 4°C overnight. Ni-Sepharose 6 Fast Flow resin (GE Healthcare) was equilibrated with wash buffer and then incubated with the cleaved protein mixture for 60 min at 4°C. The resin containing the bound SUMO tag was pelleted in a microcentrifuge at 2,000 × g for 5 min, and the supernatant containing the native gp16 was recovered for use in biological assays. Prior to use in DNA packaging assays, gp16 was diluted to the appropriate concentration (see below) in 1× TM buffer (25 mM Tris, pH 7.6, 5 mM MgCl₂).

DNA packaging assay.

The in vitro DNA packaging assay is based on a DNase protection assay and was performed as described previously (23, 26). Briefly, proheads or RNA-free proheads reconstituted with the 120-base or the 174-base pRNA (8.3 nM), DNA-gp3 (4.2 nM; whole-length or restriction-digested DNA-gp3), and gp16 (166 to 208 nM) were mixed together in 0.5× TMS buffer in 20 μl and incubated for 5 min at room temperature. ATP was then added to 0.5 mM to initiate packaging, and the mixture was incubated for 15 min. DNase I was added to 1 μg/ml, and the mixture was incubated for 10 min to digest unpackaged DNA.

(i) Agarose gel analysis of DNA packaging.

After DNase digestion, EDTA (final concentration, 25 mM) and proteinase K (final concentration, 500 μg/ml) were then added to the reaction mixture, and the mixture was incubated for 30 min at 65°C to inactivate the DNase I and release the protected, packaged DNA from particles. The packaged DNA was analyzed by agarose gel electrophoresis.

(ii) Sucrose gradient analysis of DNA packaging.

[³H]DNA-gp3 was used as the packaging substrate. After DNase digestion of the packaging reaction, the sample was diluted 12- to 15-fold in 0.5× TMS buffer and loaded on top of a linear 5 to 20% sucrose gradient in 0.5× TMS buffer. The gradients were spun in an SW55 rotor at 35,000 rpm for 30 min at 20°C. The tube was punctured on the bottom, 16-drop fractions were collected, and the number of disintegrations per minute in each fraction was measured by liquid scintillation counting.

To stabilize DNA-filled heads that had not terminated packaging, adenosine 5′-[γ-thio]triphosphate (γ-S-ATP) was added to 250 μM prior to DNase addition. γ-S-ATP at 1 μM was also included in the gradient (27).

(iii) DNA competition packaging assay.

To assess competition between ϕ29 and M2 DNAs as the packaging substrate, proheads were made limiting and the packaging reaction was scaled up. Whole-length ϕ29 DNA-gp3 (4.2 nM) or M2 DNA (4.2 nM) or a 1:1 mixture of ϕ29 and M2 DNAs (8.4 nM) was mixed with proheads reconstituted with the 120-base or the 174-base pRNA (4.2 nM) and gp16 (166 to 208 nM) in 0.5× TMS buffer in 100 μl, and the mixture was incubated for 5 min at room temperature. ATP was then added to 0.5 mM to initiate packaging, and the mixture was incubated for 10 min. DNase I was added to 1 μg/ml, and the mixture was incubated for 10 min to digest unpackaged DNA. Proteinase K (final concentration, 500 μg/ml) and EDTA (final concentration, 10 mM) were added to the reaction mixture, and the mixture was heated at 65°C for 30 min. The reaction mixture was then extracted with an equal volume of phenol-chloroform, followed by two chloroform extractions. The aqueous phase was removed, 1/10 volume of 3 M sodium acetate was added, and the DNA was precipitated by addition of an equal volume of isopropanol and incubation at −20°C. The precipitate was pelleted by centrifugation in a microcentrifuge at 13,000 × g for 30 min. The pellet was washed twice with 0.5 ml 70% ethanol, and the pellet was air dried. The pellet was resuspended in water and then cut with EcoRI in the manufacturer's buffer at 37°C for 2 h. The DNA was analyzed on a 0.8% agarose gel.

Ordered heteromeric pRNA rings.

