The bacteriophage phi 29 head-tail connector shows 13-fold symmetry in both hexagonally packed arrays and as single particles.

The symmetry of the phi 29 head-tail connector is controversial: several studies of two-dimensional arrays of the connector have found a 12-fold symmetry, while a recent study of isolated particles has found a 13-fold symmetry. To investigate whether a polymorphism of the structure might explain these different results, electron microscopy and image analysis were used to study both isolated connectors and particles in hexagonally packed arrays. The hexagonally packed arrays have a P1 symmetry, and the connectors displayed 13 subunits both in the arrays and as isolated single particles. While we do not observe a polymorphism between connectors in two-dimensional arrays and as isolated particles, data show that the connectors can exist with either 12 or 13 subunits. A three-dimensional reconstruction of our 13-fold connector was generated by combining an averaged side-view projection with the known symmetry. The structure of rosettes of the connectors formed in the presence of phi 29 prohead RNA (pRNA) was also examined. These rosettes contain five connectors arranged about a single connector in the center, and this arrangement may reflect an essential role of the pRNA in mediating a symmetry mismatch between either a 12- or 13-fold symmetric connector and a putative fivefold symmetric prohead portal vertex into which the connector fits.     

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.     

Structural changes of bacteriophage phi29 upon DNA packaging and release

Cryo-electron microscopy three-dimensional reconstructions have been made of mature and of emptied bacteriophage phi29 particles without making symmetry assumptions. Comparisons of these structures with each other and with the phi29 prohead indicate how conformational changes might initiate successive steps of assembly and infection. The 12 adsorption capable 'appendages' were found to have a structure homologous to the bacteriophage P22 tailspikes. Two of the appendages are extended radially outwards, away from the long axis of the virus, whereas the others are around and parallel to the phage axis. The appendage orientations are correlated with the symmetry-mismatched positions of the five-fold related head fibers, suggesting a mechanism for partial cell wall digestion upon rotation of the head about the tail when initiating infection. The narrow end of the head-tail connector is expanded in the mature virus. Gene product 3, bound to the 5' ends of the genome, appears to be positioned within the expanded connector, which may potentiate the release of DNA-packaging machine components, creating a binding site for attachment of the tail.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: mapping and functional analysis of the head fiber gene.

A set of mutants of Bacillus subtilis bacteriophage phi29 unable to synthesize the head fiber protein has been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Infectious phage are produced during restrictive infection. We have focused on mutant sus 8.5(900) because the mutation is suppressible by both the su(+3) and su(+44) hosts, and it can be mapped by three- and four-factor crosses. After restrictive infection with mutant sus 8.5(900), a fragment about 70% of the size of the normal fiber is produced as well as particles that are fast-sedimenting in sucrose gradients relative to phi29(+). These particles have the buoyant density of particles with the fibers removed and have the absolute plating efficiency of phi29(+). Fiber protein is absent from prohead as well as virion. A second set of mutants produces fiber protein with a slightly altered electrophoretic mobility. This type of fiber protein is either present or absent on both prohead and virion. A third class of mutants, typified by 914, produces a "normal" fiber, but a major head protein of altered electrophoretic mobility. After infection by this mutant, the fiber is absent from both prohead and virion, and the biological and physical properties of the 914(-) particle are similar to those of particles produced after infection of the su(-) host by sus8.5(900). These observations suggest that the head fiber is not an essential component of the prohead or virion and that the assembly process is efficient in the absence of fiber protein. Three- and four-factor genetic crosses have established the order sus8(769)-8(914)-sus8.5(900)-sus9(756) and indicate that cistrons 8 and 8.5 code for the major head protein and head fiber protein, respectively.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: preliminary isolation and characterization of intermediate particles of the assembly pathway.

Three classes of particles have been identified in restrictive phi 29 suppressor-sensitive (sus) mutant infections of Bacillus subtilis, including DNA-containing heads or phage, prohead, and empty heads. Pulse-chase labeling experiments indicate that the prohead, the first particle assembled in 14-infected cells, is converted to DNA-filled heads and phi 29. In addition to the proteins Hd, P10, and F found in mature phi 29, the prohead contains a "core" protein P7 that exits as the prohead matures and appears to recycle during subsequent rounds of prohead assembly. Prohead-like structures accumulate in UV-irradiated cells and are present in restrictive infections with sus mutants of cistrons 9 and 16. Empty heads are observed only when infection results in the formation of DNA-containing particles; this and other evidence indicates that the empty heads are probably not true intermediates. Phage phi 29 assembly apparently occurs by a single pathway in which neck and tail components interact to stabilize the completed DNA-containing head.     

