Bacteriophage HK97 Head Assembly: A Protein Ballet

ADVANCES IN VlRUS RESEARCH, VOL.50

BACTERIOPHAGE HK97 HEAD ASSEMBLY: A PROTEIN BALLET Roger W. Hendrix and Robert L. Duda Pittsburgh Bacteriophage Institute and Department of Biological Sciences University of Pittsburgh Pittsburgh, Pennsylvania 15260

I. Introduction A. Questions Addressed B. Overview of HK97 Head Assembly 11. Initial Steps A. GroEL Chaperonin: Required for Head Assembly in Vivo B. Folding of HK97 Head Protein into Hexamers and Pentamers in Vitro 111. Shell Assembly A. Assembly of Prohead I in Viuo B. Assembly of Prohead I in Vitro C. Specification of Shell Size D. Comparisons with Assembly of P22, T7, T4, and h in Vitro IV. Proteolysis: The Remodeling of Prohead I into Prohead I1 A. Prohead I1 Origins and Structural Differences from Prohead I B. Control of Proteolysis by gp4 C. Assembly-Activated Proteolysis: Comparison with T4 and h V. Scaffolds, Proteolysis, and the Initiation of Head Assembly A. Questions Concerning whether the HK97 delta Domain Is a Scaffold B. The Functions of Scaffold Proteins VI . Expansion of the HK97 Capsid A. Induction of Expansion in Vzuo and in Vitro B. Changes Induced during Expansion VII. Cross-Linking of Head Proteins in HK97 A. Early Observations of HK97 Head Protein Cross-Linking B. Induction of Cross-Linking in Vitro by Expansion C. Biochemistry and Topology of Cross-Linking D. Kinetics of the Cross-Linking Reaction E. Creation of Protein Chain Mail by Interlinking before Cross-Linking F. Chain Mail and Cross-Linking in Other Phages and Elsewhere G. Possible Enhancement of Capsid Stability by Chain Mail Interlinking H. Interdigitating Proteins: A Theme in Viral Assembly VIII. Questions about Phage Head Expansion IX. Conclusions References

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I. INTRODUCTION A. Questions Addressed Bacteriophage virions, like all biological structures, are assembled from their molecular parts. Among biological structures, bacteriophages have the virtue of being exceptionally amenable to experimental investigation. As a consequence, studies of bacteriophage assembly have revealed not only many specific details of how bacteriophage virions arise from their parts, but also more than their share of insight into the general principles of biological assembly. For example: How do specific interactions between individual protein subunits lead uniquely to a several hundred-subunit structure of precisely determined size and shape, when in principle the subunits should be capable of making a wide range of different structures? How do “assembly catalysts” of various sorts work? What are the roles of the structural transitions that proteins undergo during the process of assembly? How are they propagated through the structure in an ordered way? How are they coordinated into an overall strategy of assembly? What are the properties of the assembled structure that arise as a consequence of these assembly strategies? These and many related questions of assembly and structure have been addressed (and sometimes answered) in more than 30 years of study of phage assembly. More recently, experiments with bacteriophage HK97 head assembly, the subject of this article, have produced a new set of insights and have as well provided new perspective on some of the work in other phage systems. The major head subunits of HK97, gp5, undergoes a remarkable number of covalent modifications and conformational changes during its maturation. These changes are tightly coordinated both spatially and temporally as the protein moves through the head assembly pathway in the company of, and interacting with, 414 copies of itself plus smaller numbers of a few other proteins and a DNA molecule. As we learn more about it, this whole performance comes increasingly to resemble an elaborate ballet, with proteins entering and leaving the stage, moving in coordination, changing shape in response to their fellow dancers, linking arms, and joining hands. This article takes the reader through the full performance of the HK97 ballet as viewed through our best optics, compares the HK97 program with those of other viruses and other proteins, and speculates on the causes and purposes of the individual steps and what they reveal about how proteins work. The mechanisms used by HK97 to control head shell assembly and maturation appear in many ways to differ from those used by

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other phages, but these differences can be reconciled with a set of common themes that govern the action and function of scaffolds, proteolysis, and expansion in bacteriophage capsid assembly. In comparing the results from studies of HK97 with those from other phage systems, one hope is to learn which features of the assembly process are shared among the different phages, and therefore possibly represent more fundamental features of the process. In addition, by learning what the differences are-that is, how different phages accomplish the same biological end by different biochemical mechanisms-we can hope to learn something about what functional variations are accessible through time to a coevolving set of genes. Finally, the differences in mechanism used by different phages to accomplish a common functional part of the assembly process can in some cases give a clearer impression of what the “true” purpose of that part of the process is than would be available from consideration of a single phage in isolation. Our discussions of scaffoldingfunction and capsid stabilization given below, for example, may fall in this last category. The validity of this approach depends on the underlying assumption that the different phages under consideration-in this case, phages with double-stranded DNA (dsDNA) genomes-are descended from a common ancestral gene pool, that is, that the head assembly mechanisms are variations on an ancestral theme rather than independently invented solutions to the same problem. This assumption, although not known rigorously to be correct, is receiving increasing support from striking functional similarities among phages (including, for example, many features of structure and assembly described here) as well as from relationships among the phages inferred from similarities of gene organization and of DNA and protein sequence.

B. Overview

of

HK97 Head Assembly

Bacteriophage HK97 is a temperate Escherichia coli phage of the lambdoid phage family. It was isolated from pig dung in Hong Kong and first described in 1980 (Dhillon et al., 1980). In overall morphology HK97 is similar to the well-known phage A. HK97 has an isometric head and a long flexible tail (Fig. 1). Early attempts to characterize the virion proteins revealed the first hints that, despite the superficial similarity, HK97 is strikingly different from A in having all of its major capsid protein covalently crosslinked into large oligomers (Popa et al., 1991). More recently, a detailed description of the HK97 head assembly pathway has emerged from studies of the genetics, biochemis-

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FIG1. Bacteriophage HK97. Shown here is an electron micrograph of the virion of HK97, a temperate phage of E. coli. The sample was negatively stainedowith uranyl acetate after adsorption to an unsupported carbon film. Scale bar: -500 A.

try, structure, and assembly of the HK97 capsid (Conway et al., 1995; Duda et al., 1995a-c); Xie and Hendrix, 1995). There are three major protein players in HK97 head assembly ballet, gp3, gp4, and gp5, named for their adjacent genes in the HK97 genome. gp3 is the 47-kDa portal protein, gp4 is the 25-kDa putative prohead protease, and gp5 is the 42-kDa major head protein, also called the capsid or shell protein. The major head protein is processed into many forms, including the cleaved, 31-kDa form, gp5*, and a series of covalently cross-linked oligomers of gp5*. The overall assembly pathway is outlined in Fig. 2. The final product is a (DNA filled) -550-A-diameter capsid with the same icosahedral T = 7 symmetry as several other large double-stranded DNA phages (Conway et al., 19951,including A (Dokland and Murialdo, 19931, P22 (Prasad et al., 19931, and T7 (Steven and Trus, 1986). The head is composed of 71 capsomers, with 11morphological pentamers located at the corners and 60 morphological hexamers located on the faces (all derived from 415 monomers of the major head protein), and has a 12fold symmetric doughnut-shaped portal substituting for a pentamer a t one corner. The portal provides the gateway for DNA to pass into and out of the head, and as such, acts as the docking site for the DNA packaging machinery and for the attachment of the DNA injection apparatus, the tail. A summary of HK97 head assembly is presented next t o provide a framework for the more detailed comparative discussion that follows. Newly synthesized 42-kDa head protein is folded with the assistance of the GroEL/GroES chaperonins and assembles into the hexamers and pentamers (collectively termed capsomers) that are precursors of shell

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42kD head subunit

II

HK97 Head Assembl

GroEL (415)

PROHEAD I

t

PROHEAD I1

HEAD I

HEAD I1

5-mers +

-

portal (12) I

1

protease (-50) FIG2. The HK97 capsid assembly pathway. The details of the assembly pathway are described in text. The proteins required for head shell assembly are the portal ( g p 3 , 47 kDa), protease (gp4, 25 kDa), and major head subunit (gp5, 42 m a ) . A map of the genes is shown in Fig. 6 . The three-dimensional images of capsids were derived from reconstructions created by J. Conway (Conway et al., 1995).

assembly. A mixture of hexamers and pentamers of the 42-kDa gp5 head protein initiates assembly with a preformed portal (a dodecamer of gp3) and gp4, the putative protease. The shell protein (415 copies of gp5) and the presumptive protease (about 50 copies ofgp4) copolymerize into the thick-walled prohead I, with the gp4 in the interior. Prohead I, an -470-A-diameter transient intermediate, is roundish rather than angular, has a lumpy surface, and has essentially the same T = 7 symmetry as the mature head, but with a skewed hexamer shape (Fig. 3; Conway et al., 1995). The protease remains inactive until shell assembly is complete. Once cleavage is initiated, the amino-terminal 26% of all of the shell subunits (corresponding t o the interior parts of the shell) and the presumptive protease, gp4 itself, are all cleaved rapidly to small peptides. The peptides apparently exit through small pores in the new structure, prohead 11, composed of a portal and 415 copies of gp5*, the 31-kDa, cleaved capsid protein. Prohead I1 is essentially identical t o prohead I on the exterior, although the interior has been extensively remodeled by proteolysis (Figs. 3 and 4; Conway et al., 1995). By analogy with other phages we assume that DNA packaging initiates the expansion transformation of prohead I1 from its round, thick-walled shape to the thin-walled angular head I (Figs. 3 and 5; Conway et al., 1995). During expansion, a large-scale reorganization of the shell protein causes segments of capsid protein from adjacent

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FIG3. Thc HK97 three-dimensional “family photo”: three-dimensional density maps I, (b) prohead 11, and (c) head 11. Head I (not shown) is identical to head I1 a t this resolution. Surface renderings are shown of the outer surfaces (left), and inner surfaces (middle),as viewed down a threefold symmetry axis of the particles. There is little difference between the outer surfaces of prohead I and prohead 11, but the inside of prohead I has more density lining its inner surface and suspended in blobs o f density under each capsomer. Stereoviews in Fig. 4 show this more clearly. The pentameric capsomers of the proheads are regular pentagons, but the hexameric capsomo f HK97 (a) prohead

ers appear to be sheared along a twofold axis into an elongated shape. The dramatic differences between prohead I1 and head I1 are shown in more detail in Fig. 5 and discussed in text. On the right are central sections in which the highest densities are shown as black and the lowest as white. The volume enclosed by the surface renderings represents the expected protein volume. The handedness of the capsids is unknown. We have chosen to use dextro for all illustrations. (Images provided by J. Conway; see Conway et al., 1995.1 Scale bar: 100 A.

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FIG4. Remodeling the interior of a prohead by proteolysis. (a) Stereopair of the prohead I reconstruction shown in Fig. 3, but contoured a t a slightly less stringent level to visualize the thin arms of density that connect the interior blobs with the inside surface of the prohead. (b) Stereopair of prohead 11, using the same contour level as in (a). The interior blobs are missing in prohead 11. The cross-section of the prohead I1 shell is also reduced, apparently by loss of material from the inside surface. Both of these differences are the result of proteolytic removal of the “delta region” of gp5. The density just under the surface of the capsid is reduced by an interference fringe created as a consequence of phase-contrast imaging. The interference fringe may mask some of the tenuous, low-density connection between the blobs and the rim (Images provided by J. Conway; see Conway et al., 1995.) Scale bar: 100 A.