The sequences for the loop residues used to create ordered rings are based on those of Zhang et al. (28) and Guo et al. (29). The sequences for the two-pRNA system were 45-GCGA-48/85-UUGG-82 (F6 pRNA) and AACC/CGCU (F7 pRNA). The sequences for the three-pRNA system were 45-GGAC-48/85-UGCG-82 (Ab pRNA), ACGC/CGGU (Be pRNA), and GCCA/CCUG (Ea pRNA). For the ordered heteromeric rings, the 174-base pRNA was made using the F6, F7, or Ea template to create the variant RNAs; the remaining RNAs in a set were the 120-base form. The RNAs in a set were mixed in 1:1 ratios and used to reconstitute RNA-free proheads as described above. The resulting proheads with ordered rings or ordered variant rings were used in packaging experiments as described above.

RESULTS

Comparison of DNA packaging motors containing either the 120-base or the 174-base pRNA.

RNA-free proheads were reconstituted with the in vitro-transcribed 120-base pRNA (domain I only) or 174-base pRNA (domains I and II) and assayed for packaging using genome-length DNA-gp3 (Fig. 2A) or subgenomic restriction fragments of DNA-gp3 (Fig. 2B). Packaging activity was compared to that of proheads isolated from mutant-infected cells where pRNA was assembled into the head in vivo; the RNA content of these proheads is mostly the 174-base pRNA, with a small variable amount of the 120-base pRNA being present (16, 30).

FIG 2.

Comparison of DNA packaging activity of motors containing the 120-base or the 174-base pRNA. RNA-free proheads reconstituted with either the 120-base or the 174-base pRNA were assayed in the in vitro DNA packaging system using whole-length ϕ29 DNA-gp3 (A) or ClaI-digested ϕ29 DNA-gp3 (B) as a packaging substrate. Lanes P, proheads derived from infected cells, where pRNA is assembled into the head in vivo and contains the 174-base (174b) pRNA along with a small amount of the 120-base (120b) pRNA. After packaging, DNase treatment degrades unpackaged DNA, while the packaged DNA is protected inside the viral head. The packaged DNA is extracted and visualized in an agarose gel. Lanes input, the DNA added to the packaging assay; lanes −ve, a negative control where ATP was omitted from the reaction mixture. ClaI digestion yields fragments L-DNA-gp3 (left end [L]) and R-DNA-gp3 (right end [R]), which are 6,183 bp and 13,137 bp, respectively.

Both the 120-base and the 174-base pRNAs supported high levels of whole-length genome packaging, similar to the in vivo-derived prohead-pRNA complex (Fig. 2A). Using the ClaI restriction digest as the packaging substrate (6.1 kbp of the left DNA-gp3 end, 13.2 kbp of the right DNA-gp3 end), the in vivo-assembled prohead-pRNA complex preferentially packaged the left-end DNA-gp3 restriction fragment (Fig. 2B, lane 2), consistent with the demonstrated direction of packaging in ϕ29 proceeding from left to right (9). Proheads reconstituted with the 174-base pRNA also selectively packaged left ends (Fig. 2B, lane 4), similar to the in vivo prohead-pRNA complex. In contrast, the proheads reconstituted with the 120-base pRNA efficiently packaged both left- and right-end DNA-gp3 fragments, indicating a loss of specificity in DNA end selection (Fig. 2B, lane 3). Thus, the presence of domain II of pRNA conferred specificity in the packaging motor for the correct left-end orientation of DNA packaging.

Interestingly, for the proheads reconstituted with the 120-base pRNA, the motor not only was less selective for the DNA substrate, but also the overall quantity of DNA fragments packaged in the reaction was greater than that of in vivo-derived prohead-pRNA or proheads with the 174-base pRNA (Fig. 2B, lane 3 versus lanes 2 and 4). To investigate the nature of this increased quantity of packaged DNA, sucrose gradient centrifugation analysis of packaging was done, where the sedimentation of DNA-filled heads is dictated by the amount of DNA packaged in a head; i.e., the more DNA that the head contains, the faster the particle sediments (9).

