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.     

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

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

Role of φ29 connector channel loops in late-stage DNA packaging

Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have 12-fold head-tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA packaging motor, and the connector has a central channel through which viral DNA passes during genome packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Given the proximity to the translocating DNA substrate, these loops have been proposed to play a role in DNA packaging. Previous models have proposed structural motions in either the packaging ATPase or the connector channel loops as the driving force that translocates the DNA into the prohead. Here, we mutate the channel loops of the Bacillus subtilis bacteriophage φ29 connector and show that these loops have no active role in translocation of DNA. Instead, they appear to have an essential function near the end of packaging, acting to retain the packaged DNA in the head in preparation for motor detachment and subsequent tail assembly and virion completion.     

Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1

By electron microscopy and image analysis, we find that baculovirus-expressed UL6 is polymorphic, consisting of rings of 11-, 12-, 13-, and 14-fold symmetry. The 12-mer is likely to be the oligomer incorporated into procapsids: at a resolution of 16 A, it has an axial channel, peripheral flanges, and fits snugly into a vacant vertex site. Its architecture resembles those of bacteriophage portal/connector proteins.     

Classification and three-dimensional reconstruction of unevenly distributed or symmetry mismatched features of icosahedral particles

Methods for the three-dimensional reconstruction of icosahedral particles, such as spherical viruses, from electron micrographs are well established. These methods take advantage of the 60-fold symmetry of the icosahedral group. Several features within these particles, however, may deviate from icosahedral symmetry. Examples include viral genomes, symmetry mismatched vertex proteins, unique DNA packaging vertices, flexible proteins, and proteins that are present at less than 100% occupancy. Such asymmetrically distributed features are smeared in the final density map when icosahedral symmetry is applied. Here, we describe a novel approach to classifying, analysing, and obtaining three-dimensional reconstructions of such features. The approach uses the orientation information derived from the icosahedral orientation search to facilitate multivariate statistical analysis and to limit the orientational degrees of freedom for reconstruction. We demonstrate the application of this approach to images of Kelp fly Virus. In this case, each virion may have two different types of fivefold vertex. We use our approach to produce independent reconstructions of the two types of vertex.     

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.     

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.     

A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.     

The portal protein of bacteriophage SPP1: a DNA pump with 13-fold symmetry.

Electron microscopy in combination with image processing is a powerful method for obtaining structural information on non-crystallized biological macromolecules at the 10-50 A resolution level. The processing of noisy microscopical images requires advanced data processing methodologies in which one must carefully avoid the introduction of any form of bias into the data set. Using a novel multivariate statistical approach to the analysis of symmetry, we studied the structure of the bacteriophage SPP1 portal protein oligomer. This portal structure, ubiquitous in icosahedral bacteriophages which package dsDNA, is located at the site of symmetry mismatch between a 5-fold vertex of the icosahedral shell and the 6-fold symmetric (helical) tail. From previous studies such 'head-to-tail connector' structures were generally accepted to be homododecamers assembled in a 12-fold symmetric ring around a central channel. Using a new analysis methodology we have found that the phage SPP1 portal structure exhibits 13-fold cyclical symmetry: a new point group organization for oligomeric proteins. A model for the DNA packaging mechanism by 13-fold symmetric portal protein assemblies is presented which attributes a coherent functional meaning to their unusual symmetry.     

The mechanism for producing two symmetries at the head-tail junction of bacteriophages: a hypothesis

Some double-stranded DNA bacteriophages consist of DNA packaged in a proteinaceous capsid. The capsid has a DNA-enclosing outer shell (head) attached to an external projection (tail). At the head-tail junction is a ring of subunits (connector) that has either six or twelve-fold rotational symmetry, and is joined to the head at an axis of the head's five-fold rotational symmetry. The head is made of subunits in either an icosahedral array or an array consisting of two icosahedral hemispheres separated by a cylinder of subunits. During infection of a host, the head with connector is assembled as a procapsid that subsequently packages DNA and joins a tail. The mechanism for producing two symmetries at the head-tail junction has in the past been an unsolved problem. The observation that the connector of bacteriophage T7 does not nucleate assembly of the outer shell of T7's icosahedral procapsid (P. Serwer and R. H. Watson [1982] J. Virol. 42, 595-601) places a constraint on a solution for the above problem. To solve the above problem for icosahedral procapsids, it is proposed here that: (a) assembly of the outer shell of procapsids is nucleated by a six-membered ring of hexameric aggregates of the major outer shell protein, (b) the connector is assembled in the center of this ring, (c) one of the hexamers dissociates from the ring, creating a five-membered ring and forcing the connector to the inside of the outer shell. A related mechanism is proposed for nucleation of the elongated procapsid of bacteriophage T4.     

Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid

Bacteriophage T4 capsid is a prolate icosahedron composed of the major capsid protein gp23*, the vertex protein gp24*, and the portal protein gp20. Assembled on its surface are 810 molecules of the non-essential small outer capsid protein, Soc (10 kDa), and 155 molecules of the highly antigenic outer capsid protein, Hoc (39 kDa). In this study Soc, a "triplex" protein that stabilizes T4 capsid, is targeted for molecular engineering of T4 particle surface. Using a defined in vitro assembly system, anthrax toxins, protective antigen, lethal factor and their domains, fused to Soc were efficiently displayed on the capsid. Both the N and C termini of the 80 amino acid Soc polypeptide can be simultaneously used to display antigens. Proteins as large as 93 kDa can be stably anchored on the capsid through Soc-capsid interactions. Using both Soc and Hoc, up to 1662 anthrax toxin molecules are assembled on the phage T4 capsid under controlled conditions. We infer from the binding data that a relatively high affinity capsid binding site is located in the middle of the rod-shaped Soc, with the N and C termini facing the 2- and 3-fold symmetry axes of the capsid, respectively. Soc subunits interact at these interfaces, gluing the adjacent capsid protein hexamers and generating a cage-like outer scaffold. Antigen fusion does interfere with the inter-subunit interactions, but these interactions are not essential for capsid binding and antigen display. These features make the T4-Soc platform the most robust phage display system reported to date. The study offers insights into the architectural design of bacteriophage T4 virion, one of the most stable viruses known, and how its capsid surface can be engineered for novel applications in basic molecular biology and biotechnology.     