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29

Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.     

Composition and mass of the bacteriophage phi29 prohead and virion

The protein composition of the Bacillus subtilis bacteriophage phi29 prohead and virion was determined by combustion of gel bands of (3)H-labeled proteins. Copy numbers of individual proteins were calculated relative to the 12 copies of the head-tail connector protein. The mean numbers of copies of the major capsid protein in the prohead and virion were 241 and 218, respectively, approaching the 235 copies determined previously by cryoelectron microscopy. The mean numbers of copies of the dimeric head fiber on the prohead and virion were 24 and 31, respectively, demonstrating partial occupancy of the 55 fiber binding sites. Measured copies of neck and tail proteins in the virion included 11 of the lower collar, 58 of the appendage, and 9 of the tail; if the true copies of these proteins are 12, 60, and 9, respectively, the entire neck and tail of phi29 has quasi-sixfold symmetry. The mass of the fiberless prohead with pRNA was about 14.2 MDa, and the mass of the prohead determined by scanning transmission electron microscopy was consistent with the biochemical data. The mass of the fiberless virion containing the 12.8-MDa DNA genome was about 30.4 MDa. A full complement of dimeric fibers on the prohead or virion would increase the mass of the particle by about 3.2 MDa. The data complement studies relating the structure of phi29 components to dynamic functions in morphogenesis and infection.     

Phylogenetic analysis and secondary structure of the Bacillus subtilis bacteriophage RNA required for DNA packaging

An unusual RNA molecule encoded by the Bacillus subtilis bacteriophage phi 29 is a structural component of the viral prohead and is required for the ATP-dependent packaging of DNA. Here we report a model of secondary structure for this prohead RNA developed from a phylogenetic analysis of the primary sequences of prohead RNAs of related phages. Twenty-nine phages related to phi 29 were found to produce prohead RNAs. These RNAs were analyzed by their ability to replace phi 29 RNA in in vitro phage assembly, by Northern blot hybridization with a probe complementary to phi 29 RNA, and by partial and complete sequence analyses. These analyses revealed four quite different sequences ranging in length from 161 to 174 residues. The secondary structure deduced from these sequences, in agreement with earlier observations, indicated that prohead RNA is organized into two domains. The larger 5'-domain (Domain I) is composed of 113-117 residues and contains four helices. Three of these helices appear to be organized into a central stem that is interrupted by two unpaired loops and the fourth helix and loop. The smaller 3'-domain (Domain II) is composed of 40-44 residues and consists of two helices. Domains I and II are separated by 8-13 unpaired residues. Nuclease cleavage occurs readily in this single-stranded joining region, and this cleavage allows the subsequent separation of the two RNA domains. The separated Domain I is fully active in DNA packaging in vitro. The functional significance and biological role of Domain II are unknown. The phylogenetic secondary structure model provides a basis for further analysis of the role of this RNA in bacteriophage morphogenesis.     

RNA dependence of the bacteriophage phi 29 DNA packaging ATPase.

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

DNA packaging motor assembly intermediate of bacteriophage phi29

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

Implication of the prohead RNA in phage phi29 DNA packaging

RNA is an important component of many biological processes, including DNA encapsidation of bacteriophage phi29 of Bacillus subtilis. Interestingly, the prohead RNA is involved in this encapsidation, and was found in monomer, dimer, pentamer and hexamer conformations. This article presents and debates current knowledge about the prohead RNA structures, mechanisms, and roles in DNA encapsidation. A new dimer structure is presented, and its specific role in DNA encapsidation is discussed.     