The sedimentation profile for packaging using proheads reconstituted with the 174-base pRNA-containing heads showed a dominant peak in the position of heads containing a left-end 1/3-genome-length fragment (fraction 10; Fig. 3A) and a small peak of faster-sedimenting heads that packaged the larger 2/3-genome-length right-end fragment (fraction 7). These results are consistent with the packaging profile seen in the agarose gel packaging assay (Fig. 2B). For packaging with the 120-base pRNA-containing heads, the sedimentation profile showed a similarly sized peak due to left-end packaging, as well as a larger amount of right-end packaging (Fig. 3B), consistent with the findings on the agarose gel (Fig. 2B). However, two other peaks unique to the 120-base pRNA-containing heads were detected. The peak in fraction 15 was in the position of heads that did not terminate packaging properly, such that the DNA was unstably packaged and lost from the head during centrifugation (31). The other unique peak (fraction 4) contains heads that sedimented faster than heads containing the right-end DNA fragment (fraction 7).

FIG 3.

Sucrose gradient analysis of DNA packaging by motors containing the 120-base or the 174-base form of pRNA. ClaI-digested (A and B) or HpaI-digested (C) ³H-labeled ϕ29 DNA-gp3 was used as the packaging substrate in the in vitro DNA packaging system. After packaging, unpackaged DNA was digested with DNase I and the DNA-containing heads were then sedimented through a 5 to 20% sucrose gradient. DNA-filled heads migrate according to the amount of DNA that they contain. Gradient fractions were collected and counted. Sedimentation through the gradient is from right to left. (A and B) Packaging from motors containing the 174-base pRNA (A) and packaging from motors containing the 120-base pRNA (B); γ-S-ATP was added to the sample and gradient to stabilize DNA-filled heads that had not fully terminated packaging. The sizes of the DNA restriction fragments are as follows: ClaI digestion gives left DNA-gp3 of 6,183 bp and right DNA-gp3 of 13,137 bp; HpaI digestion gives left DNA-gp3 of 6,784 bp, right DNA-gp3 of 2,549 bp, and 6 internal fragments.

To stabilize the population of DNA-filled heads that lost their DNA during centrifugation, γ-S-ATP was included in the gradients to lock the motor in a DNA-bound state (27). The sedimentation profile of heads reconstituted with the 174-base pRNA was unchanged by the addition of γ-S-ATP (Fig. 3A). However, for heads reconstituted with the 120-base pRNA, the profile was altered in the presence of γ-S-ATP (Fig. 3B). Specifically, there was a reduction in the amount of radiolabel found in the unstably packaged particle position (fraction 15) and a corresponding increase in the amount of radiolabel found in the fastest-sedimenting peak (fraction 4) of the heads. This faster-sedimenting peak was unexpected, since the packaging substrates are subgenome lengths. Since ClaI digestion generates ends with single-stranded overhangs, it is possible that transient annealing of fragments led to packaging of a genome equivalent.

To exclude the possibility that heads in the fast-sedimenting position were the result of annealed sticky ends, a similar experiment was done using DNA cut with HpaI (Fig. 3C). Unlike ClaI, this digest generated blunt ends yet yielded a similarly sized left-end fragment. Additionally, the remainder of the DNA was cleaved into multiple fragments such that the left end was the largest DNA substrate. Therefore, any packaged particles that sediment faster than heads with left ends must contain more than one independent DNA fragment.

Similar to the result obtained with ClaI, the heads reconstituted with the 174-base pRNA selectively packaged the HpaI left-end fragment (Fig. 3C, fraction 9). For the heads reconstituted with the 120-base pRNA, there was a peak due to left-end packaging (fraction 9), as well as a broad peak of faster-sedimenting heads (fractions 3 to 7). A portion of this peak overlapped with the sedimentation position of heads that contain genome-length DNA (fractions 2 to 4, not shown), indicating that at least three HpaI DNA fragments (three left ends or an equivalent mass of left- and right-end DNA-gp3 fragments) must have been packaged to achieve this sedimentation position. Thus, in the absence of domain II, the motor with the 120-base pRNA undergoes multiple initiation events, packaging several DNA fragments into the head, while the presence of domain II in the motor with the 174-base pRNA provides stringency to the motor by restricting initiation to a singular, specific event.