Poliovirus RNA is released from the capsid near a twofold symmetry axis

After recognizing and binding to its host cell, poliovirus (like other nonenveloped viruses) faces the challenge of translocating its genome across a cellular membrane and into the cytoplasm. To avoid entanglement with the capsid, the RNA must exit via a single site on the virion surface. However, the mechanism by which a single site is selected (from among 60 equivalents) is unknown; and until now, even its location on the virion surface has been controversial. To help to elucidate the mechanism of infection, we have used single-particle cryo-electron microscopy and tomography to reconstruct conformationally altered intermediates that are formed by the poliovirion at various stages of the poliovirus infection process. Recently, we reported icosahedrally symmetric structures for two forms of the end-state 80S empty capsid particle. Surprisingly, RNA was frequently visible near the capsid; and in a subset of the virions, RNA was seen on both the inside and outside of the capsid, caught in the act of exiting. To visualize RNA exiting, we have now determined asymmetric reconstructions from that subset, using both single-particle cryo-electron microscopy and cryo-electron tomographic methods, producing independent reconstructions at ∼50-Å resolution. Contrary to predictions in the literature, the footprint of RNA on the capsid surface is located close to a viral 2-fold axis, covering a slot-shaped area of reduced density that is present in both of the symmetrized 80S reconstructions and which extends by about 20 Å away from the 2-fold axis toward each neighboring 5-fold axis.In its role as the intermediate that links one round of infection with the next, a virus particle protects the viral genome during passage from cell to cell and from host to host, it specifically recognizes and binds to target cells, and it delivers the viral genome into the appropriate compartment in the target cell. For enveloped viruses, which have their own external membranes, fusion of the viral membrane with a host membrane presents a conceptually simple mechanism for delivery of the genome or nucleoprotein into the cytoplasm. For nonenveloped viruses, the viral particle must provide the machinery necessary for either the entire virion, a nucleoprotein complex, or the viral genome to cross a membrane. This process remains poorly understood. Poliovirus provides an excellent model system for probing the mechanisms used for genome translocation. As the type member of the Picornavirus family and the etiological agent of poliomyelitis, poliovirus has been well characterized biochemically and genetically (42), its cell entry pathways have been well characterized (5, 15, 30, 52), and a number of cell entry intermediates have been identified and are accessible for structural studies (2-4, 7, 8, 18, 34, 38, 42, 55, 56).The capsid of the mature poliovirion (160S particle) consists of 60 copies of each of the four coat proteins VP1, VP2, VP3, and VP4 (which is myristolated at its amino-terminal glycine [13]) and encloses a 7.5-kbp positive-sense RNA genome. The outer surface of the capsid has a number of major features, including star-shaped mesas at its 5-fold axes, 3-fold propeller-like protrusions, canyon-like depressions surrounding each of the 5-fold mesas, and depressions at the 2-fold axes (30, 31).Poliovirus infection is initiated when the virus binds to the host-cell-surface poliovirus receptor (called Pvr or CD155) (41), triggering a conformational change of the native capsid into an altered particle called the A particle or 135S particle (18, 19). The 135S particle has been shown to be expanded by about 4% (2, 7), is infectious (16, 33), and is believed to be a productive intermediate in viral entry (30, 33). This conformational change results in the externalization of the small myristoylated capsid protein, VP4 (18), and of the amino-terminal extension of VP1 (which includes a conserved amphipathic helix) (23). Both of these externalized polypeptides then associate with membranes (17, 23). In subsequent steps, the viral genome is released from the capsid and translocated across a membrane (probably an endosomal membrane [5]) to gain access to the cytoplasm, leaving behind an end-state empty capsid shell (called the 80S particle). The trigger for RNA release and the mechanism of genome translocation are both poorly understood (30, 52).Electrophysiology and mutational experiments have shown that the externalization of VP4 and of the amino terminus of VP1 is associated with the formation of channels in membranes (17, 49, 50) and, furthermore, that point mutations in threonine 28 of VP4 can either eliminate (T28G) or alter (T28V, T28S) the ability to form channels and either eliminate (T28G) or slow (T28V, T28S) the kinetics of productive RNA release (17). These observations have led to the hypothesis that the viral polypeptides insert into host cell membranes during infection and rearrange to form channels that permit the viral genome to pass through the membrane, thereby gaining access to the cytoplasm (7, 17, 49, 50).Speculation about the sites of externalization of the viral peptides and of the viral genome began soon after the structures of mature rhinovirus and poliovirus were determined crystallographically 25 years ago (31, 44). In both structures there is a solvent-filled channel running along each 5-fold axis. This channel is closed off at the outer surface of the capsid by polypeptide loops and on the inner surface by a plug that is formed by five intertwined copies of the amino terminus of VP3, forming a parallel beta tube (31, 44). In poliovirus this tube is flanked on its inner surface by five copies of a three-stranded beta sheet in which the outermost two strands come from a beta hairpin at the amino terminus of VP4 and the innermost strand comes from residues at the extreme amino terminus of VP1 (20). The presence of this channel, together with its proximity to peptide segments that were known to be externalized upon receptor attachment, and analogies with other viruses led to a model in which both the peptides and the viral RNA are externalized via the channel at the 5-fold axis (25, 45). At that time, an alternative model for the egress of polypeptides was proposed, based on an analogy with the externalization of the amino-terminal extensions of capsid proteins in expanded states of the topologically similar T=3 plant viruses (26, 32, 43, 47) and on genetic and biochemical studies of mutations that affect cell entry and capsid stability in poliovirus (14, 39, 54). In the latter model, the peptides were proposed to exit from the base of the canyon and then proceed along the outer surface toward the 5-fold peak (43, 47). Both models suggested that five copies of each of the externalized peptides would interact in some way to form a pore in the membrane