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FIG5. The expansion transformation of the HK97 prohead surface. Stereopairs of prohead I1 (a) and head I1 (b) show the striking changes in size, surface relief, and local symmetry that take place as a result of the expansion transformation. The surface becomes smooth and the hexamers change t o regular hexagons from their elongated sheared form in the proheads. The pentamers a t the corners also change from regular pentagons to star-shaped pentagrams. (Images provided by J. Conway; see Conway et al., 1995.) Scale bar: 100 A.

capsomers to pass arm-over-arm to link the neighboring capsomers via interlocking loops of polypeptide chains, using movements that resemble the actions required in an (unhquare dance. The same massive conformation change brings residues Lys-169 and Asn-356 from adjacent subunits in each reorganized capsomer into an active site, where they react to form covalent bonds. The covalent bonds link the

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adjacent subunits into closed chains of five or six subunits-covalent protein rings. Because the polypeptides from adjacent rings (or capsomers) have previously intertwined, the adjacent rings are catenated into a large protein mesh called protein or viral chain mail. The addition of cross-links creates the final form of the protein shell, head 11,which is on its surface identical in appearance to head I (Conway et al., 1995).

11. INITIAL STEPS

A. GroEL Chaperonin: Required for Head Assembly in Vivo Simple plating experiments show that some of the missense mutants in the E. coli groEL gene, originally isolated because they block growth of phage A, prevent plaque formation by HK97. Examination by electron microscopy of lysates of such groEL mutants infected by wild-type HK97 shows an abundant crop of morphologcally normal tails but few heads. The bulk of the head protein is found instead in the fastsedimenting fraction of the cell lysate, presumably in “inclusion bodies.” This is similar to what is seen in an infection of a groEL mutant by phage T4, and as in the T4 case suggests that the HK97 head protein fails to fold correctly in the absence of wild-type GroEL and instead aggregates into the inclusion bodies. This view of the role of GroEL is supported by in uitro folding experiments. HK97 capsid protein (gp5) that has been unfolded in guanidinium chloride (GuHC1) can be refolded efficiently in the absence of added protein factors following dilution out of the GuHC1, but only if it is dilute, cold (0-4”C), and in the presence of 10% glycerol (Xie and Hendrix, 1995). At higher concentrations and temperatures, gp5 aggregates rapidly following dilution out of GuHC1, but aggregation can be prevented and correct folding allowed by the presence of GroEL and GroES proteins plus ATP. As with other proteins whose folding requires the complete GroE chaperonin system, it can be shown that GroEL alone makes a rather stable complex with the unfolded (or most likely, partially folded)gp5, and that gp5 is released and can be detected in folded form on addition of GroES and ATP. The stable complex of gp5 and GroEL clearly contains a single subunit of gp5 and a 14-subunit oligomer of GroEL (Ding et al., 1995), but the folded form of gp5 that is detected is not a monomer but a mixture of pentamers and hexamers. These oligomeric forms appear to be obligatory intermediates in capsid assembly (see Section 111,B). Although it is not possible on present evidence to rule out rigorously the possibility that formation of the pentameric and hexameric oligomers is mediated directly by the GroE proteins, several lines of evidence seem more

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compatible with the view that GroE releases the folded gp5 monomer, which then associates spontaneously with other monomers to make the pentamers and hexamers. Among these are the fact that the same pentamers and hexamers are produced from in vitro folding of gp5 in the absence of GroE proteins and the observation described below that pentamers and hexamers can be interconverted in vitro in the absence of GroE proteins. The use of the GroE system to fold the major subunit of phage capsids is probably common-possibly even universal, although it has been demonstrated explicitly for only three phages beside HK97. The situation with T4 appears quite similar to that of HK97, in that when T4 infects a groEL mutant, the major capsid subunit, gp23, fails to fold correctly and is found aggregated in inclusion bodies (Georgopoulos et al., 1972). T4 apparently does not require the cellular GroES protein but instead encodes its own GroES analog in the form of gp31 (van der Vies et al., 1994). Attempts to isolate mutants of the Salmonella typhimurium groE genes that block capsid assembly of P22 have not been successful (S. Casjens, unpublished). However, with missense mutants in the P22 capsid subunit gene (gene 5 ) that cause folding defects, the protein can be rescued from aggregation by overproduction of the E. coli GroE proteins in the P22-infected S. typhimurium cell (Gordon et al., 1994). This is, at a minimum, consistent with P22 gp5 interacting with the GroE chaperonins to achieve correct folding in much the same way as for T4 gp23 and HK97 gp5. The situation with phage A is less clear. As with T4 and HK97, mutants of the groE genes block production of functional A capsids, and as with T4, second-site suppressors of the groE mutants can be selected in the gene encoding the major capsid subunit of A, gene E (Georgopoulos et al., 1973). (In fact, these mutants are the source of the “IT’in the name groE; Georgopoulos et al., 1973). However, in a A wild-type infection of a groE- cell, the gpE does not aggregate into inclusion bodies but instead assembles aberrantly into tubes, spirals, and other capsid-related monsters (Hendrix and Casjens, 1975; Hohn et al., 1975). One possible explanation for this difference might be that the mutant GroELs deliver the gpE to a slightly different fold than the normal one, which allows assembly of gpE into a lattice but with relaxed control on the size and shape of the resulting structures. Alternatively, it may be that the direct effects of the groE mutation on h are not on gpE but on some other proteinb) involved in capsid assembly, whose incorrect folding would interfere with accurate gpE assembly. Facts that might support this view include (1)that second-site suppressors ofgroE mutants are also found in gene B, which encodes the A portal (Georgopoulos et al., 1973; Sternberg,

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1973), and (2) that many of the mutants in gene E that suppress groE mutants are of a type (suppressed amber mutants) expected t o produce a gpE with the wild-type sequence but in reduced quantity relative to the other head proteins.

B. Folding of HK97 Head Protein into Hexumers and Pentamers in Vitro The pentamers and hexamers of gp5 can be produced by in vitro folding of denatured gp5 as described above and also by dissociation from prohead I shells through the agency of high concentrations of salt (e.g., 2 M KC1) or sugar (e.g., 40% glucose). In either case, the pentamers and hexamers correspond to the morphological capsomers present in the capsid, and by either route they are produced in roughly the same 6:l mass ratio (hexamers t o pentamers) that is found in the T = 7 capsid. The fact that the same gp5 protein can participate in forming either pentamers or hexamers gives the first indication of the conformational flexibility possessed by this protein. This flexibility is further emphasized by the finding that pentamers and hexamers can be interconverted in vitro by adjusting solvent conditions, which argues strongly that it really is the same protein that assumes the two conformations required for making pentamers and hexamers, and against any model that postulates a covalent modification of the protein (e.g., phosphorylation) that directs it into one oligomeric form or the other. The conditions for in vitro conversion require the presence of moderate concentrations of denaturants-0.2 M GuHCl or 0.5 M NaSCNprobably to facilitate dissociation and reassortment of subunits into oligomers. In the presence of denaturant alone at neutral pH, -90% of the protein ends up in hexamers, and if 1%dimethylformamide is added at pH 5.2 along with the denaturant, -90% of the protein converts to pentamers. In either case the pentamer-to-hexamer ratio is stable when the protein is moved into a neutral nondenaturing buffer (Xie and Hendrix, 1995). Although much remains t o be done to understand these reactions in detail, a useful working model that is consistent with existing data postulates that the gp5 monomer, when freshly folded by GroE or dissociated in vitro by denaturant, is in equilibrium between two conformations, one suited for pentamers and one for hexamers. The position of the equilibrium is determined by solvent conditions. The monomers assemble into pentamers and hexamers in a ratio determined by the ratio of the two conformations among the monomers. According to this scheme, we imagine that the structure of the protein is tuned so that under solvent conditions prevailing in the infected cell, the ratio of the

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two conformational forms leads to the appropriate ratio of pentamers and hexamers for assembling a normal-sized (7’= 7) capsid. In accord with this view, mutant forms of gp5 that fail to assemble capsids have been identified in which the ratio of pentamer to hexamer capsomers assembled i n vivo is strongly biased in one direction or the other. To date there is no clear parallel to the HK97 gp5 pentamers and hexamers among other phage systems. For P22, for which i n uitro assembly of the capsid is well studied, it seems clear that the capsid assembles directly from the monomer subunit, and there is no evidence for formation of intermediate oligomers. Note that for P22, as for HK97, the subunits in the assembled capsid are arranged in groups of five or six. However, the correct subunit conformations to occupy different positions in the capsid structure are apparently selected (or imposed) at the time of shell assembly, without the intermediate step of organizing conformations of subunits in capsomers. For T4, the evidence is not clear on what the pathway of shell assembly is in this regard, but it seems likely (see Section II1,D) that there may be pentamers and hexamers as in HK97. Even if so, however, T4 has solved the problem of how to have both pentamers and hexamers for building the capsid by encoding a second protein, gp24, to make the pentameric “corners” of the capsid, with the bulk of the capsid composed of hexamers of the major capsid subunit, gp23. Thus, although it may well be that T4 assembles its capsid from preassembled pentamers and hexamers like HK97, those two oligomers would be made of two distinct proteins rather than two conformations of the same protein as for HK97.

ASSEMBLY 111. SHELL A. Assembly of Prohead I in Vivo The general features of the HK97 head assembly pathway were deduced largely from two experimental paradigms: the first was to analyze the intermediates that accumulate when amber mutants are grown under nonpermissive conditions (Duda et al., 1995a; Popa et al., 19911, and the second was to express the HK97 capsid genes in different combinations (with or without mutations) using high-efficiency plasmid expression systems (Duda et al., 199513). A highly productive effort to image all structures in the pathway using cryoelectron microscopy has yielded valuable insights from three-dimensional reconstructions of prohead I, prohead 11, head I, and head I1 at resolutions of 2531 (Figs. 3-5; Conway et al., 1995). The earliest large intermediate discovered was prohead I, composed of a portal and 415 copies of the

A

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unprocessed 42-kDa shell protein, which accumulated in cells infected with HK97amU4 or HK97amE2. These mutations were later shown Similar by genetic complementation to be in gene 4 (Duda et al., 1995~). structures (without a portal) were assembled in cells expressing a single phage gene, gene 5, from a plasmid (Duda et al., 1995a). These results suggested that gene 4 controls proteolysis and, more importantly, that the major capsid protein contains all of the information for efficient and accurate assembly of the HK97 capsid shell, a conclusion that did not fit with results from other phages, where a separate factor, the scaffold protein, was required for accurate and efficient assembly. A comparison of the gene order of the capsid genes of several of the more or less well-characterized phages, shown in Fig. 6, shows that the gene order is generally either portal, scaffold, shell, or portal, protease, scaffold, shell. It was noticed early that for many phages, genes cluster by function and that those genes whose products interact are often adjacent (see, for example, Epstein et al., 1964; Stahl and Murray, 1966; and a summary of this concept in Casjens and Hendrix, 1988). Some phages have the portal out of order, or have additional genes, but HK97 seems to be a radical departure in that there is neither a scaffold protein, nor a candidate for a scaffold gene immediately upstream from the capsid protein gene.