Discrimination between ϕ29 and related phage genomes.

Since pRNA imparts specificity for selection of where to begin packaging, it may also provide selectivity regarding which DNA among the DNA molecules in the host cell to package. Phages closely related to ϕ29 also have genomes with terminal proteins, and their genomes encode pRNAs at positions similar the position where the pRNA is encoded by the ϕ29 genome (21). These related pRNAs have sequence homology ranging from 30 to 50%, yet they retain a similar domain I/domain II secondary structure organization. Since domain II of pRNA is involved in recognition of the left end of ϕ29 DNA, it may also provide specificity for the selection of ϕ29 DNA instead of related phage DNAs. To test this, proheads reconstituted with the 120-base or the 174-base pRNA were assayed for packaging using either ϕ29 DNA, the related phage M2 DNA, or a ϕ29-M2 DNA mixture as the packaging substrate. The DNAs were either digested with EcoRI before packaging to assess the orientation of DNA packaging initiation or digested after packaging to allow identification of the origin of DNA packaged in competition assays.

Using predigested DNA substrate, heads with the 120-base pRNA packaged left-end DNA-gp3, right-end DNA-gp3, and even some internal EcoRI ϕ29 DNA fragments (Fig. 4A, lane 2); a similar result was obtained using M2 DNA fragments as a substrate (Fig. 4B, lane 2). As expected, proheads reconstituted with the 174-base pRNA were selective for the ϕ29 EcoRI left-end fragment (Fig. 4A, lane 3), and this selectivity extended to a preference for the left-end M2 DNA-terminal protein fragment as well (Fig. 4B, lane 3).

FIG 4.

DNA packaging of ϕ29 and M2 DNAs. Motors containing the 120-base or the 174-base pRNA were assayed for packaging using ϕ29 or M2 DNA as the packaging substrate. To test the orientation of DNA packaging, the DNA substrate was EcoRI-digested ϕ29 DNA (A) or EcoRI-digested M2 DNA (B). (C) To examine the competition between ϕ29 and M2 DNAs in packaging, whole-length DNAs were packaged and then subsequently extracted and digested with EcoRI to identify the source of packaged DNA. Lanes 3 and 4, digests of ϕ29 and M2 DNA, respectively.

To test for the ability to discriminate between related DNAs, a mix of ϕ29 and M2 whole-length DNAs was used as the packaging substrate, and proheads were made limiting in the reaction mixture so that there would be competition for heads. The packaged DNA was then extracted from the heads and cleaved with EcoRI to identify the origin of the DNA that had been packaged on the basis of the cleavage pattern. For proheads with the 120-base pRNA, the fragment pattern of packaged DNA showed that both ϕ29 and M2 DNAs were packaged with similar levels of efficiency, indicating little specificity for selection of the native DNA substrate (Fig. 4C, lane 1). In contrast, for proheads with the 174-base pRNA, the digestion pattern showed that ϕ29 DNA was the preferred substrate; there was some packaging of M2 DNA (lane 2), but at a reduced efficiency compared to that of proheads with the 120-base pRNA (lane 1). Thus, the presence of domain II in the motor provided specificity for the selection of ϕ29 DNA, but this was not absolute, as a low level of M2 DNA was still packaged in the presence of native ϕ29 DNA substrate.

Copy number in the motor.