that was contiguous with one of the 5-fold channels, thus providing a way for RNA to be released from the virion at a 5-fold axis of symmetry. No data yet exist to specify what specific structural roles VP4 and the amino terminus of VP1 might play in forming pores and serving as membrane anchors. However, both the electrophysiology data (cited above) and the greater sequence conservation of VP4 suggest that its role in pore formation may be the more central (17, 49, 50).To further elucidate various steps along the infection pathway, cryo-electron microscopy (cryo-EM) reconstructions have been determined for a number of cell entry intermediates of poliovirus and rhinoviruses, and their resolutions have been improved over time (2, 3, 7, 28, 38). Structures of the complexes of polioviruses and major-group rhinoviruses with the ectodomains of their respective receptors have confirmed earlier models that suggested that the canyon is the receptor-binding site and have begun to suggest how receptor binding might lead to receptor-induced conformational rearrangements (3, 56). Cryo-EM and cryo-electron tomography structures (cryo-ET) of a poliovirus-receptor-membrane complex (using a novel receptor-decorated liposome model [51]) confirmed that initial receptor binding brings the surface of the 5-fold mesa into close proximity with the membrane and appears to produce an outward distortion of the outer leaflet of the membrane in its area of closest approach to the virus particle (4, 8).Structures have also been determined for the soluble 135S and 80S particles of poliovirus, formed by heating the virus at 50°C (135S) or 56°C (80S) in hypotonic buffers, and for the 80S particles of rhinovirus 14 and 16, formed by exposing virus to acidic pH. All of the biological and immunological evidence that is currently available indicates that the particles prepared in vitro and used for structural studies are indistinguishable from the particles that are released from the cell surface during infection (6, 53). These structures have allowed the models for peptide release and genome release to be extended and refined (7, 38) and indeed have confirmed that VP1 exits from the particle surface at the base of the canyon and climbs up the side of the 5-fold mesa. However, contrary to the assumptions of the earlier models, the 10-Å structures of the poliovirus 135S and 80S particles show that the amino end of the amino-terminal extension of VP1 does not remain associated with the mesa. Instead, it forms an alpha-helical bridge that stretches across the canyon and binds to the large EF loop of VP2, a surface projection that appears as a 3-fold propeller blade (7, 38).Until recently, the mechanism of RNA release (during the 135S-to-80S transition) has been largely a matter of conjecture. We can infer that the RNA must exit via a single site on the virion surface, to avoid entanglement with the capsid (particularly as entanglement has never been observed in electron micrographs), though the mechanism by which a single site is selected (from among 60 equivalents) is unknown. All models presented to date have assumed that the RNA is released from the channel at the 5-fold axes (2, 3, 7, 8, 25, 27, 28, 30, 42, 45). However, in the icosahedrally constrained 10-Å structures of both the poliovirus 135S and 80S particles (7, 38), the apparent intactness and stability of the 5-fold mesa argues against the 5-fold axis being the site of RNA egress, given that the diameter of the opening, as seen in those structures, would be insufficient to accommodate RNA, even if the “plug” formed by the intertwined amino termini of VP3 was displaced. Moreover, both structures revealed significant thinning between 2-fold-related pentamers in the vicinity of the 2-fold axes. Most convincingly, large holes (easily sufficient to accommodate RNA) were seen at and near the 2-fold axes in the atomic model of the late-80S structure. This coincided with an open hole in the reconstruction, when viewed at a contour level that left most of the remainder of the capsid intact. This evidence was suggestive, but not definitive, as a number of other openings were present, particularly in the interfaces between protomers. Furthermore, the behavior of the capsid structure in the immediate vicinity of the unique site of RNA exit is likely to be different from what we see in the icosahedral average, which is dominated by the remainder of the capsid.In the course of solving icosahedrally symmetric cryo-EM structures for the poliovirus end-state 80S empty capsid particle (7, 38), we were surprised to find that RNA was frequently visible near the capsid and that in a subset of about 5% of the sampled virions, RNA was seen on both the inside and outside of the capsid, apparently caught in the act of exiting. This was an exciting development, as images of viral RNA release had never previously been reported. We were able to improve the resolution to ∼10 Å by classifying the projected images into two groups: an early 80Se particle that was more prevalent in the population after a shorter heating time and a late 80Sl particle that was seen more often when the heating time was increased. The amount of RNA density remaining in the interior appears to be continuously variable in both classes, suggesting that release is gradual. Of the 5% subset of particles clearly caught in the act, almost all belonged to the 80Se class. Our interpretation was that the 80Se class may represent particles in which exiting RNA is still engaged with the capsid machinery and traversing the capsid, while the 80Sl class (in which much of the capsid resembles the 135S form more closely in structure) represents particles with the RNA disengaged, possibly after nuclease cleavage. More than two structural classes may be present, but at the current resolution, we could not distinguish them.The present report addresses the question of what we can learn about the details of RNA release from an asymmetric cryo-EM reconstruction, based on the 540-particle caught-in-the-act subset, and independently from cryo-electron tomographic reconstructions of a similarly prepared sample. In each projected particle image or subtomogram, preliminary orientation parameters are first determined from an icosahedrally symmetric calculation, and in a second stage, the symmetry is broken by choosing 1 of the 60 symmetry-equivalent orientations. Both methods have yielded similar information, at about 50-Å resolution, concerning the footprint of the RNA on the virion surface, which demonstrates that RNA is released from an asymmetric site at the base of the canyon near a particle 2-fold axis and not at the channel at the 5-fold axes, as suggested by previous models. Additionally, the demonstrated success of the methodology provides us with a blueprint for resolving the molecular details of the RNA-capsid interaction in future experiments.     