B. Assembly of Prohead I in Vitro Additional evidence for this surprising lack of a scaffold protein in HK97 comes from studies of HK97 prohead I dissociation and reassembly in uitro (described in Xle and Hendrix, 1995).Prohead I was found to dissociate in 2 M salt, and further, the dissociated protein was in the form of a mixture of hexamers and pentamers of capsid protein, in the 6:l ratio expected for capsomers from a T = 7 structure. The dissociated mixture of capsomers at about 1-2 mg/ml could reassemble efficiently and accurately into shells indistinguishable from prohead I on addition of polyethylene glycol (PEG; 6% PEG 8000) and a divalent cation (10 mM Ca2+1. Further experiments showed that hexamers and pentamers of capsid protein could be interconverted in vitro by adjusting the solvent conditions. The ability to alter the ratio of hexamer to pentamer in an assembly reaction allowed an experiment to ask if the size (T number) resulting from assembly a t different ratios would give different-size structures-as might have been predicted from the fact that shells of different T numbers have different pentamer-to-hexamer ratios (Caspar and Klug, 1962). Exclusively T = 7 structures were

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assembled over the range of input ratios used; the efficiency of assembly was maximal at the correct 6:l ratio and fell sharply as the ratio was shifted in either direction. This supports the view that the information for determining HK97 capsid size is intrinsic to the 42-kDa shell subunit alone. This result provides the clearest evidence that pentamers and hexamers are true intermediates in the assembly reaction, because if they were able to dissociate or otherwise rearrange themselves before assembly, the sharp dependence on the pentamer-to-hexamer ratio would not be expected (Xie and Hendrix, 1995). In contrast to the rather clear case for in uitro assembly, there has been no direct demonstration that hexamers and pentamers of shell protein are the actual in uiuo precursors of HK97 prohead assembly, but indirect evidence suggests that this is the case. Because the shell protein forms hexamers and pentamers immediately after release from a chaperone, with no detectable monomer (see above), it seems likely that this would happen in uivo. Unassembled capsomers actually do accumulate in cells expressing gp4 and a mutant variant of the HK97 head protein gp5-N356D (in which residue 356 is changed from asparagine to aspartate). The addition of 10 mM Mg2+and 10%PEG to crude extracts containing the N365D variant capsomers can evidently force the mutant capsomers to assemble and cleave into prohead II-like shells that contain the cleaved 31-kDa form of the shell protein (Duda et al., 199513). Preliminary experiments to find the earliest HK97 intermediates strongly suggest that pentamer and hexamer are present in uiuo before any shells are found (P. Costa, R. Duda, and R. Hendrix, unpublished observations).

FIG6. Head genes, function, and gene order among phages. The genes required for head shell assembly in a variety of well-known bacteriophages are shown in physical gene maps drawn to scale. The (known or proposed) function of each gene is indicated by the pattern filling the boxes representing genes, as indicated by the key at the bottom of the figure. The order portal, (protease), scaffold, capsid protein is almost universally conserved. The genes that encode (putative or known) proteases are indicated by the abbreviation “p’ase.” The genes encoding proteins (or parts thereof) that are largely removed or digested during maturation of the prohead are indicated by a dark-toned bar above the gene; these represent the broadest definition of scaffold or core proteins. The genes encoding internal proteins of the prohead that are retained in the mature capsid are indicated by an open bar above the gene. The sid gene of P4 encodes a unique external shape-limiting scaffold protein. The gene sequences are available in public databases.

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C. Specification of Shell Size If the input ratio of hexamers to pentamers does not account for the accuracy with which T = 7 structures are assembled, some other cause for accurate assembly must be sought. A simple idea is that the bonding properties of the capsomers are such that they will only fit together in the correct way to build a T = 7 structure. However, individual subunits at different locations in a T = 7 capsid necessarily make somewhat different contacts with their neighbors. For this scheme to work, therefore, the subunits would need to have enough of a bonding repertoire to assemble a T = 7 structure but not be able to muster the perhaps only slightly different repertoire that would allow assembly of a larger or smaller shell. This model cannot be ruled out at present; without more detailed structural information than is currently available, it is difficult to state in sufficiently specific terms to allow an experimental test. A more appealing, if similarly untested, model postulates the existence of discrete assembly intermediates made of several capsomers. These assembly intermediates are able to join to each other through repeated identical sets of bonding interactions in such a way that T = 7 capsids are produced uniquely. The model makes use of specific geometric properties of T = 7 capsids and of the HK97 prohead structure, both of which can be seen in the reconstructed cryoelectron microscopy (cryo-EM)image in Fig. 3. Note, first, that each hexamer contacts exactly 1 pentamer, which means that the entire T = 7 structure can be disassembled conceptually into 12 “superpentons,” each consisting of 1 pentamer surrounded symmetrically by 5 hexamers (Conway et al., 1995). These superpentons are the postulated assembly intermediates, 12 of which would assemble into a T = 7 capsid through equivalent sets of bonding interactions. The other feature of the structure in Figs. 3 and 5 that plays a role in the model is the shape of the hexamers. Unlike the pentamers, which show a clear fivefold symmetry, the hexamers are strongly twofold symmetric and appear to be related to a sixfold symmetric hexamer by a pronounced skewing along a plane passing through the center of the hexamer. An important component of this assembly model is the concept of conformational control of assembly. Specifically, in this case it is proposed that the hexamer skewing occurs in response to binding of the hexamer to the pentamer during assembly of the superpenton intermediate (Conway et aZ., 1995). The skewing would serve as a signal of the state of assembly of the hexamer, eclipsing the binding sites that would allow further (inappropriate) interaction with pentamers and exposing the binding sites that enable

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25 1

the hexamer, as part of a superpenton, to join to the other superpentons. Phages P22 and h both have T = 7 symmetry and both have asymmetric hexamers in their proheads like HK97 (Dokland and Murialdo, 1993; Prasad et al., 1993). The occurrence of asymmetric substructures in places that symmetric components are expected in viral structures is common among viruses (Steven et al., 1997) and, although not understood, the asymmetry may be part of a mechanism to specify capsid size, as proposed.

D. Comparisons with Assembly of P22, T7, T4,and h in Vitro Prohead-like shells of phages P22 (Fuller and King, 1982; Prevelige et al., 1988, 1993) and T7 (Cerritelli and Studier, 19961, both with T = 7 symmetry, have also been assembled i n uitro and both reactions require the addition of a separate scaffold protein for correct, accurate assembly. The P22 assembly reaction is radically different from that of HK97. P22 prohead assembly requires only monomeric coat protein (P22 gp5) at about 1 mg/ml and a minimum ratio of about 0.1-0.2, or excess, of scaffolding protein (P22 gp8, which may be a monomer or small oligomer). There is no requirement for additional factors, such as cations or PEG (Prevelige et al., 1988). So, in contrast to HK97, the P22 shell assembles from monomers, not oligomers, although a pentamer of P22 coat protein is inferred in the initiation of P22 procapsid assembly from a fifth-order dependence of the initial rate of reaction on capsid protein concentration (Prevelige et al., 19931, and there is some evidence that a coat-scaffold heterooligomer may exist under reaction conditions (Prevelige et al., 1988). No clear evidence has been found for the existence of stable hexamers or pentamers of P22 capsid protein (Prevelige et al., 1988); conversely, there is no evidence for the existence of a stable monomer of the HK97 capsid protein. The only stable monomeric forms of the HK97 shell protein observed to date are unfolded-either bound to GroEL or denatured in detergent (Ding et al., 1995; Xie and Hendrix, 1995). T7 prohead assembly was carried out in uitro using independently expressed and purified head protein (T7 gp9) and scaffolding protein (T7 gpl0) and was found to share different features of both the HK97 and P22 in uitro reactions (Cerritelli and Studier, 1996). The T7 head protein will assemble in prohead shells at about 1mg/ml in the presence of scaffolding protein at a minimum ratio of about 0.1, but only in the presence of added PEG (20% PEG 200) or dextran. The head protein did not exclusively form proheads; all reactions also produced “polycap-

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sids,” an aberrant, mostly tubular form of the head protein that is also found in addition to proheads in uiuo when the portal or internal proteins are missing (Roeder and Sadowski, 1977). The scaffold protein was found to be in a monomeric form, like the scaffold of P22, but the capsid protein was in the form of a n oligomer, or a mixture of oligomers, possibly hexamers and pentamers like HK97 head protein. The oligomeric state of the T7 head protein is unclear. A gel-filtration experiment was interpreted to indicate that the protein was tetrameric (Cerritelli and Studier, 1996); however, hexamer and/or pentamer would seem to better fit the gel-filtration data and make more sense biologically. Do these in uitro reactions accurately reflect the in uiuo reactions? It must be emphasized that a capsid built without a portal is a biological dead end and none of the three in uitro reactions described include this crucial element of capsid assembly. Furthermore, the HK97 reaction can occur without the incorporation of the putative protease, the P22 reaction occurs without two internal injection proteins (a third protein, gp16, does incorporate in uitro; Thomas and Prevelige, 19911, and the T7 reaction occurs without the four proteins of the complex T7 “core,” a large protein cylinder, coaxial with the portal, that is required for DNA injection (Steven and Trus, 1986). The T4 prohead, a much more complicated structure than the three discussed so far (Black et al., 1994), has also been reassembled in uitro (van Driel and Couture, 1978a,b) using a mixture of dissociated protein derived from purified proheads defective in proteolysis, usually carrying a mutation in the T4 proteinase gene 21. Assembly is highly dependent on ionic strength and temperature, and conditions were found that would reconstitute particles with the approximately correct composition, size, and morphology. Further examination of the oligomeric state of several major components purified from the mixtures revealed that under assembly-promoting conditions, the major head protein (T4 gp23) was partitioned between free hexamers and hexamers assembled into tubular polyheads, the major scaffold protein (T4 gp22) was partitioned between small oligomers and long ribbons, while the minor capsid corner protein (T4 gp24) was exclusively in the form of pentamers (van Driel, 1980).Studies of the form of T4 shell protein involved in polyhead assembly suggest that hexamers are a n assembly intermediate (Caldentey and Kellenberger, 1986). These results suggest that hexamers and pentamers of head proteins may be the relevant assembly substrate for T4, HK97, and T7, but not P22. Bacteriophage h proheads have also been assembled in uitro (Murialdo and Becker, 1977, 1978). Unlike the studies described above, the reactions have mostly been carried out in crude extracts of mutant-

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infected cells, but, in contrast to the other in vitro studies, the proheads were assayed on the basis of their biological function-their ability to package DNA and make viable phage-not only on their ability to form a particle. The basis of the assembly reaction is the mixing of two extracts, one containing unassembled shell protein (A gpE ), and another extract that donates the scaffold protein (A gpNu3), a preassembled portal dodecamer of A gpB (the initiator complex), and A gpC. Shell protein must be donated from an extract defective in scaffold protein in order to be active in the in vitro assembly reaction, because the shell protein will assemble efficiently into defective prohead-like particles, aberrant unclosed shells, and/or tubular polyheads in the presence of the scaffold (Hendrix and Casjens, 1975; Hohn et al., 1975). In contrast with HK97 there is a scaffolding protein that is required for efficient assembly but is not present in the mature phage particle. The shell protein is present in donor extracts as a small oligomer, but it is not known whether a monomer or possibly hexamers and pentamers are the active form (Murialdo and Becker, 1977). The role of the scaffold protein in prohead assembly is confounded by the fact that A gpC and A gpNu3 are made from overlapping genes in the same reading frame (Shaw and Murialdo, 1980), with the scaffold protein sharing the last 132 amino acid residues with A gpC (Ziegelhoffer et al., 1992). The overlap has interesting implications for assembly interactions; for example, the unique N-terminal end of A gpC might bind to the A portal whereas the C-terminal end may coassemble with the A gpNu3 scaffold. All mutations in gene Nu3 may also affect gene C function, making it a mind-bending adventure t o carefully filter the interpretations in some A prohead literature from before 1980, including two excellent reviews (Hohn and Katsura, 1977; Murialdo and Becker, 1978).

