Three-dimensional reconstruction of bacteriophage phi 29 neck particles at 2 X 2 nm resolution

The three-dimensional structure of the head-to-tail connecting region of bacteriophage phi 29 has been studied by analysing two-dimensional, hexagonal ordered aggregates of negatively stained viral necks to a resolution of 2 X 2 nm. These necks are composed of two proteins, p10 and p11; p10 being the connector protein. A 12-folded and a 6-folded axially symmetric domain are present in the specimen. The 12-folded domain is the larger part of the structure; it consists of 12 subunits associated in pairs. These subunits appear to be more closely paired towards the centre, where only six subunits are resolved forming the 6-folded domain. The pairs of subunits present an important twist between the 12-folded and the 6-folded areas. A conical hole is formed at the centre of the structure. This hole is more open at the 12-folded domain than at the level of the possible zone of interaction between p10 and p11, where it is almost closed. Protein p11 is very poorly represented in the reconstruction, probably due to lack of staining. The structure described for the phi 29 neck has many of the attributes expected for an active device involved in bacteriophage DNA encapsidation.     

Domain architecture of the bacteriophage phi29 connector protein.

Viral connectors are essential components of the DNA packaging machinery. They interact with nucleic acids and other viral components to translocate DNA inside the viral head. We have attempted to locate the different structural and functional domains of the phage Phi29 connector using a combination of approaches to generate different antigenic probes. Complexes of native connectors with either monoclonal or monospecific antibodies were studied by immunoelectron microscopy and image averaging methods. The data were merged in a model of the connector domain structure at 2-3 nm resolution. This epitope mapping provides a general outline of the folding architecture of the connector polypeptide, following a complicated threading that places the amino and carboxyl-terminals in close alignment in the narrower domain at 2-3 nm from the top of the connector. The appendages are built up by a long and highly immunogenic sequence (amino acid residues 153 to 206). The RNA binding domain forms part of the top of the narrow conical area of the connector, a flexible region that undergoes structural changes during viral morphogenesis. The DNA binding domain is located not far away, 2-3 nm below, in the outer side of the narrow conical part. The precise location of the functional domains of the connector, as well as their relative positions provide the first experimental framework for understanding the connector function.     

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.     

Conformational changes in bacteriophage phi 29 connector prevents DNA-binding activity

In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.     

DNA conformational change induced by the bacteriophage phi 29 connector.

Translocation of viral DNA inwards and outwards of the capsid of double-stranded DNA bacteriophages occurs through the connector, a key viral structure that is known to interact with DNA. It is shown here that phage phi 29 connector binds both linear and circular double-stranded DNA. However, DNA-mediated protection of phi 29 connectors against Staphylococcus aureus endoprotease V8 digestion suggests that binding to linear DNA is more stable than to circular DNA. Endoprotease V8-protection assays also suggest that the length of the linear DNA required to produce a stable phi 29 connector-DNA interaction is, at least, twice longer than the phi 29 connector channel. This result is confirmed by experiments of phi 29 connector-protection of DNA against DNase I digestion. Furthermore, DNA circularization assays indicate that phi 29 connectors restrain negative supercoiling when bound to linear DNA. This DNA conformational change is not observed upon binding to circular DNA and it could reflect the existence of some left-handed DNA coiling or DNA untwisting inside of the phi 29 connector channel.     

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.     

Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29

A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of phi29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi29 DNA packaging 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.     

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.     

Structure of Bacillus subtilis bacteriophage phi 29 and the length of phi 29 deoxyribonucleic acid

Anderson, D. L. (University of Minnesota, Minneapolis), D. D. Hickman, and B. E. Reilly. Structure of Bacillus subtilis bacteriophage phi29 and the length of phi29 deoxyribonucleic acid. J. Bacteriol. 91:2081-2089. 1966-Bacillus subtilis bacteriophage phi29 were negatively stained with phosphotungstic acid. The head of phi29 has a hexagonal outline with a flattened base, and is about 315 A wide and 415 A in length. The virus has an intricate tail about 325 A in length. Twelve spindle-shaped appendages are attached to the lower of two collars which comprise the proximal portion of the tail. The distal 130 A of the tail axis has a diameter of about 60 A and is larger in diameter than the axis of the upper portion of the tail. Comparison of electron microscopic counts of phi29 with plaque-forming units indicated that about 50% of the microscopic entities were infective. Phenol-extracted phi29 deoxyribonucleic acid (DNA) molecules were prepared for electron microscopy by the cytochrome c film technique of Kleinschmidt et al. Measurement of contour lengths of DNA molecules from three preparations gave skewed distributions of lengths with observed modal class values ranging from 5.7 to 5.9 mu. Assuming that phi29 DNA is a double helix in the B form, the corresponding molecular weights would be 10.9 x 10(6) to 11.3 x 10(6) daltons. The largest DNA molecules would have a volume of 1.9 x 10(7) A(3) which is about 25% greater than the estimated 1.4 x 10(7) A(3) internal volume of the phage head.     

Determinants of bacteriophage phi29 head morphology

Bacteriophage phi29 requires scaffolding protein to assemble the 450 x 540 A prolate prohead with T = 3 symmetry end caps. In infections with a temperature-sensitive mutant scaffolding protein, capsids assemble predominantly into 370 A diameter isometric particles with T = 3 symmetry that lack a head-tail connector. However, a few larger, 430 A diameter, particles are also assembled. Cryo-electron microscopy shows that these larger particles are icosahedral with T = 4 symmetry. The prolate prohead, as well as the two isometric capsids with T = 3 and T = 4 symmetry, are composed of similar pentamers and differently skewed hexamers. The skewing of the hexamers in the equatorial region of proheads and in the T = 4 isometric particles reflects their different environments. One of the functions of the scaffolding protein, present in the prohead, may be to stabilize skewed hexamers during assembly.     