Having established a role for domain II in the initiation phase of packaging, we sought to determine the effect of the pRNA copy number on this process. Since the components of the phage packaging motors operate as rings, the question of whether a full complement of domain II in the motor is necessary to impart the observed specificity and stringency during initiation arises. Indeed, proheads with pRNA assembled in vivo contain some of the 120-base pRNA that is generated during prohead purification (16), yet they still retain specificity (Fig. 2), suggesting that a full complement of domain II may not be necessary. To assess the effect of the domain II copy number directly, we created ordered heteromeric rings of pRNA comprised of combinations of the 174-base and the 120-base pRNAs. Although the native pRNA ring consists of identical molecules, it has been shown that functional heteromeric rings can be assembled from a combination of two or three different pRNAs by engineering the bases involved in the intermolecular interaction (Fig. 5A) (28, 29). Each type of pRNA in a set has noncomplementary loops and is thus inactive on its own, but the loop sequences are complementary to a second (or third) type of modified pRNA. When all RNAs in a set are present, an ordered ring is created on the head via the complementary loops, thus restoring packaging activity. This design allows introduction of an additional mutation/variant of interest into one of the pRNAs in a set, thereby creating an ordered mutant ring (Fig. 5B). Thus, in a given population of proheads, use of the two-RNA system would yield heads with either 2 or 3 mutants/variants in the pRNA ring (present in a 1:1 ratio), whereas the three-RNA system would yield heads with 1 or 2 mutants/variants in the ring (present in a 1:2 ratio).

FIG 5.

Ordered heteromeric pRNA rings containing the 174-base and the 120-base pRNAs. (A) Schematic depicting how bases involved in the intermolecular base pairing are engineered with mutant sequences to create ordered pRNA rings. Wt, wild type. (B) The creation of ordered mutant rings involves introduction of a mutation/variant into one of the RNAs (spiked RNA) of either a two- or three-pRNA ordered set. The two-RNA system (green, blue) yields a population of proheads with 2 or 3 variant RNAs (in a 1:1 ratio) in the ring. The three-RNA system (green, blue, red) yields a population of proheads with one or two variant RNAs (in a 1:2 ratio) in the ring. (C) DNA packaging using the ordered heteromeric rings of the 174-base and the 120-base pRNAs. The 174-base pRNA was the variant, with the other RNA(s) of the set being comprised of the 120-base pRNA. Lanes 2 (120-base pRNA) and 3 (174-base pRNA), homomeric rings; lanes 4 (120-base pRNA), 5 (174-base pRNA), and 8 (120-base pRNA), ordered double or triple rings with RNAs of the same length; lanes 6 and 7, ordered double 174-base–120-base heteromeric rings; lane 9, ordered triple 174-base–120-base heteromeric rings.

Using these ordered systems, the selected variant RNA was the 174-base form, while the other RNA(s) in the sets was the 120-base form of pRNA, and the effect of these heteromeric rings on packaging was determined (Fig. 5C). As controls, ordered rings that were fully the 174-base or the 120-base form were assessed; these ordered rings had the same respective packaging phenotype as homomeric rings with the native sequence in the loops (for the 120-base pRNA, compare lane 2 to lanes 4 and 8; for the 174-base pRNA, compare lanes 3 and 5). For the 174-base–120-base heteromeric rings, both types of ordered sets showed left-end specificity, similar to heads with homomeric 174-base pRNA rings (compare lane 3 to lanes 6, 7, and 9). Furthermore, there was no increase in overall packaging efficiency, as seen with homomeric rings of the 120-base pRNA (lane 2), indicating that the stringency of the initiation process was also maintained. Together, this suggests that just 1 or 2 copies of domain II are sufficient to impart the left-end specificity and stringency for a single packaging initiation event.

DISCUSSION

The packaging motor of bacteriophage ϕ29 shares many commonalities with the motors of other dsDNA phages and the herpesviruses, yet it is distinct, in that there is an RNA component, in addition to the protein components of the packaging motor (6). A goal has been to determine the role of an RNA as an essential part of a force-generating motor. While it has been known for the last 25 years that pRNA is comprised of two domains (21), the role of domain II has been elusive due to the lack of a clear phenotype associated with its presence (16). Comparison of the DNA packaging activity of proheads containing either the 120-base pRNA or the 174-base pRNA showed similar levels of DNA packaging (Fig. 2A), consistent with previous reports (16). However, the use of subgenomic lengths of DNA as packaging substrates revealed that the presence of domain II determines the correct left-to-right orientation of DNA packaging (Fig. 2B) by excluding right-end DNA-gp3 from the population of DNAs packaged. Further, since pRNA is a component of the prohead, the specific selection of DNA for packaging takes place on the prohead, in contrast to other phage systems, such as bacteriophage lambda, where genome selection occurs on the DNA level, as the terminase complex first binds sites in the DNA prior to interaction with the head (32). If the polarity of packaging was determined via a DNA-gp3-ATPase interaction prior to prohead binding, then the presence or absence of domain II would not impact the specificity of DNA fragment selection; this was clearly not the case.