IV. PROTEOLYSIS: THEREMODELING OF PROHEAD I INTO PROHEAD I1

A. Prohead 11 Origins and Structural Differences from Prohead I Prohead 11, the proteolytic products of prohead I maturation, was first found in cells infected with mutant HK97arnC2 (Popa et al., 1991). The prohead I1 that accumulates contains a portal, but no DNA, so the mutant appears to be defective in DNA packaging, although the mutated gene has not been identified. Similar prohead I1 shells lacking a portal are produced when the HK97 putative protease (gp4) and the head protein (gp5) are expressed from a plasmid (Duda et al., 1995b). Although there is a slight increase in diameter, prohead I1 is nearly identical to prohead I on the outside [compare the three-dimensional

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(3D) structures shown in Fig. 3; and see Conway et al., 19951, but has profound differences in most other physical and biochemical properties. The extensive remodeling of the interior of the HK97 prohead can be seen by comparing the stereoimages of the interiors of prohead I and prohead I1 shown in Fig. 4 (see also Conway et al., 1995). In prohead 11, the amino-terminal 102 amino acids (the “delta domain”) of all 420 copies of the head protein, and all of the putative protease, gp4, have been removed (Duda et al., 1995a). Comparison of the masses of proheads I and I1 by scanning transmission electron microscopy (STEM) measurements suggests that the missing protein has been digested to peptides and lost from the structure (Conway et al., 1995); a mass loss is also suggested by a decrease in sedimentation rate for prohead I1 relative t o prohead I (R. L. Duda and R. W. Hendrix, unpublished). When a prohead I1 surface map is made using a stringent density threshold, small holes are found near the three fold axes; if these holes are real, they may provide convenient escape ports for the digested peptides (Conway et al., 1995). In prohead I1 the shell is thinner than in prohead I. This can be seen by comparing the central sections through proheads I and I1 in Fig. 3 or the stereoviews in Fig. 4;evidently the interior surface of the prohead has been excavated by proteolysis (Conway et al., 1995). Also missing are the “blobs”or “islands” of interior density that can be seen in the prohead I image. These interior blobs are tenuously connected t o the shell by thin arms of density that can be traced in the density map. All density in the region just under the inside surface of the shell is artificially reduced by the phase-contrast interference fringe in the images; this contributes to making the connecting density “arms” even less obvious in the images presented because the surfaces outline only those segments of density above a threshold. Any interior segments of density may also be underrepresented in the reconstructions, if some fraction of the delta domain is disordered, or does not conform to the symmetry imposed during the reconstruction process. The data strongly indicate that the delta domain of the head protein is partly on the interior surface and also in organized domains well within the interior of the prohead. The delta domain is predicted to have significant ahelical content, and further predictions support the idea that the interior density blobs might be connected to the capsid rim by thin antiparallel coiled coils (Conway et al., 1995). Between the delta domain and the protease (see Section IV,C) it seems likely that the entire inside volume of prohead I is occupied by protein, albeit at a rather low density. Prohead I1 is different from prohead I in its biochemical properties. In agarose gel electrophoresis experiments, prohead I1 migrates slightly

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faster than prohead I, suggesting a subtle change in surface properties. Prohead I1 is, in different ways, both more and less stable than prohead I. Whereas both proheads are sensitive t o trypsin digestion and share a resistant carboxyl-terminal fragment (beginning at residue 167),prohead I1 is resistant to chymotrypsin, which cleaves the 42-kDa prohead I shell protein to about 30 kDa. Prohead I1 remains assembled under conditions that dissociate prohead I into capsomers, such as treatment with 2 M salt or 20% glucose, or chromatography on DEAl3-cellulose. Citriconylation at pH 9 is the only treatment tried to date that dissociates prohead I1 into soluble capsomers (R. Duda, unpublished). On the other hand, prohead I1 is like a cocked mouse trap with regard to head expansion, which is triggered by any of a variety of mostly abusive conditions, such as exposure to denaturants or organic solvents. Prohead I1 also expands spontaneously during storage, more rapidly if the portal is present (see below and Duda et al., 1995a). Most conditions that a biochemist would use to denature prohead I1 for further analysis trigger partial or full expansion during treatment and usually also induce the formation of covalent cross-links, as described in Section VI1,B. Merely placing a prohead I1 sample into sodium dodecyl sulfate (SDS) gel sample buffer induces cross-linking-fortunately this can be prevented by rapid denaturation followed by precipitation of samples with cold trichloroacetic acid (Duda et al., 1995a).

B. Control

of

Proteolysis by gp4

The proteolytic activity that converts prohead I to prohead I1 is clearly controlled by gp4, the putative protease. Although there is no direct proof that gp4 is the actual HK97 prohead protease, genetic evidence strongly supports this idea. Phage mutants that cannot cleave are complemented in uiuo by a plasmid that expresses only gp4 (Duda et al., 1995~1,and the plasmid that produces prohead I differs from the one that produces prohead I1 only by a frameshift mutation near the start of gene 4 (Duda et al., 1995b). In addition, the 25-kDa gp4 protein has been detected in pulse-labeling experiments, but it has never been found in purified prohead 11, suggesting that it autodigests when prohead cleavage is completed (Duda et al., 1995a). The specificity of HK97 shell protein cleavage is known for only a single site, the site that produces the mature 31-kDa form, although examination by pulse-labeling experiments with wild-type and mutant forms of shell protein reveals three transient intermediates, suggesting a progressive digestion at several sites (Duda et al., 1995b). It is tempt-

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ing to speculate that the cleavage is to some degree sequence specific, because the known cleavage is between residues K103 and S104 and the dipeptide sequence KS appears three more times in the delta domain of shell protein, four times in the carboxyl-terminal end of the putative protease, and nowhere else in any of the head proteins (Conway et al., 1995). A variant of the head protein with a n amino acid substitution at the cleavage site (K104L) is still cleaved to the correct size (Duda et al., 1995b),suggesting that cleavage is not entirely sequence specific, but perhaps context specific. An alternative t o the hypothesis that gp4 is the actual protease is the possibility that gp4 may activate the shell to undergo self-cleavage. Some viral capsid proteins catalyze their own cleavage (Zlotnick et al., 1994), and the RecA protein mediates the cleavage of repressor proteins (Little, 1984). However, the observation that cleavage occurs a t multiple sites seems easier to reconcile with the view that gp4 is the actual protease than with the view that cleavage happens a t each site by an autocatalytic mechanism.

C. Assembly-Activated Proteolysis: Comparison with T4 and h The proteolytic processing of HK97 head proteins seems t o be entirely dependent on assembly, similar to the situation found for phage T4 (Black et al., 1994). The T4 protease gene 21 encodes two proteins, one 27.5-kDa protein (P21L) and a second 21.5-kDa protein (P21S) starting at codon 45, but although both are required to complement a T4 gene 21 mutant phage, neither seems to have any proteolytic or inhibitory activity when expressed in the absence of phage infection (Hintermann and Kuhn, 1992). The active T4 prohead proteinase (T4PPase) is an 18.5-kDa enzyme produced from an inactive zymogen (P21L) that is incorporated into T4 proheads; this same T4PPase can cleave many of the T4 head proteins i n uitro that are cleaved in uivo and also cleaves and activates the zymogen (Showe et al., 1976a,b). The best data on the location of the inactive T4 gp21 protease places it in the kernel, or center of the prohead, much like a cherrystone inside a cherry (van Driel et al., 1980).An astounding i n situ activation of prohead cleavage was achieved when T4 proheads that lack T4 gp24 (the pentamer shell protein that occupies the corners) were induced to cleave by the addition of the missing corner protein (Onorato et al., 19781, suggesting that T4 gp24 and the completion of the shell are the crucial factors for activation. The HK97 protease stays inactive when unassembled capsomers accumulate in cells expressing gp4 and a mutant variant of the HK97

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head protein, gp5-N356D, but efficient assembly and cleavage will take place if a cell extract is concentrated with PEG in the presence of Mg2+ and salt (i.e., under in uitro assembly conditions) (Duda et aZ., 1995b). This further supports the idea that HK97 maturation cleavage requires assembly. A linker-insertion mutant variant of gp4 (gp4-Ml02) produces a protein that is incorporated into prohead I, but does not allow cleavage, suggesting that the mutant protein is indeed a dead protease that is still competent to assemble (D. Lau, R. W. Hendrix, et aZ., unpublished, 1997). About 50 copies of the gp4-M102 protein were found to be present in purified M102 prohead I, suggesting that 50 active proteins constitute the approximate cohort that is transiently present in the normal precursor to prohead 11.Examination of the same gp4-M102 prohead I by conventional negative-stain electron microscopy (Fig. 7) suggests that these few copies of gp4 form a structure in the

FIG7. Comparison of HK97 prohead I containing protease with empty prohead I. Electron micrographs of two varieties of HK97 prohead I reveal a structure that appears to fill the central cavity of prohead I containing an inactive form of the protease, gp4. Shown are negative-stain electron micrographs of (a) prohead I containing a variant form of gp4 that is incorporated, but defective in cleavage (insertion mutant M102; D. Lau, K. Martincic, X. Xie, R. Duda, and R. Hendrix, unpublished, 19971, and (b) prohead I made without gp4. Micrographs were prepared as described previously (Duda et al., 1995a). Scale bar: 1000 A.

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center of the particle. These proheads seem to crack open i n situ in interesting ways, revealing a prominent stain-excluding central region surrounded by a darker halo. Prohead I that is completely missing gp4 does not share these unusual features. The stain-excluding blob seems t o remain intact in many of the particles that have completely fallen apart, and in some cases this central blob appears to remain, while the rest to the prohead has dissociated. We suggest that this central structure is composed of the proteolytically inactive form of gp4. Like the scaffold of other phages (as well as the prohead protease of phage T4), it may partially fill up the central volume of prohead I. The bacteriophage A scaffold protein gpNu3 is also cleaved after assembly of the A prohead is complete. The likely candidate for the protease is the unique N-terminal part of minor protein gpC, which is coexpressed in frame with the scaffold protein gpNu3. The A gpC protein has not been shown directly to be a protease, but amber mutants in gene C assemble normal-looking proheads that do not undergo cleavage of either the scaffold, or the A portal, gpB (Hendrix and Casjens, 1975). Sequence comparisons indicate that A gpC shows significant similarity to a subset of known proteases (Baird et al., 19911, including two from E. coli. The signal for A prohead cleavage seems to be the completion of correct assembly ofthe prohead (Hendrix and Casjens, 1975).Besides mutants in gene C the only mutations that block A cleavage are host groE mutations, which fail to make an assembly-competent portal and make proheads that incorporate inactive A gpC protein.

V.

SCAFFOLDS,

PROTEOLYSIS, AND THE INITIATION OF HEAD ASSEMBLY

A. Questions Concerning whether the HK97 delta Domain is a Scaffold What is the function of the HK97 delta domain, the amino-terminal 103 residues of the HK97 shell protein that are removed during the processing of prohead I to prohead II? The delta domain may be the functional equivalent of a scaffold protein that is genetically fused to the major head protein. All identified phage scaffold proteins are just upstream of the major shell protein (Fig. 6). Like a typical scaffold protein the delta domain is not needed after initial assembly is complete, so it can be digested away. Curiously, the HK97 delta domain is predicted to be largely a helical in structure, an unexplained shared feature of many phage scaffold proteins. It appears that the HK97 shell protein may assemble efficiently and accurately in the

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absence of a separate scaffold precisely because an assemblypromoting scaffold function is built in.

