Control of Virus Assembly: HK97 “Whiffleball” Mutant Capsids Without Pentons

doi:10.1016/j.jmb.2005.02.045

J. Mol. Biol. (2005) 348, 167–182

Control of Virus Assembly: HK97 “Whiffleball” Mutant Capsids Without Pentons Yiyong Li1, James F. Conway2, Naiqian Cheng3, Alasdair C. Steven3 Roger W. Hendrix1 and Robert L. Duda1* 1

Department of Biological Sciences, University of Pittsburgh, PA 15260, USA 2

Institut de Biologie Structurale J-P Ebel, Grenoble, France 3

Laboratory of Structural Biology, NIAMS, National Institutes of Health, Bethesda MD 20892, USA

The capsid of Escherichia coli bacteriophage HK97 assembles as a 420 subunit icosahedral shell called Prohead I which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature Head II shell. Prohead I have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. We report here the properties of a mutant of the capsid protein, E219K, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wildtype Prohead I, when the mutant structure is dissociated into capsomers in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into “Whiffleballs”, particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer. q 2005 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: bacteriophage; HK97; capsid structure; virus assembly; subunit oligomerization

Introduction Viruses with spherical capsids can have as few as 60 protein subunits, clustered as 12 pentamers and arranged with icosahedral symmetry. A few viruses such as plant satellite viruses1 and the parvoviruses2 do use 60-subunit, strictly equivalently bonded capsids, but most viruses have genomes far too big to fit into the diminutive capsids that are possible with only 60 subunits. In the Caspar and Present address: Y. Li, 35 Millwood Drive, Shrewsbury, MA 01545, USA. Abbreviations used: TCA, trichloroacetic acid; cryoEM, cryo-electron microscopy; IPTG, isopropyl-b-Dthiogalactopyranoside. E-mail address of the corresponding author: [email protected]

Klug strategy of quasi-equivalence3 a single capsid protein can be sufficiently flexible in shape or flexible in the manner of its interactions to allow the same protein to assemble into hexamers that are nearly equivalent to pentamers in internal bonding, and that form hexamer–pentamer and hexamer– hexamer interactions that are nearly equivalent to each other. Such quasi-equivalent interactions allow for hexamers and pentamers to form capsids that are larger than those built from pentamers alone, but regular closed particles can only be made in discrete sizes, constrained to those described by Triangulation numbers (T numbers) TZ1, 3, 4, 7, 9, 12, 13, etc.3, where the number of pentamers is fixed at 12, and the number of hexamers is (TK1)!10. It is clear that some spherical viruses use variations on this strategy to assemble larger capsids; for example, T44,5 and Adenovirus6,7 use

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

168 distinct proteins to constitute the hexamers and pentamers, while papovaviruses assemble pentamers at both the 5-coordinated and 6-coordinated lattice sites. However, there are a considerable number of viruses that employ hexamer–pentamer clustering in capsid construction. These include double-stranded DNA bacteriophages (TZ4, TZ7, TZ13 and perhaps other T-numbers), and the herpesviruses (TZ16). For these viruses, the question arises: how is a particular T number specified, with the attendant specification of the numbers of hexamers and pentamers and their correct spatial arrangements? Many of them, including the herpesviruses and the phages T4, T7, l, P22, SPP1, and f29, employ an assembly co-factor known as a scaffolding protein to facilitate correct assembly. The scaffolding protein assembles into a core inside the procapsid shell during shell assembly but is removed from the structure during subsequent maturation,8–10 sometimes by proteolysis. In the absence of scaffolding protein, the major capsid protein assembles into abnormal structures which range in shape from long tubes (polyheads: T45,11; T712,13) and unclosed curved particles (monsters: P2214; l15,16; f2917) to smaller capsids (P2214). Besides its crucial role in vivo, scaffolding protein is also required for correct capsid shell assembly under in vitro conditions where this has been tested.18–20 While scaffolding proteins play a crucial role in capsid assembly, the major capsid proteins themselves are of course important, and missense mutants in capsid protein genes also influence assembly and capsid size, as expected and demonstrated, for example, in T421,22 and l.23,24 Despite this detailed knowledge, the exact mechanism for T number and capsid size determination for the tailed dsDNA bacteriophages is still poorly understood. Bacteriophage HK97 has a TZ7 capsid25 with subunits of a single major capsid protein forming both hexons and pentons. 25,26 HK97 capsid assembly has been characterized genetically and biochemically.27,28 The pathway for HK97 capsid assembly is outlined in Figure 1(a). Assembly requires three protein gene products: gp3, the portal protein that forms the vertex through which DNA is packaged and to which a tail is attached, gp4, the maturation protease, and gp5, the major capsid protein. Assembly begins when a dodecamer of gp3 and about 60 copies of gp4 coassemble with 60 hexamers and 11 pentamers of gp5 to form a transient, round Prohead I inside which the protease cleaves each gp5 (42 kDa) to gp5* (31 kDa) and itself to peptides, forming Prohead II.26 Prohead II remains round, but is thinned by cleavage of internal peptides, which escape.25 DNA packaging induces Prohead II to mature by changing conformation, expanding in size and forming active sites that cross-link gp5 subunits to each other to form a stable network in Head II, the mature angular capsid.25,27,29,30,31 In the laboratory, HK97 procapsids can be assembled without portals in vivo by expressing the major capsid protein gp5 to form Prohead I, or