The orientation of DNA packaging is likely key to the viability of the virus. Indeed, the dsDNA phages in general have a polarity of packaging (1). The left-to-right orientation of DNA movement in packaging for ϕ29 is later reversed upon ejection, where the right end of the DNA enters the host cell first (33). Studies have shown that DNA ejection in ϕ29 is only partially driven by the internal pressure inside the packaged head (10, 11). After the right end enters the cell, genes 16.7 and 17 at the extreme right end of the genome are transcribed and the resulting proteins serve to pull the remainder of the ϕ29 DNA into the host cell. Failure to select the left end during the packaging initiation phase may yield heads where the right end of the DNA is not in the correct orientation to enter the cell first, resulting in an abortive infection.

Identification of the packaging start site is a role associated with the small subunit of the terminase complex in the other dsDNA phages, through recognition of either a packaging signal in the DNA (pac site) or, in the case of phage lambda, binding sites near the cohesive end sequence (cos site) (1, 2). Several atomic structures of small terminases have recently been solved, including a dimer form of the DNA-binding domain (34) and ring structures with various stoichiometries ranging from 8- to 12-mers (35–38). Models for DNA recognition include wrapping of DNA around the ring structure or threading of the DNA directly through the central channel. How an RNA molecule would carry out the same function remains to be determined. Since pRNA operates as a ring in the packaging motor, it could accommodate both the wrapping and threading models of genome recognition. Furthermore, the fact that pRNA is encoded by the extreme left end of the genome, where packaging begins, suggests the possibility of a transient RNA-DNA hybrid between domain II and its coding sequence in the DNA as a selective mechanism. A putative RNA-DNA hybrid would require only one copy of domain II to impart specificity, consistent with the results from the ordered heteromeric pRNA rings that revealed that only one or two copies of domain II are needed to impart a left-end specificity of packaging (Fig. 5C). Indeed, for several head-full packaging phages, the pac site for DNA cleavage to generate termini for packaging is located in the small terminase-coding sequence (39). An alternative possible determinant of packaging orientation is the terminal gp3 proteins, covalently bound to each 5′ end of the DNA. The packaging ATPase gp16 has been shown to interact specifically with gp3-containing restriction fragments, coiling both left- and right-end DNA-gp3 restriction fragments into faster-sedimenting forms (24). DNA recognition in this case would require discrimination between the right- and left-end terminal proteins. Given that the terminal protein on each end is also an essential component of a protein-primed DNA replication mechanism for ϕ29 (40), it seems less likely that they provide a discriminating difference between DNA ends.

The left-end specificity imparted by domain II held for the relative M2 DNA as well, with the 174-base pRNA showing specificity for the left end of M2 DNA, whereas the 120-base pRNA packaged left-end, right-end, and some internal M2 DNA fragments (Fig. 4A and B). The selectivity between native DNA and phage relative DNA was not absolute (Fig. 4C). It is possible that greater discrimination between substrates may occur in vivo for ϕ29, or it may represent similarities between the motor components in these two related phages. The sequence identity between the ϕ29 and M2 terminal proteins is 62%, and that between domain II of their pRNAs is 69%.