B. The Functions of Scaffold Proteins

1. Catalysis of Shell Assembly Why do phages require a separate scaffold protein t o assemble capsids? It seems clear that much of the information needed for correct assembly of some phages is actually built into the shell proteins themselves. In the absence of a scaffold, shell assembly may be inefficient or largely inaccurate, but some phage shell proteins are still capable of assembling correctly sized, closed shells from the shell protein alone in uiuo, including those of A (Hohn et al., 1975), P22 (Earnshaw and King 1978), and the P2P4 capsid protein (Marvik et al., 1995). In contrast, the T7 shell protein does not assemble (Cerritelli and Studier, 1996; Roeder and Sadowski, 1977) whereas the shell protein of 429 makes aggregates of poorly organized capsid-like structures (Hagen et al., 1976; Lee and Guo, 1995) in the absence of scaffold. The dominance of the shell protein in regulating capsid size is clearly demonstrated by mutants in the A shell protein that form small heads (Katsura, 1983). These small-head mutants make small proheads (2' = 4 vs T = 7), which incorporate a smaller number (-60 vs -180 copies) of scaffold protein, A gpNu3, and can successfully package DNA, if an appropriately sized genome is available for packaging. Phage P4 apparently uses a different mechanism, an external shape-limiting scaffold (P4 gpsid ), t o make smaller heads from the P2 shell protein, but the internal scaffold for P2 and P4 is the same (Marvik et al., 1994, 1995). 2. Control of the Internal Content of Proheads The lesser amounts of A scaffold protein incorporated into smaller proheads in the A small-head mutants suggest a direct relationship between prohead volume and number of scaffold molecules. Measurements of the incorporation of half-length P22 scaffold molecules into normal-sized P22 procapsids strongly suggest that twice the normal number (-400 vs -200) of short scaffold molecules participate in assembly (L. Sampson and s.Casjens, unpublished). Both of these results support the hypothesis that an important role of scaffolds is to exclude the highly concentrated cytoplasmic components of the host that might later interfere with DNA packaging, because excluding material during assembly would be more efficient than exporting it following assembly

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(Casjens, 1997; Earnshaw and Casjens, 1980). Under this model the HK97 shell protein delta domain and the internal putative protease together would provide the cytoplasm-excluding scaffold or “stuffing” function for HK97, and both are removed by proteolysis. Such a stuffing function might be a fundamental common purpose for a variety of cleaved and uncleaved scaffold proteins. There are apparently no examples of host proteins that are incorporated into phage heads, suggesting that exclusion of host proteins is important. Some host or phage proteins might be actively toxic during packaging or injection, and the bulk solids present in the cytoplasm, estimated at 0.3-0.4 g/ml (Zimmerman and Trach, 19911,would very likely prevent a full genome from being packaged unless they are removed. Few experiments address whether the volume of internal proteins might compete with the volume available for DNA packaging in a phage head. Phage T4 normally packages a headfull of DNA that is dependent on head volume and includes a complete genome plus a 3300-bp terminal redundancy that is apparently crucial for viability (Mosig and Werner, 1969; Singer and Parma, 1987). Members of a family of duplication mutants (Hp17 mutants) of T4 that have about 4000 bp of extra genomic DNA were isolated on the basis of increasing the expression of poorly expressed phage gene 17 umber mutants in an ochre-suppressing host (Wu and Black, 1987). These mutants could be isolated only in the presence of additional mutation(s1 that eliminate the incorporation of a phage-encoded internal protein (T4 gpalt), equivalent to about 1000 bp of DNA in volume (Wu and Black, 1987). One interpretation of these results is that the extra volume needed for viable packaging of these oversized T4 chromosomes was made available by eliminating a quantity of nonessential internal protein. A growing consensus from the in vitro studies outlined above and from genetic studies seems to be that the major function of a scaffolding protein is to act as a kind of cochaperone that coordinates the correct assembly of monomers or hexamers and pentamers into the correct closed shape. Formally the HK97 results disagree with this, but the scaffold might be part of the shell protein. The results from the A smallhead mutants, from the P22 half-length scaffold studies, and from the T4 Hp17 mutants described above suggest that Casjens’ cytoplasmexcluding stuffing role of scaffold function is a fundamental property of scaffolds. If scaffolds share this common function then an effective way to ensure that the stuffing is incorporated is to require the presence of the stuffing to facilitate shell protein polymerization. Scaffolds appear to play a central active role in selecting specific proteins for incorporation into proheads in addition to their passive

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control of prohead content by exclusion of host material. Results from phages T4 and P22 support this hypothesis. Specific T4 proteins are incorporated into T4 capsids to participate in assembly or for injection into the host (Black et al., 1994). The protein packaging signal of a T4 internal protein, T4 gpIPII1, has been identified and used to direct the incorporation of genetically engineered fusions of gpIPIII with pgalactosidase or other proteins into T4 heads, from which they are subsequently injected into the host (Hong and Black, 1993a,b). Specific ts mutants in the T4 major scaffold protein gene 22 have been found to be defective in the recruitment of minor T4 core proteins gpalt and gpIPIlI into the T4 head (Paulson et al., 19761, strongly suggesting that the major scaffold protein of T4 controls the incorporation of minor prohead proteins. Similar ts mutants in the P22 scaffold protein gene also fail to incorporate the minor proteins into proheads (Bazinet and King, 1988).

3. Recruitment of a Single Portal and Initiation of Prohead Assembly The results of in vivo and in vitro experiments from a variety of phages show that the scaffold has a crucial role in initiating shell protein assembly. No stable head structures are assembled in the absence of scaffold in phage T7 (Roeder and Sadowski, 1977), or in phage 429 (Hagen et al., 1976;Lee and Guo, 1995).T4 shell protein assembles in the absence of the major scaffold T4 gp22, but tubular polyheads instead of shells are formed and only after a significant delay required for the shell protein concentration to reach a concentration about fivefold higher than that required to initiate normal prohead assembly (Laemmli and Eiserling, 1968). For P22 the rate of shell formation in the absence of the P22 scaffold is virtually zero when measured at early times (Bazinet and King, 1988), although a low level of heterogeneous closed shells composed solely of shell protein is eventually formed (Earnshaw and King, 1978). As described above, the h shell protein remains largely unassembled (and assembly competent) when the scaffold is eliminated by an amber mutation (Murialdo and Becker, 1977). In addition to specifying what components are enclosed within capsids, the scaffold, owing to its requirement to initiate assembly, also plays a crucial role in recruiting the portal into the head shell. For tailed phages, capsids without portals cannot become mature virions, so the mechanisms used to ensure the incorporation of one and only one portal are essential. Results summarized above from several phage systems show that the scaffold is required for the incorporation of all minor proteins into shells, including the portal. Genetic evidence also

ROGER W. HENDRIX AND ROBERT L. DUDA

strongly implicates the scaffold in the incorporation of a portal into proheads in some cases. A specific ts mutant of the scaffold protein of P22 fails to recruit the portal and all other minor proteins into head shells, a1though it efficiently catalyzes shell assembly with normal kinetics under nonpermissive conditions (Bazinet and King, 1988). Stocks of two T4 scaffold gene ts mutants contained 2 to 6% two-tailed phages when grown under permissive conditions (Paulson et al., 1976); stocks of a T4 gene 22 amber mutant grown on an amber-suppressing host contain similar numbers of two-tailed phages (R. L. Duda and F. Eiserling, unpublished, 1997), strongly suggesting that the T4 gp22 scaffold protein has a role in correct portal recruitment. 4. Speculations on the Evolution of Scaffold Protein and Prohead Proteases

If a major role of the scaffold is to exclude the cytoplasmic components, how did the various properties and functions of scaffolds evolve? There seems to be a logical continuum of types of scaffold ranging from simple t o complex. A primordial phage that built a prohead with large holes in it to allow cytoplasmic components to exit during DNA packaging might not need a scaffold, but large holes in the prohead might lead to structural weaknesses in the mature virion. A recruited scaffold might then allow such holes t o be smaller, but large enough to allow the scaffold to exit. The P22 scaffold apparently exits from holes in the prohead hexamers that “close” during maturation (Prasad et al., 1993). Another method to get the scaffold out is t o cut it to small pieces by proteolysis-for the pieces to exit would probably require only very small holes, such as those that may exist in HK97 prohead 11. Proteolysis of scaffolds is common, and under a scaffold-cleavageplan the protease is an essential component, structurally as well as enzymatically. A protease might also digest “accidentally” incorporated cytoplasmic proteins, depending on its specificity. Scaffold genes are often genetically coupled to the shell protein by being adjacent genes. This allows the possibility of rapid coevolution, and might even lead to partial or full fusions of scaffold function with head shell protein function, or even with protease function. This is the possibility proposed for HK97, and it may be a likely mechanism for the inclusion of major head shell protein cleavage into the assembly pathway of many phages. We have indicated prohead proteins (or parts thereof) that exit or are removed by cleavage in Fig. 6, and in all cases the genes are tightly linked. With a scaffold integrated into an assembly mechanism it becomes possible to evolve new functions. Is the delta domain of the T4 shell protein, the N-terminal66 residues of T4 gp23

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that are removed during maturation, a part of the scaffold that became fused to the shell? Is it instructive in this regard to consider T4 gene 24, which encodes the minor shell protein that forms pentamers and occupies the capsid vertices. Comparison of the sequences of T4 gp23 and T4 gp24 suggests that their genes may be related by a duplication and drift mechanism. The regions of homology between the T4 shell and corner proteins occur mostly in three regions that align all of T4 gp24 with all but the delta region of the T4 shell protein. T4 gp24 seems to be missing the delta region, suggesting that the hypothetical gene duplication that created it occurred before the acquisition of the delta region by gene 23, or the delta region was later lost. If capsid maturation proteases are indeed an integral part of scaffolds, a mechanism must ensure that the protease, in a n inactive form, is incorporated into the structure. Several possible mechanisms include regulated coassembly with the major scaffold (T4, HK97?), genetic fusion with scaffold (P2, P4?), genetic overlap and coassembly with scaffold (A?), and genetic fusion with the shell protein.

VI. EXPANSION OF THE HK97 CAPSID A. Induction of Expansion in Vivo and i n Vitro Assembly of a biologically active prohead creates the substrate for DNA packaging, the next step in bacteriophage maturation (for reviews see Black, 1989; Earnshaw and Casjens, 1980). Although a prohead is required to initiate DNA packaging, the reaction can finish only if the prohead expands, because the enclosed volume of the prohead is too small to package a full genome. For all of the phage systems in which it has been studied, expansion is closely coupled to DNA packaging (Casjens and Hendrix, 1988). Bacteriophage h DNA packaging induces expansion in vitro if a large DNA fragment (44.5% of the genome) is packaged, but not if a small fragment (11.4% of the genome) is used (B. Hohn, quoted in Earnshaw and Casjens, 1980). These results support the idea that expansion is not required to initiate packaging, and further suggest that DNA packaging may actually induce expansion. Little is known about the details of HK97 DNA packaging, but the general model that expansion is triggered by DNA packaging is assumed to hold. Conditions t o expand proheads i n uitro have been found for several phages and most require treatment with a detergent or denaturant. HK97 prohead I1 can be induced to expand by a variety of treatments (Duda et al., 1995a) that include freezing and thawing, exposure to low

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pH in salt, to urea at low pH, to organic solvents, or to the detergent sodium dodecyl sulfate (SDS). SDS treatment will also induce prohead expansion for phages P22 (Earnshaw et al., 1976) and T7 (Serwer and Pichler, 19781, and phage T4 for polyheads (Onorato et al., 1978). The SDS-expanded P22 shells lose the portal and internal proteins, and detergent-expanded T7 shells apparently experience similar losses of internal proteins and portal. Urea treatment of A proheads induces expansion (Earnshaw et al., 1979; Kunzler and Hohn, 1978). HK97 prohead I1 expands spontaneously during storage, but shells made without a portal are distinctly more stable (R. L. Duda, unpublished), suggesting that the portal may have a role in initiating expansion. Expansion seems to be a cooperative reaction; in most cases the entire particle rapidly switches state with no accumulation of obvious intermediates. Expansion in T4 giant proheads seems to propagate in a wave along the structure and this is viewed as the paradigm for phage capsid expansion-that a small cluster of subunits changes to the expanded state and induces neighboring subunits to do the same via cooperative allosteric interactions, until expansion is complete (Steven and Carrascosa, 1979; see also Section VIII). Because treatments that induce expansion might also cause partial denaturation of capsid proteins, it is likely that inducing treatments artificially induce a few subunits or capsomers to switch to the expanded state, and once this happens, the rest of the capsid expands cooperatively.