HK97 “Whiffleball” Capsids without Pentons

together with the protease gp4 to produce Prohead II composed of cleaved capsid protein gp5*.27 Thus, HK97 does not require a separate scaffolding protein for correct assembly. Instead, a disposable part of the major capsid protein, the N-terminal 102 amino acid residues or delta domain, is thought to play the role of a scaffolding protein that is removed proteolytically by gp4 after assembly is complete.25 The proteolytic cleavage does not occur unless gp4 and gp5 co-assemble into Prohead I. A crystal structure has been determined for Head II30,32 and the ˚ cryoEM Head II X-ray model has been fit into a 12 A 33 structure of Prohead II in large part by rigid body rotations of the core of the subunits. Prohead II and Prohead I are quite similar in structure (except of course for the missing delta domains),25 so the HK97 system offers the potential for relating biochemical and structural aspects of assembly and maturation at a quite detailed level. Hexamers and pentamers of gp5 accumulate when an assembly-defective missense mutant in gene 5 is expressed (mutant N356D27), suggesting HK97 capsids assemble from a mixture of hexamers and pentamers, instead of from monomers, as has been shown for P22.18 Indeed, HK97 procapsids can be assembled in vitro from hexamers and pentamers of gp5.34 The gp5 hexamers and pentamers (or capsomers, collectively) used for the assembly experiments were produced by dissociation of Prohead I. Striking findings of the in vitro assembly experiments were that the yield was greatest when the ratio of pentamers to hexamers matched the ratio for TZ7 particles (12 : 60) and that only normal-sized particles were made; no smaller or larger particles were found, even when the ratio of hexamers to pentamers was unbalanced. These experiments imply that assembly of procapsids under these conditions requires both hexamers and pentamers and that their structures and their interactions together choreograph correct assembly. In the course of attempting to further dissect how HK97 capsomers assemble into proheads, as well as other aspects of capsid assembly and maturation, we have isolated single amino acid mutants of gp5 and surveyed their phenotypic effects. One of these mutants, E219K, appeared to have properties that could illuminate capsid assembly. In particular, assays for capsid protein complexes suggested that E219K procapsids might assemble without pentamers, which was unexpected in light of known features of the HK97 capsid assembly pathway. Glutamate 219, the locus of the mutation, is almost entirely buried in the interior of the gp5 subunit, and points towards the interior of the capsid in the X-ray structure of HK97 Head II.32 Here we report a detailed examination of the E219K phenotype. Figure 1(b) summarizes the in vitro experiments presented below and both Figure 1(a) and (b) are annotated to point out some of the biochemical properties of HK97 proheads and other particles examined as an aid to the reader.