Detailed architecture of a DNA translocating machine: the high-resolution structure of the bacteriophage phi29 connector particle.

The three-dimensional crystal structure of the bacteriophage phi29 connector has been solved and refined to 2.1A resolution. This 422 kDa oligomeric protein connects the head of the phage to its tail and translocates the DNA into the prohead during packaging. Each monomer has an elongated shape and is composed of a central, mainly alpha-helical domain that includes a three-helix bundle, a distal alpha/beta domain and a proximal six-stranded SH3-like domain. The protomers assemble into a 12-mer, propeller-like, super-structure with a 35 A wide central channel. The surface of the channel is mainly electronegative, but it includes two lysine rings 20 A apart. On the external surface of the particle a hydrophobic belt extends to the concave area below the SH3-like domain, which forms a crown that retains the particle in the head. The lipophilic belt contacts the non-matching symmetry vertex of the capsid and forms a bearing for the connector rotation. The structure suggests a translocation mechanism in which the longitudinal displacement of the DNA along its axis is coupled to connector spinning.     

Structure of the head-tail connector of bacteriophage φ29

The head-to-tail connecting region of bacteriophage φ29 has been studied by isolating neck-tail complexes from disrupted phage. These complexes can be isolated with appendages (from wild-type phage) or without appendages (from phage mutant sus12). Treatment of the neck-tail complex without appendages with urea or guanidinium hydrochloride releases the tail protein (p9) from the neck complex (proteins p10 and p11). Electron micrographs of φ29 necks show that they are composed of two collars and a thin axial tube. There is an internal hole along the longitudinal axis, from the upper collar to the thin tube.Image-processing analysis of electron micrographs of two-dimensional crystals of necks shows that the neck of phage φ29 consists of 12 external units and an internal area of apparent 6-fold symmetry, with a hole in the centre.     

Controlling bacteriophage phi29 DNA-packaging motor by addition or discharge of a peptide at N-terminus of connector protein that interacts with pRNA

Bacteriophage phi29 utilizes a motor to translocate genomic DNA into a preformed procapsid. The motor contains six pRNAs, an enzyme and one 12-subunit connector with a central channel for DNA transportation. A 20-residue peptide containing a His-tag was fused to the N-terminus of the connector protein gp10. This fusion neither interfered with procapsid assembly nor affected the morphology of the prolate-shaped procapsid. However, the pRNA binding and virion assembly activity were greatly reduced. Such decreased functions can be switched back on by the removal of the tag via protease cleavage, supporting the previous finding that the N-terminus of gp10 is essential for the pRNA binding. The DNA-packaging efficiency with dimeric pRNA was more seriously affected by the extension than with monomeric pRNA. It is speculated that the fusion of the tag generated physical hindrance to pRNA binding, with greater influence for the dimers than the monomers due to their size. These results reveal a potential to turn off and turn on the motor by attaching or removing, respectively, a component to outer part of the motor, and offers an approach for the inhibition of viral replication by using a drug or a small peptide targeted to motor components.     

Deletions at the N terminus of bacteriophage phi 29 protein p6: DNA binding and activity in phi 29 DNA replication

Deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial Met, have been introduced into the N terminus of the phage phi 29 protein p6. The activity of such proteins in the in vitro phi 29 DNA replication system, their capacity to interact with the phi 29 DNA ends, and their interference with the wild type (wt) protein p6 activity have been studied. The initiation activity of protein p6 decreased considerably when 5 as were deleted and was undetectable when 13 aa were removed. The mutant proteins were unable to specifically interact with the phi 29 DNA ends. These results indicate the need of an intact N terminus for the activity of protein p6. However, such N-truncated proteins inhibited both the specific binding of the wt protein p6 to the phi 29 DNA ends and its activity in phi 29 DNA replication.     

DNA poised for release in bacteriophage phi29

We present here the first asymmetric, three-dimensional reconstruction of a tailed dsDNA virus, the mature bacteriophage phi29, at subnanometer resolution. This structure reveals the rich detail of the asymmetric interactions and conformational dynamics of the phi29 protein and DNA components, and provides novel insight into the mechanics of virus assembly. For example, the dodecameric head-tail connector protein undergoes significant rearrangement upon assembly into the virion. Specific interactions occur between the tightly packed dsDNA and the proteins of the head and tail. Of particular interest and novelty, an approximately 60A diameter toroid of dsDNA was observed in the connector-lower collar cavity. The extreme deformation that occurs over a small stretch of DNA is likely a consequence of the high pressure of the packaged genome. This toroid structure may help retain the DNA inside the capsid prior to its injection into the bacterial host.     

Mapping of temperature sensitive mutants of bacteriophage phi 29

Summary Temperature-sensitive mutants of eleven complementation groups of phage 29 have been mapped by means of two-factor crosses. The results show the existence of a single non-circular linkage map. Cistrons expressed early after infection are clustered at the left end of the map.     

Role of RNA in bacteriophage phi 29 DNA packaging

A novel bacteriophage phi 29 RNA of 174 nucleotides is essential for the in vitro packaging of the DNA-terminal protein complex into proheads. The RNA, bound to the prohead portal vertex (connector), participates in assembly and function of the DNA translocating ATPase and in recognition of the DNA left-end during the course of the packaging reaction. The RNA is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is both highly conserved and unique among small RNAs.