Defining molecular and domain boundaries in the bacteriophage phi29 DNA packaging motor

Cryo-electron microscopy (cryo-EM) studies of the bacteriophage phi29 DNA packaging motor have delineated the relative positions and molecular boundaries of the 12-fold symmetric head-tail connector, the 5-fold symmetric prohead RNA (pRNA), the ATPase that provides the energy for packaging, and the procapsid. Reconstructions, assuming 5-fold symmetry, were determined for proheads with 174-base, 120-base, and 71-base pRNA; proheads lacking pRNA; proheads with ATPase bound; and proheads in which the packaging motor was missing the connector. These structures are consistent with pRNA and ATPase forming a pentameric motor component around the unique vertex of proheads. They suggest an assembly pathway for the packaging motor and a mechanism for DNA translocation into empty proheads.     

DNA packaging and delivery machines in tailed bacteriophages

Several symmetric and asymmetric reconstructions of bacteriophage particles have recently been determined using electron cryo-microscopy and image reconstruction, and X-ray crystal structures of phage particles and particle-associated gene products have also been solved. In the past two years, the asymmetric structures of four different phages, T7, epsilon15, P22 and phi29, were determined at resolutions sufficient to visualize details of the machinery for DNA packaging and delivery, as well as the organization of the double-stranded DNA within the particles. Invariably, the portals, through which DNA enters and leaves the particle, have 12-fold symmetry, occupy a pentavalent site in the capsid and, along with tail machine accessory proteins attached to it, are fixed in a specific orientation relative to the rest of the capsid.     

A three-dimensional cryo-electron microscopy structure of the bacteriophage phiKZ head

The three-dimensional structure of the Pseudomonas aeruginosa bacteriophage phiKZ head has been determined by cryo-electron microscopy and image reconstruction to 18A resolution. The head has icosahedral symmetry measuring 1455 A in diameter along 5-fold axes and a unique portal vertex to which is attached an approximately 1800 A-long contractile tail. The 65 kDa major capsid protein, gp120, is organized into a surface lattice of hexamers, with T = 27 triangulation. The shape and size of the hexamers is similar to the hexameric building blocks of the bacteriophages T4, phi29, P22, and HK97. Pentameric vertices of the capsid are occupied by complexes composed of several special vertex proteins. The double-stranded genomic DNA is packaged into a highly condensed series of layers, separated by 24 A, that follow the contour of the inner wall of the capsid.     

Maturation of the head of bacteriophage T4. I. DNA packaging events

Pulse-chase experiments in wild-type and mutant phage-infected cells provide evidence that the following particles called prohead I, II and III are successive precursors to the mature heads. The prohead I particles contain predominantly the precursor protein P23 and possibly P22 (mol. wt 31,000) and IP III (mol. wt 24,000) and have an s value of about 400 S. Concomitantly with the cleavage of most of P23 (mol. wt 55,000) to P231 (mol. wt 45,000), they are rapidly converted into prohead II particles which sediment with about 350 S. The prohead II particles contain, in addition to P231, the major constituents of the viral shella—a core consisting of proteins P22 and IP III. In cell lysates, prohead I and prohead II particles contain no DNA in a DNase-resistant form and are not bound to the replicative DNA. We cannot, however, positively rule out the possibility that these particles may have contained some DNA while in the cells.The prohead II particles are in turn converted into particles which sediment with about 550 S after DNase treatment (prohead III). During this conversion about 50% of normal DNA complement becomes packaged in a DNase-resistant form, and roughly 50% of the core proteins P22 and IP III are cleaved. In lysates the prohead III particles are attached to the replicative DNA. The prohead III particle appears to be the immediate precursor of the full mature head (1100 S). Cleavage of protein P22 to small polypeptides and conversion of IP III IP III1 are completed at this time. No precursor proteins are found in the full heads. Studies with various mutant phage showed that the prohead II to III conversion is blocked by mutations in genes 16 and 17 and that the conversion of the prohead III particles to the mature heads is blocked by mutations in gene 49. Cleavage of the head proteins, however, occurs normally in these mutant-infected cells. We conclude that the cleavage of the major component of the viral shell, P23, into P231 precedes the DNA packaging event, whereas cleavage of the core proteins P22 and IP III appears to be intimately linked to the DNA packaging event. Models relating the cleavage processes to DNA encapsulation are discussed.     

Characterization of the small RNA of the bacteriophage phi 29 DNA packaging machine.