B. Changes Induced during Expansion For all of the large dsDNA phages, the magnitude and complexity of changes that take place during expansion clearly show that head shells are complex biological machines and not just simple containers. “Conformational change” seems inadequate t o describe the transformation of the roundish, thick-walled proheads into thin-walled, angular structures with flat icosahedral faces. For HK97 (Figs. 3 and 8; Conway et al., 1995), P22 (Prasad et al., 1993), and A (Dokland and Murialdo, 1993),the transformation has been documented a t relatively high resolution in three-dimensional reconstructions from cryoelectron micrographs. The magnitude of the expansion is strikingly demonstrated in Fig. 8, where HK97 prohead I1 is shown to fit inside of the expanded head 11. Estimates of the increase of volume due to expansion can be made from negative-stain electron micrographs, and more reliably from three-dimensional reconstructions from unstained frozen hydrated

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FIG8. Maturation changes in internal volume and thickness of the HK97 head shell. HK97 prohead I1 is shown to scale inside of head I1 to emphasize the radical changes that occur during the expansion transformation. The shell thickness decreases from -46 A to about 35 A o r less. The enclosed volume approximately doubles. Scale bar: 100 A.

-

samples. It seems that the change in internal volume varies considerably: 50%increase for P22,100% increase for HK97, and 150%increase for A. This large variation in the change in volume on expansion suggests that the value of the increase is not an important feature of a common mechanism. More than just the internal volume changes on expansion (Figs. 3 and 8; Conway et al.; 1995). The thickness of the HK97 shell changes from about 50 to 35 A or less. The overall shape changes from roughly spherical to icosahedron-like with flat faces. The surface relief changes

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from the knobby capsomer-studded proheads I and I1 to the relatively smooth head 11. Although there is less surface relief in the expanded heads I and I1 than in proheads I and 11, there are noticeable changes in the surface features that are identified as capsomers. The pentons change shape from a pentagon t o a starlike form, and the hexons change from the twofold symmetric “sheared hexamers” into a shape that actually has sixfold rotational symmetry, which may reflect a change from nonequivalent to quasi-equivalent bonding. The sheared shape of the hexamers in proheads may have a special size-determining role early in assembly (as suggested above);in the mature head the subunits may need to have nearly equivalent interactions to become maximally stable, including forming cross-links. Proheads of two other T = 7 phages, P22 (Prasad et al., 1993) and A (Dokland and Murialdo, 1993), have slightly oval hexons that also become sixfold symmetric on expansion. Changes in secondary structure have been monitored by biophysical methods in other phages, but not HK97. Whereas expansion produces a substantial change in the bulk amounts of secondary structure types in T4 (Steven et al., 1990),little change was seen in P22 (Prevelige et al., 1993) or A (Kawaguchi et al., 1983). Substantial unfolding and refolding of subunits could occur in all cases during expansion, but not be evident by comparing the starting and ending states. The topological relationships among the capsid proteins in the final state of the HK97 head (see Section VI1,E) suggest that unfolding and refolding on at least a small scale occur during HK97 capsid maturation. VII. CROSS-LINKING OF HEAD PROTEINS IN HK97

A. Early Observations of HK97 Head Protein Cross-Linking HK97 was the first bacteriophage reported to have capsid shells entirely composed of covalently cross-linked head shell subunits (Popa et al., 1991).Unlike A and nearly all previously studied bacteriophages, the HK97 phage particles do not contain an abundant 30- to 50-kDa protein derived from hundreds of independent copies of a major head protein. Instead, most of the capsid protein is in large oligomers that will not even enter the stack of an SDS-polyacrylamide gel (Popa et al., 1991), implying that much of the mature head protein is tightly bound into enormous complexes. Early studies (Popa et a.?., 1991) showed that there was a prominent 42-kDa protein produced during HK97 infection and that a 42-kDa protein was also present in a prohead-like structure (prohead I; Duda et al., 1995a) produced by an amber mutant (amU4, later shown to be in gene 4, the putative protease gene; Duda et al., 1995b). The 42-kDa

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protein appeared to be the pi ecursor to the high molecular weight head proteins. A different prohead-like structure found in lysates of another amber mutant (amC2) contained only cross-linked head protein in the early studies (Popa et al., 1991), but was later found to accumulate prohead I1 containing the 31-kDa, cleaved major head protein (Duda et al., 1995a). The cross-linked version found in the early studies was a result of in uitro cross-linking induced by chloroform that had been used to aid cell lysis. The interpretation of many of the early HK97 capsid maturation experiments was complicated by ignorance of the head protein cleavage and the rather late discovery that both the 42and 31-kDa proteins in proheads I and I1 were able to cross-link spontaneously into a series of high molecular weight forms in SDS sample buffer before heating. Precipitation of HK97 head proteins with cold trichloroacetic acid (TCA) before denaturation in detergent was found to prevent the artifactual cross-linking.

B. Induction of Cross-Linking i n Vitro by Expansion Efforts to break apart the large HK97 protein complexes with denaturants revealed, instead, that many denaturing treatments induced expansion (as described in the previous section) and also induced crosslinking of HK97 proheads (Duda et al., 1995a). When we monitored expansion by native (nondenaturing) agarose gel electrophoresis and cross-linking by SDS-polyacrylamide gel electrophoresis, as shown in Fig. 9, we found a close correlation between expansion and crosslinking. In a different experiment we clearly showed that expansion precedes cross-linking by taking advantage of the observation that treatments that induced expansion transformation also slowed crosslinking. Chloroform induces expansion rapidly in a population of proheads, but cross-linking occurs slowly when chloroform is present. Removal of chloroform by dilution allows the fraction of proheads that have already expanded to cross-link rapidly to completion, while unexpanded heads remain essentially monomeric (Duda et al., 1995a).

C. Biochemistry and Topology of Cross-Linking All of the high molecular weight forms of the HK97 gp5 major head protein are the consequence of a single type of chemical cross-link. This bond links a lysine (K169)of one head protein subunit to an asparagine (N356) on an adjacent subunit within each hexamer and pentamer in the HK97 head shell (with the loss of ammonia). The chemical identity

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ROGER W. HENDRIX AND ROBERT L. DUDA SDS Polyacrylamide gel

t(min)=O 0.5 1.5 3

5

10 15 30 60 120240300

FIG 9. In oztro expansion and cross-linking of HK97 proheads. HK97 prohead I1 was induced t o expand and cross-link in uitro by stirring with chloroform as described previously (Duda et al., 1995a). Samples were taken a t the indicated times and prepared for electrophoretic analysis using an SDS-polyacrylamide gel (top) to monitor crosslinking, or a native (nondenaturing) agarose gel (bottom)to monitor expansion. SDS gel samples were precipitated with trichloroacetic acid before denaturation. Agarose gel samples were diluted 10-foldto reduce the chloroform concentration and stop the reaction.

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of the covalent bond was determined in a series of peptide-mapping experiments comparing prohead I1 with head I1 (Duda et aZ., 1995a). Peptides were made by digesting samples sequentially with cyanogen bromide and either of two proteases. In each case a large peptide was shown to be modified by cross-linking and change its chromatographic elution properties. Amino-terminal sequencing of the donor and crosslinked peptides identified the regions of the protein involved: One residue linked was Lys-169, and the other was likely to be Asn-356. The lysine-to-asparagine bond and the loss of ammonia were confirmed by mass spectrometry. All cross-linking of HK97 head protein is abolished by a single mutational change of Lys-169 to tyrosine (K169Y). The protein that contains this single amino acid substitution assembles correctly into prohead I, cleaves normally into prohead 11, and even undergoes the expansion transformation to the angular head I, but does not cross-link to become head 11. The lysine-to-asparagine cross-linking reaction is outlined in Fig. 10. The details of the reaction mechanism are unknown, although we assume that the active sites that catalyze the reaction are created

CH2

I

Lysine 169

CH2 I

0

NH2

\J

Aspauagine 356

L

I

-

CH2

I

CH2

I

CH2

+N&

I

NH

I

c=o I

FIG10. The lysine-to-asparagine cross-linking reaction. The substrates and products of the cross-linking reaction catalyzed by HK97 head protein (from Duda et al., 1995a). The cross-link joins residues from different adjacent subunits.

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within the protein shell by the expansion transformation. The reaction appears not to require any external factors. The prohead is in a sense the enzyme, substrate, and major product for the reaction (ammonia should also be a product). The reaction is easily performed in uitro, normally starting with prohead 11, consisting entirely of the cleaved 31-kDa g ~ 5subunits ~' (the portal protein is not required). What is the evidence for the geometry of the cross-linking? Virtually all of the subunits in each HK97 mature capsid are cross-linked. This includes the 12 pentamers at the vertices and the 60 hexamers on the faces. All of the monomer protein is converted to higher molecular weight forms. In the mature capsid and in preparations that have been induced to cross-link the completion in uitro the smallest forms are the linear pentamer and hexamer. In comparisons of capsid protein before and after cross-linking by peptide mapping, the cross-link-bearing peptide quantitatively replaces the precursor peptide. Because the capsids are highly symmetric and all of the subunits participate in cross-links, the symmetry of the links must match some of the (real or quasi-) symmetry of the capsid. These are the fivefold axes of the pentamers, the quasi-sixfolds of the hexamers, the threefolds in the center of the icosahedral faces, and the quasi-threefolds that surround them, the twofolds between hexamers at the edges, and the quasi-twofolds between hexamers. The products of in uitro cross-linking reactions give a clear indication that the symmetry of the covalent bonds follows only the symmetries of the hexamers and pentamers. During the reaction there is a progressive appearance of dimer, trimer, tetramer, pentamer, and hexamer (see Fig. 91, indicating an essentially equal probability of joining one or more cross-linked pairs into oligomers forming all o r part of five-or six-membered circles. The logical end points of sequential linking of hexamers and pentamers are circular hexamers and pentamem, and, indeed, we were able to show that these do exist (Duda, 1997) and appear in SDS gels as two bands that migrate just above the position of the linear hexamer (indicated in Fig. 9).

D. Kinetics

of

the Cross-Linking Reaction

Additional insight into the cross-linking process comes from measuring the intensities of the bands in a gel such as that in Fig. 9 to give a quantitative representation of the in uitro cross-linking kinetics. The left panel of Fig. 11 shows a set of data derived in this way. The right panel of Fig. 11shows the results predicted from a mathematical model in which it is assumed that cross-links form randomly in time and in position without regard to the presence or absence of other cross-links

271

BACTERIOPHAGE HK97 HEAD ASSEMBLY Theotetical cross-linking hnerss

Mcasured cross-iinkmg kinecics

'

m

1

me,

2 me,

Bmer 4 me,

0

5

10

Mm

(6 ?