The presence of domain II also imparted stringency to the packaging event, restricting the motor to just one initiation event. The fact that, in the absence of domain II, the motor with the 120-base pRNA packaged multiple DNA fragments to nearly fill the head was surprising. Whereas other phages, such as T4 and SPP1, package a head full of DNA that is processed from a concatemeric DNA substrate generated during replication (1, 2), for ϕ29 the amount of DNA packaged is determined by the unit-length replicative DNA-gp3; hence, there is no innate requirement to sense how much DNA has been packaged, yet in the absence of domain II, the motor function was reminiscent of a head-full-type mechanism. How the 174-base pRNA provides a constraint on the motor to exclude multiple initiation events is unknown; whether the physical presence of domain II is restrictive or, rather, imparts a subtle effect on the conformation of the ring of ATPases remains to be determined.

Taken together, the data show that domain II plays an important role in the initiation phase of DNA packaging (Fig. 1A). Whereas the 120-base form (which contains domain I only) is sufficient for all steps to yield infectious virions in vitro (16), the presence of domain II (the 174-base pRNA) imparts to the process the stringency and selectivity that are likely essential in vivo. Similarly, terS of T4 phage has been reported to be dispensable in in vitro studies, yet it provides an essential function in vivo (1). While initiation is clearly occurring in the absence of domain II (and, by extension, the small terminase), it remains to be determined how this differs from the event that occurs with the presence of domain II that imparts the stringency to the process. Additionally, the ability to retain translocation activity and relax the stringency of substrate selection allows us to use pRNA as a tool in packaging studies. For instance, experiments employing modified DNA substrates or certain ATPase mutants may require relaxed initiation properties to allow us to probe translocation; here the choice of proheads with the 120-base pRNA may facilitate packaging, whereas the 174-base form would restrict the reaction.

During the packaging process, the dsDNA phages must complete a series of common tasks in order to successfully complete this stage in virion assembly (Fig. 6) (1, 2). The correct DNA substrate must be selected and oriented for packaging; in most of the phages, this is accomplished by the small subunit of terminase, whereas in ϕ29, domain II of pRNA provides selectivity (Fig. 6C, green shading of pRNA). The ATPase must assemble to the head; in the other phages, the large subunit of the terminase interacts directly with the connector and perhaps the head shell, whereas in ϕ29 it is the prohead binding domain of pRNA (Fig. 6C, blue shading) that binds directly to the head, interacting with the shell protein (14) and the connector (17–19). Prohead binding thereby positions the A-helices of pRNA (Fig. 6C, red shading) to serve as the scaffold for assembly of the gp16 ATPase ring on the prohead (25). The translocation process itself is driven by the packaging ATPase common to all motors. Finally, single-molecule studies of packaging show that the motors slow down as the head fills with DNA (3, 41), implying communication of the head-filling state to the motor. Again, for the other dsDNA phages, the ATPase and connector that are in direct contact would facilitate communication, whereas in ϕ29, since the connector/shell and ATPase do not contact each other, communication is likely mediated by the pRNA superhelices that connect them (42). Taken together, although the pRNA component is distinct to the motors of ϕ29 and its relatives, functions provided by pRNA are indeed encoded by both the small and large terminase components of the other phage motors. As the packaging field continues to dissect some of the more subtle aspects of motor function, such as communication, the pRNA component may offer an advantage in such studies, in that it is easily manipulated independently of the other motor components, thereby providing a means of disrupting communication while maintaining a wild-type ATPase and connector.

FIG 6.

Schematic of packaging motors for the dsDNA phages. (A) Generalized schematic for the DNA packaging motor in the dsDNA phages. The portal protein is the structure equivalent to the ϕ29 connector. terL is comprised of the ATPase and endonuclease functional domains, and terS is the DNA-binding component. (Reprinted from reference 2 with permission of the publisher.) (B) The DNA packaging motor of ϕ29, with the connector in green, the pRNA ring in magenta, and the ring ATPase gp16 in blue. The figure is adapted from reference 42. (C) Functional domains of pRNA as detailed in the legend to Fig. 1. Blue, the prohead binding domain; red, the A-helices of pRNA that comprise the protruding spokes that serve as scaffold for the ATPase; green, domain II, which provides specificity and stringency to the motor for the initiation phase of DNA packaging.