The prohead connector of the bacteriophage luminal diameter 29 DNA packaging machine was reconstructed with the small RNA that regulates DNA packaging in vitro. The complete sequence of the 120 nucleotide RNA proved its origination from the promoter PE1(A1) of the left early region of phi 29 DNA, the end packaged first during assembly. The prohead RNA was clearly distinct from eubacterial 5S rRNA in sequence and composition.     

Cleaving the prohead RNA of bacteriophage phi 29 alters the in vitro packaging of restriction fragments of DNA-gp3

In vitro packaging of restriction fragments of the bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) in the defined system was dependent on prohead RNA. Truncated prohead RNAs were obtained by in situ RNase A digestion, isolated and sequenced. Proheads having the intact 174 base RNA were compared to proheads having RNAs of 120, 95, 71, 69 or 54 bases for the capacity to package the DNA-gp3 left and right ends and internal (non-end) fragments generated by the restriction enzymes EcoRI, HpaI and BstNI. Proheads with the 174 or 120 base RNAs packaged both left and right ends; internal fragments were packaged more efficiently by proheads with the 120 base RNA. Proheads with the 95 base RNA packaged DNA-gp3 left ends and internal fragments efficiently, but lost the capacity to package right ends. Only internal fragments were packaged by proheads with the 71 base RNA, and proheads having 69 or 54 base RNAs were inactive. RNA-free proheads were effectively reconstituted with purified 174 and 120 base RNAs to produce particles similar in biological activity to the proheads from which the RNAs were isolated. The 95 base RNA was the smallest RNA of the group that could reconstitute the prohead and direct fragment packaging, although packaging was inefficient. Alteration of the specificity of DNA fragment packaging with truncated prohead RNAs has delineated RNA domains that function in DNA-gp3 recognition and prohead binding.     

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.     

Topology of the components of the DNA packaging machinery in the phage phi29 prohead

Chromosome condensation inside dsDNA viral particles is a complex process requiring the coordinated action of several viral components. The similarity of the process in different viral systems has led to the suggestion that there is a common underlying mechanism for DNA packaging, in which the portal vertex or connector plays a key role. We have studied the topology of the packaging machinery using a number of antibodies directed against different domains of the connector. The charged amino-terminal, the carboxyl-terminal, and the RNA binding domain are accessible areas in the connector assembled into the prohead, while the domains corresponding to the 12 large appendages of the connector are buried inside the prohead. Furthermore, while the antibodies against the carboxyl and amino-terminal do not affect the packaging reaction, incubation of proheads with antibodies against the RNA binding domain abolishes the packaging activity. The comparison of the three-dimensional reconstructions of bacteriophage phi29 proheads with proheads devoid of their specific pRNA by RNase treatment shows that this treatment removes structural elements of the distal vertex of the portal structure, suggesting that the pRNA required for packaging is located at the open gate of the channel in the narrow side of the connector.     

The three-dimensional structure of a DNA translocating machine at 10 A resolution.

BACKGROUND: Head-tail connectors are viral substructures that are very important in the viral morphogenetic cycle, having roles in the formation of the precursor capsid (prohead), DNA packaging, tail binding to the mature head and in the infection process. Structural information on the connector would, therefore, help us to understand how this structure is related to a multiplicity of functions. RESULTS: Recombinant bacteriophage phi29 connectors have been crystallized in two-dimensional aggregates. An average projection image and a three-dimensional map have been obtained at 8 A and 10 A resolution, respectively, from untilted and tilted images of vitrified specimens of the two-dimensional crystals. The average projection image reveals a central mass surrounding a channel with 12 appendages protruding from the central mass. The three-dimensional map reveals a wide domain surrounded by 12 appendages that interact with the prohead vertex, and a narrow domain that interacts with the bacteriophage tail. At the junction of the two domains, 12 smaller appendages are visualized. A channel runs along the axis of the connector structure and is sufficiently wide to allow a double-stranded DNA molecule to pass through. CONCLUSIONS: The propeller-like structure of the phi29 connector strengthens the notion of the connector rotating during DNA packaging. The groove formed by the two lanes of large and small appendages may act as a rail to prevent the liberation of the connector from the prohead vertex during rotation.