,

I

15

0

5

10

15

MI"

FIG11. Cross-linking reaction kinetics. HK97 prohead I1 was induced to expand and cross-link in uitro, and run in an SDS gel as described in the caption to Fig. 9. The stained gel bands were quantified using a raster scanning laser densitometer and twodimensional gel analysis software. The values were normalized t o the value for the portal protein and plotted in arbitrary units (left). A set of equations to estimate the number of subunits per shell in different oligomeric forms during HK97 cross-linking was modeled in a spreadsheet, assuming that cross-links would form randomly with respect to position and a t a rate independent of the presence or absence of cross-links in other positions, and the results were plotted (right).

in the vicinity-that is, noncooperatively. Of the disagreements between the theoretical and measured curves, two differences arise from oversimplifications in the mathematical model. First, the higher final levels of hexamer and pentamer in the theoretical calculations are due to the fact that the model does not account for the loss of (linear) pentamer and hexamer t o circular and larger forms. Second, the initial lag in the measured kinetic data is due to the -30-sec half-time for shell expansion (because cross-linking requires prior expansion), which is also not represented in the model. Beyond these differences the two sets of curves are rather similar. We believe the results rule out any highly cooperative models of cross-link formation in favor of a closer

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ROGER W. HENDRIX AND ROBERT L. DUDA

approximation to the purely stochastic model represented in the right panel of Fig. 11. The lower levels of the intermediate oligomers seen in the measured data do suggest a small degree of cooperativity: This is the result that would be predicted if the initial bond joining two monomers into a dimer formed with roughly twofold slower kinetics than subsequent bonds. The delayed appearance of hexamers in the measured data relative to the model is less easily interpreted but it does suggest a deviation from the simple predictions of the stochastic model in the final stages of hexamer formation. The fact that the crosslinking reaction nonetheless conforms a t least approximately with a noncooperative model is compatible with the observation that there is no detectable change in the protein structure associated with crosslink formation, a s judged by the similarity of the cryo-EM images of head I and head 11.

E. Creation of Protein Chain Mail by Interlinking before Cross-Linking The identification of a cross-link between head protein subunits seemed to explain the smaller cross-linked forms of the head protein, but not the large species that formed multiple bands a t the top of SDS gels and the material that could not penetrate even a stackinggel. We initially thought that there must be a second unique covalent bond that joined the hexamers into larger forms, such as trimers of hexamers. However, repeated detailed searches for this “secondary cross-link” failed. It was not initially obvious that we had failed to account for circular oligomers in our experiments-that the logical outcome of sequentially linking circularly symmetric hexamers should yield six linear forms and one circular form (also five linear and one circular form for pentamers). Where would circular forms run in a gel? Could the circular forms be some of the bands that we could not assign to linear forms? Asking these questions led to the realization that circular forms could be interlinked to form multiply interlinked circles (see Fig. 12). In a large symmetric capsid the end point of this topological linking would be large complexes of catenated circular proteins that resembled the chain mail armor worn by ancient warriors in Europe (Katabira in Japan, kuijia in China). The protein chain mail hypothesis explained how a single type of covalent cross-link could produce all ofthe unexplained large oligomers, ifit occurred in proteins that had previously intertwined, arm-over-arm, as it were, before cross-linking. The material that failed to enter gels was likely t o be an enormous complex that might contain nearly all of the 400 polypeptides in the capsid, and the unusual species of high molecular

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Alternate Cross-linking Topologies

FIG12. Two models to explain the cross-linking of HK97 capsid proteins. In each model the circles represent covalently closed circular polymers (hexamers) of the HK97 head protein gp5”. In the chain mail model the circles are catenated to create topological oligomers. In the two cross-link model a second set of covalent cross-links (represented by short lines) joins adjacent circles a t several places t o create large oligomers.

weight proteins that we observed during in uitro cross-linking experiments were likely to be the expected family of multiply catenated covalent circles of hexamers and pentamers. The two alternative models of cross-linking are shown schematically in Fig. 12. The most obvious property of independent protein circles in the chain mail model is that they are covalently bonded only to other proteins within the circle, so that if the circle is broken, the topological link to other circles is broken and a “linear”form of the full circle can be released. A prediction of the model is that during partial proteolysis of chain mail networks, a major fraction of the earliest products released would be once-cut linear versions of the covalent, circular hexamers and pentameters proposed to be the components of chain mail. In the alternative model with two different kinds of covalent link, the results would be radically different. The two cross-links would bond each circle to more than one adjacent circle, so no uniform products will be released during proteolysis, only heterogeneous, multiply branched species that would not form discrete bands in gels. For the actual experiments, we denatured fully cross-linked head I1 particles in 2% SDS, diluted them to 0.1% SDS, and incubated with a series of dilutions of protease. Figure 13 shows the results of one such experiment using Staphylococcus aureus VI protease (similar results

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ROGER W. HENDRIX AND ROBERT L. DUDA

[enzyme] 0 0.40.8 t

2 4

Ligh

o 15 30 60 izoz40

t=(sec)

o 15 30 60 i z o z 4 0 t=(sec)

FIG13. In uitro unlinking of HK97 chain mail by proteolysis and by sonication. HK97 head I1 was denatured and treated to break the polypeptides randomly either by digesting with protease in (a), or by treatment with a probe sonicator in (b) and (c). In (a) the samples were denatured in sodium dodecyl sulfate (SDS), diluted, treated with the indicated concentration of protease for 15 min, precipitated with TCA, denatured, and run in an SDS-polyacrylamide gel. In (b) and (c) samples were denatured in 6 M GuHCl, sonicated for the indicated time, precipitated with acetone, denatured, and run in a n SDS-polyacrylamide gel in (b), or in an agarose gel containing 0.1% SDS in (c). The starting material is largely composed of protein chain mail. As the chain mail is digested or broken, linearized hexamers and pentamers predominate among the products. A complex series of bands containing catenanes are present during midpoints of each reaction. The migration positions of circular forms indicated. The methods have been described in detail (Duda, 1997).

were obtained using chymotrypsin). The chain mail is essentially invisible at the start of the experiment, because only a fraction of it penetrates into the top of the stacking gel. As predicted by the chain mail model, early prominent products are the linear hexamer and pentamer. As the amount of digestion increases, the linear hexamer and pentamer bands remain the most prominent. The material that does not initially penetrate the gel is broken down into smaller forms that form a smear in the stacking gel and a complex array of slowly migrating bands. Small species corresponding roughly to the tetramer, trimer, dimer, and monomer also appear, and eventually all forms are digested into small peptides, implying that all proteins are accessible to the protease. The two bands above the linear hexamer and pentamer are the circular forms of pentamer and hexamer (Duda, 1997).The more slowly migrating species were found to release circular hexamers on redigestion (Duda, 19971, showing that these are indeed the multiply catenated rings of protein predicted to exist by the chain mail model. The unlinking experiment was repeated using sonication to break the peptide bonds, producing similar results (Fig. 13).

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We were able to demonstrate that the material that does not enter SDS gels is composed of large chain mail complexes by showing that these same complexes do appear as bands if a denatured SDS gel sample is run in an agarose gel. The bands of chain mail complexes in agarose gels disappeared and were replaced by faster migrating bands, as expected, in unlinking experiments (Fig. 13; Duda, 1997). We also found these same slow-migrating bands when denatured samples derived from intact HK97 phage were electrophoresed in agarose (Fig. 14). Most of the protein released from phage appears to be in the form of chain mail.

F. Chain Mail and Cross-Linking in Other Phages and Elsewhere Protein chain mail may actually be rather common in phage capsids. Since the initial report of cross-linking in HK97, a number of other phages with cross-linked major capsid proteins have been reported. The best documented cases are in reports that have compared Nterminal amino acid sequences of virion proteins with predicted properties of phage open reading frames. These include E. coli phage HK022 (lysogenic) (R. Hendrix and G. Hatfull, unpublished), mycobacteriophage L5 (lysogenic) (Hatfull and Sarkis, 1993) and its close relative D29 (lytic) (M. Ford, G. Sarkis, R. Hendrix, and G. Hatfull, unpublished), and two lactococcal phages r l t (lysogenic)(van Sinderen et al., 1996) and c2 (lytic) (Lubbers et al., 1995). All of these phages appear to lack a prominent monomeric major head protein, and instead have a set of prominent high molecular weight proteins with a common Nterminal amino acid sequence that in each case corresponds to a much smaller open reading frame in the phage genome. In HK022, L5, D29, and r l t , the high molecular weight bands of oligomeric head proteins appear to be pentamer and hexamer species, as in HK97. In the case of HK022, L5 and D29, and c2 some higher oligomers have been observed that behave similarly to the circular and catenated forms of HK97 head protein. For HK022, L5, D29, and c2 there are indications of very high molecular weight material, which barely enters into gels. Together these observations suggest that these phages do have crosslinked protein and protein chain mail. The HK022 and HK97 capsid protein amino acid sequences are identical (M. Ford, R. Hendrix, and G. Hatfull, unpublished), so the chain mail-like properties of HK022 capsid protein are no surprise. Most of the phages known t o have cross-linked capsid protein have large icosahedral heads, except for phage c2, which has a smaller pro-

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ROGER W. HENDRIX AND ROBERT L. DUDA

FIG14. Chain mail complexcs are released from whole phage particles. Phage particles were treated to release protein free from DNA, boiled in SDS gel sample buffer, and electrophoresed in a n agarose gel without SDS. Samples of native and SDS-boiled head I and head I1 were included as controls. Head I and head I1 exhibit the expected single bands (head I moves farther because it is more highly charged). Boiled head I shows only fast-moving small oligomers. Boiled head I1 and disrupted whole phage both show a slowly migrating band that is characteristic of denatured chain mail complexes (see Fig. 13). Electrophoresis conditions were as described previously (Duda, 1997; Duda et al., 1995a).

late head and rather small (22-kbp) genome. The cross-linked head proteins of phage c2 are also unusual-the major cross-linked bands appear to be trimer and hexamer of a 31-kDa major head protein that is probably cleaved from a larger 56-kDa precursor (Lubbers et al., 1995). We hypothesize that the geometry of this phage might explain the unusual oligomers observed, if c2 has an elongated T = 4 icosahedral shape. The hexamers in T = 4 structures are significantly distorted owing to bending at the twofold axes that bisect them and form the