A symmetry mismatch at the site of RNA packaging in the polymerase complex of dsRNA bacteriophage phi6

The polymerase complex of the enveloped double-stranded RNA (dsRNA) bacteriophage phi6 fulfils a similar function to those of other dsRNA viruses such as Reoviridae. The phi6 complex comprises protein P1, which forms the shell, and proteins P2, P4 and P7, which are involved in RNA synthesis and packaging. Icosahedral reconstructions from cryo-electron micrographs of recombinant polymerase particles revealed a clear dodecahedral shell and weaker satellites. Difference imaging demonstrated that these weak satellites were the sites of P4 and P2 within the complex. The structure determined by icosahedral reconstruction was used as an initial model in an iterative reconstruction technique to examine the departures from icosahedral symmetry. This approach showed that P4 and P2 contribute to structures at the 5-fold positions of the icosahedral P1 shell which lack 5-fold symmetry and appear in variable orientations. Reconstruction of isolated recombinant P4 showed that it was a hexamer with a size and shape matching the satellite. Symmetry mismatch between the satellites and the shell could play a role in RNA packaging akin to that of the portal vertex of dsDNA phages in DNA packaging. This is the first example of dsRNA virus in which the structure of the polymerase complex has been determined without the assumption of icosahedral symmetry. Our result with phi6 illustrates the symmetry mismatch which may occur at the sites of RNA packaging in other dsRNA viruses such as members of the Reoviridae.     

Some observations of a correlation between the symmetry of large heavy-atom complexes and their binding sites on proteins

Two large, electron dense heavy-metal complexes were found to bind to crystals of heavy riboflavin synthase. The crystallographic analysis by difference Fourier methods shows that the tungsten cluster compound [(W3O2(O2CCH3)6]2+, which has trigonal symmetry, binds to a site on the 3-fold axis of the protein. The heteropolytungstate compound [NaP5W30O110]14-, which has pentagonal symmetry, binds to a site on the 5-fold axis of the protein.     

Structural organization of authentic,mature HIV-1 virions and cores

Mature, infectious HIV-1 particles contain a characteristic cone-shaped core that encases the viral RNA and replication proteins. The architectures of mature virions and isolated cores were studied using cryo-electron microscopy. The average size ( approximately 145 nm) of the virion was unchanged during maturation. Most virions contained a single core but roughly one-third contained two or more cores. Consideration of the capsid protein concentration during core assembly indicated that core formation in vivo is template-mediated rather than concentration-driven. Although most cores were conical, 7% were tubular. These displayed a stacked-disc arrangement with 7-, 8-, 9- or 10-fold axial symmetry. Layer line filtration of these images showed that the capsid subunit arrangement is consistent with a 9.6 nm hexamer resembling that previously seen in the helical tubes assembled from purified capsid protein. A common reflection (1/3.2 nm) shared between the tubular and conical cores suggested they share a similar organization. The extraordinary flexibility observed in the assembly of the mature core appears to be well suited to accommodating variation and hence there may be no single structure for the infectious virion.     

Molecular weight of adenovirus serotype 2 capsomers a new characterization

We have determined the molecular weight of some of the adenovirus serotype 2 structural proteins: penton, penton base and fibre. Physical techniques, namely neutron scattering and hydrodynamical measurements, indicate that the penton base is a trimer. This is confirmed by analysis of the virion composition based on quantitative gel scanning. This finding implies either that other proteins (e.g. protein IIIa) are essential in the architecture of the fivefold vertex of the virion, or that the usual assumption that icosahedral symmetry involves identical interactions related to the symmetry of the virion does not hold.     

Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity.

The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles.     

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.     

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.