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edges of the icosahedral head. These bent hexamers may only be able to cross-link into trimers, whereas the nondistorted hexamers around the long axis of the capsid are able to form hexamers. The geometry of phage c2 is not known, so the proposed elongated T = 4 geometry is possible. Protein chain mail may also exist in other macromolecular systems where large-scale cross-linking occurs. The flagellar hook protein of the spirochete Teponema phagedenis has been shown to form a ladder of cross-linked oligomers i n uiuo (Limberger et al., 1994). The form of the cross-links in the T. phagedenis protein has not been examined in enough detail t o ascertain whether circular oligomers exist or whether the proteins are catenated, but the large size of the complexes suggests a chain mail-like topology as a plausible explanation for the pattern observed. The large coiled ribbon-like protein structures of bacterial R-bodies, found in the Caedibacter symbionts of Paramecia and some pseudomonads have a highly cross-linked structure that could be composed of small oligomers linked into a type of protein chain mail (Heruth et al., 1994;Pond et al., 1989).The large multimeric serum glycoprotein, von Willebrand factor, has a large number of intrachain disulfide bonds (Marti et al., 1987).The dimers that form the larger oligomers are also linked into large, biologically active multimers by additional disulfide bonds (Dong et al., 1994). It is possible that the large oligomers are not directly bonded into covalent chains, but are indirectly linked by topological interlocking of disulfide-bridged loops. Circles of nucleic acid are used to build a different kind of biological chain mail from the kinetoplast DNA of trypanosomid parasites, where planar networks of topologically linked DNA have been clearly demonstrated (Chen et al., 1995). There are many examples of covalent cross-linking via glutamineto-lysine cross-links catalyzed by transglutaminases; these occur in connective tissue proteins (Simon and Green, 19881, in blood-clotting reactions (Chen and Doolittle, 19711, and elsewhere (Folk, 1980; Wold and Moldave, 1984). The transglutaminases use an activated thiol ester as part of their reaction center (Greenberg et al., 1991). We hypothesized that the single cysteine residue in HK97 gp5 might perform a similar function, but HK97 cross-linking proceeds when this residue is changed to serine (Duda et al., 1995b). There do not seem to be any other examples of the lysine-to-asparagine link we have identified in HK97, although a variety of unexpected covalent cross-links have been identified in proteins, including, for example, the lysine-to-tyrosine link found in a lysyl oxidase (Wang et al., 1996). The putative h protease gpC also participates in a covalent cross-linking or fusion reaction

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during A capsid maturation (Hendrix and Casjens, 1974). Each gpC polypeptide is cleaved and fused with a gpE polypeptide to generate the X1 and X2 fusiodcleavage proteins that are found in mature capsids (Hendrix and Casjens, 1975). The mechanism and function of the A fusiodcleavage reaction are still not understood (but see Murialdo and Ray, 1975).

G. Possible Enhancement of Capsid Stability by Chain Mail Interlinking Phages T4 and A each add “decoration proteins” to their outer surface after expansion to stabilize their mature capsids. The T4 gpsoc protein adds at the local threefold positions on the capsids surface whereas T4 gphoc adds to the centers of hexamers, and both stabilize the resulting structure (Ross et al., 1985; Steven et al., 1992). The A gpD protein adds at the local threefold positions on the completed capsid, and the addition significantly stabilizes the capsid (Imber et al., 1980;Sternberg and Weisberg, 1977). The name protein chain mail that we have assigned to the network of catenated HK97 capsid proteins implies that this material acts as a sort of armor for the phage. This reasonable suggestion may indeed be true, but no proof is available. It is not hard to imagine that a highly interlocked fabric of protein could be stable to chemical stress. If the chain mail even slightly increased the resistance of HK97 to destructive environmental stress, it might lead to a significant selective advantage. Few experiments address this issue and none of them definitively.

H. Interdigitating Proteins: A Theme in Viral Assembly The intimate wrapping of one polypeptide or domain around another is a common feature of protein structure. In the E. coli trp repressor protein dimer, large domains of the two chains clasp each other as in a hand-shake (Schevitz et al., 1985).The interdigitation of polypeptide strands is especially common in other virus structures. In tomato bushy stunt virus, extended chains form a /3 annulus that ties the structure together (Olson et al., 1983). The basic dimer unit of the bacteriophage MS2 capsid has an interlocked structure (Valegard et al., 1990). The adenovirus hexon also has interdigitating subunits (Roberts et al., 1986).The high-resolution structure of simian virus 40 (SV40)revealed that the capsomers of this virus were held together almost entirely by interdigitating polypeptide strands that cross capsomer boundaries t o

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participate in secondary structure elements with subunits from different capsomers (Liddington et al., 1991). Parts of the carboxyl-terminal strand of cowpea chlorotic mottle virus also interpenetrate to hold capsomers together (Speir et al., 1995). In many of the protein structures described above, polypeptides often extensively fold or wrap about other chains, in a manner we infer for the HK97 head proteins as they exist in protein chain mail. The frequent appearance of interdigitating strands in viral structure implies that this may be a common means to organize large complex structures from a collection of subunits. It remains to be seen what level of intersubunit interaction occurs in HK97. VIII. QUESTIONS ABOUT PHAGE HEADEXPANSION It has been suggested that one function for expansion is to increase the volume available for DNA packaging, but why not build a capsid of the correct volume to begin with? If the function of this capsid transformation (expansion) is to create a more stable, stronger container, why does the conformational change have t o be so extensive and why does the size increase? Why does an expanded capsid invariably become thinner, with less apparent surface area of contact between subunits, if one of the goals of expansion is to become more stable? A simplistic model might predict that a shell of the correct size could be built and then altered by conformational changes to give increased contacts and greater stability. The observation that all of these evolutionarily distant proteins from a variety of bacteriophages undergo a common transformation suggests that there must be a common basis for the events. The presence of chain mail in HK97 capsids implies that during expansion segments of the capsid protein from adjacent capsomers become intertwined and interlocked. In HK97 the additional intracapsomer subunit crosslinking makes the interlocking obvious because it allows aspects of the protein topology to be deduced from easy electrophoresis experiments, instead of having to wait for the difficult solution of the high-resolution X-ray crystallographic structure. We postulate that this same sort of intertwining and interlocking may occur in other expanding phage capsids and be the fundamental reason for the shared transformation. Perhaps the thinner shell of the expanded capsids is stronger because it has an interlocking, intertwined interface instead of simple contact surfaces to bind the structure together. Why would a conformational transformation that resulted in the interlocking of subunits be manifested as a cooperative or concerted

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expansion reaction? A major reason that expansion and not just conformational change occurs might be that expansion decreases the volume of cytoplasm that needs to be excluded from the interior and decreases the mass of scaffold protein needed to accomplish this. A more fundamental reason may lie in the nature of the change and a requirement that the entire structure be built first so that the change can take place in all subunits rapidly and cooperatively. Giant elongated proheads of T4 have been “caught in the act” of cleavage and expansion and reveal zones of three states separated in space along a single particle (Steven and Carrascosa, 1979). The transformations seem to be propagated in waves along the long axis of these structures and appear to be rapid, because the frequency of partially transformed particles was low. The transition zones between unexpanded and expanded states in the giant capsids were narrow, also suggesting a rapid and cooperative change. These results and conclusions should be applicable to HK97 and to capsid expansion reactions in general. The HK97 expansion is rapid (less than 1min in uitro; Dudaet al., 1995a)and apparently cooperative. The formation of a topologically knotted or intertwined protein structure must, by its very nature, pass through a (partly unfolded?) state in which the two strings forming a knot are free in order for them to wrap around each other. Such a partly unfolded state would likely have a short half-life and be susceptible to decay or side reactions if one partner required for the interactions were missing, thus requiring that all of the subunits destined to be in the final structure be present before expansion. This argument provides a rational explanation for why a prohead must have all subunits in place before expansion; all of the dancers must find their partners and positions before the dance can begin. The changes that take place during the expansion transformation may be akin to “domain swapping,” a mechanism proposed for the formation of oligomeric proteins from monomers (Bennett et al., 1994, 1995). Expansion seems to be a poor name for the maturation transformation of phage proheads, in which expansion itself does not seem to be the goal of the process, but is the most easily observable result. Phage 429 is one example in which the overall size of the DNA-filled head is apparently not significantly different than the size of the prohead. The 429 prohead with internal scaffold protein is more rounded than the angular, flat-bottomed mature capsid. Herpes simplex virus (HSV) may not seem related, but the HSV has a procapsid precursor that undergoes a transformation analogous to that of the phage proheads, but without significant expansion in size (Newcomb et al., 1996; Trus et al., 1996). The HSV procapsid, like many phage proheads, contains an assembled

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scaffold with protease and has a rounded appearance and rather porous shell, in which the capsomers are distorted and have little contact except via the bridging of triplex proteins (Trus et al., 1996). After activation of proteolysis and removal of the scaffold, the procapsid undergoes the transformation into the mature angular capsid with few penetrating holes and highly interconnected capsomers.

IX. CONCLUSIONS When biologists first began to think about how a structure like a bacteriophage virion might assemble from its macromolecular parts, it seemed to be mostly a problem of mustering enough specific bonding interactions among all of the participating components and giving them time to find one another and stick together. The individual proteins were generally seen as rigid and unchanging objects, much like the bricks with which one might assemble a house. Our current view, as described in this article, is a much more dynamic one, especially at the level of the conformational flexibility of individual proteins. Considering only the structural transitions we have characterized in HK97 head shell assembly, the conformational versatility we can ascribe to the head subunit is already quite remarkable. The subunit initially assembles alternatively into two kinds of oligomers, pentamers and hexamers, and those assemble into shells. At some point during that process the hexamers become dramatically skewed, presumably requiring additional, substantially different conformations and intersubunit interactions. When shell assembly is complete, the protease is activated-we might imagine through a conformational signal from the head subunit-and the protease proceeds to digest the N-terminal region of the head subunits, as well as itself. The result of the digestion is not only to change the size of the head protein but also to cause changes in the strength of intersubunit interactions in the shell. Next comes the conformational change of expansion, which completely remodels the shape of the subunits and their interactions with their neighbors. Finally, each subunit makes covalent cross-links to two of its neighbors. We hope it is clear to the reader why we think of head assembly as more analogous to a ballet than to a masonry project. At this point in the analysis most of the effort in the HK97 studies has been directed at describing the choreography-what are the structures in the pathway and what is the nature of the transitions between them? Questions relating to the functional role of each of the steps-for example, why is it necessary to assemble the protein in one conforma-

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tion and then convert it in place t o a different final conformation (rather than simply assembling the protein in the final conformation directly)?-are answered rather more speculatively at this point. It is something of a matter of faith that when enough has been learned about HK97 head assembly, each of the steps will be understood not only with regard to what motions the molecules are undergoing, but also with regard to how each step contributes to steps that follow, and how each step depends on what has gone before-in short, how all steps of the pathway are coordinated to efficiently produce the final product, the phage head. In addition to studying HK97 directly, an important route to understanding the functional significance of the assembly pathway and its various parts is to make comparisons with other phage assembly systems, as we have attempted to do here. Although it is becoming increasingly clear that the structural genes of the dsDNA phages share common ancestry, there are many differences in detail embedded in the overall similarity of their assembly pathways. Comparisons of different phages at these points, where a common function is accomplished by different mechanisms, can allow inferences about the real significance of those steps to the overall assembly process; such inferences would often be difficult or impossible to make on the basis of information from one phage alone. Questions relating to the role of scaffolds or the role of protein processing during assembly are good examples of this. The studies of HK97, as for other phages, have benefitted enormously from combining information from genetic and biochemical studies with structural information. Thus the cryo-EM reconstructions shown here begin to give a tangible reality to the changes in subunit shape and interaction that constitute the steps of the ballet. Because none of the available structures yet extend to atomic resolution, we are not yet able to describe the actions of these proteins at that level of detail, However, such high-resolution structures are just over the horizon for HK97 and some of the other dsDNA phages, and we can look forward to increasingly detailed and complete appreciation of the phage assembly ballet in the near future.

ACKNOWLEDGMENTS We thank our colleagues and collaborators for many fruitful discussions on these topics, particularly Alasdair Steven, Sherwood Casjens, and Jack Johnson and their research groups. We especially thank James Conway for providing the cryo-EM reconstruction images shown here and Sherwood Casjens for supplying unpublished results. Work in our laboratory is supported by NIH Grant GM47795.

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