HK97 “Whiffleball” Capsids without Pentons

169

Figure 1. HK97 capsid assembly pathway and a schematc of in vitro experiments. (a) The HK97 capsid assembles from the major head protein, gp5 (42 kDa) and minor proteins gp3 (47 kDa portal protein), and gp4 (25 kDa maturation protease). The initial steps require the host chaperones GroEL and GroES, and ATP. Prohead I is the first large intermediate and converts to Prohead II when gp5 is cleaved from 42 kDa to 31 kDa under the control of gp4, which is incorporated into Prohead I during assembly. The N-terminal portion of gp5 that is removed by proteolysis from the interior of Prohead I is called the delta domain. Prohead II sediments more slowly than Prohead I due to the reduced mass. Prohead II expands into Head II, at the time of DNA packaging. During expansion, reactive sites are created that cross-link each of the gp5 subunits to adjacent subunits. The dodecameric portal protein complex is a grommet-like structure located on one vertex and forms the site of DNA packaging and tail attachment. HK97 proheads and heads lacking portals have been studied extensively and are produced when only gp5 or gp4 and gp5 are expressed from plasmids in bacteria. (b) Graphical summary of the flow of materials for the in vitro dis-assembly and re-assembly experiments starting with Prohead I made from wild-type gp5 or E219K mutant gp5. Relevant properties of various particles, such as gel mobility, are noted in the Figure and are further described in the the text. PI, Prohead I.

Results E219K anomaly: only one type of capsomer We examined the basic assembly properties of the E219K mutant capsid protein in cell extracts using three gel systems for which the behavior of the wild-type protein is well characterized.26,27,34 SDS gels were used to monitor expression and processing, while non-denaturing gels were used to diagnose the presence of assembled protein complexes, such as capsomers and proheads. Capsid assembly was driven using a plasmid that normally produces HK97 Prohead II from wild-type capsid proteins and the HK97 proteins were preferentially radio-labeled after host protein synthesis was inhibited, so that only HK97 proteins were visualized on gel autoradiograms. The E219K

mutant protein was expressed together with the wild-type protease and compared to controls on three gels. Figure 2(a) shows an SDS-PAGE gel of trichloroacetic acid (TCA) precipitated whole cells; the mutant protein appears predominantly as a 42 kDa gp5 band, but there is also a band at the position of the 31 kDa gp5* (proteolytically processed) form of the protein. Since gp5 cleavage depends on assembly of Prohead I,27 this result shows that the mutant protein is competent to assemble, and to co-assemble with protease. However, the relative amount of gp5* in the mutant is substantially less than in the wild-type control, arguing that the mutant is kinetically deficient in assembly or processing or both. Induced radio-labeled cells were lysed and Figure 2(b) compares the E219K mutant extract to control extracts on a non-denaturing agarose gel.

170

HK97 “Whiffleball” Capsids without Pentons

Figure 2. Gel analysis of radiolabeled E219K lysates: no pentamers detected. Cultures of host BL21(DE3) containing expression plasmids derived from pT7Hd2.9 (or pV0) were selectively radiolabeled, as described in Materials and Methods so that mainly the HK97 proteins are seen in autoradiograms. Three plasmids were compared: the wild-type plasmid (pV0) makes both gp5 and gp4, the ProteaseK plasmid (pVB) makes only gp5, and the E219K plasmid (pV0-E219K) makes the E219K mutant gp5 and wild-type gp4. (a) Cultures were directly precipitated with TCA, denatured and run in an SDS polyacrylamide gel. (b) Lysates were prepared as described in Materials and Methods and run in a non-denaturing 1% agarose gel. (c) Lysates were run in a nondenaturing polyacrylamide gel to reveal hexamer and pentamer capsomer bands. Note that only one band was found for the E219K sample and it runs at the position of wild-type hexamers.

The mutant had a strong band in the capsomer position (hexamers and pentamers are not resolved), running slightly slower than the wildtype capsomer band, possibly because of the charge difference in the mutant. In the part of the gel where we expected proheads and heads, there were two bands. The faint faster band is E219K Prohead II, which runs at the same position as wild-type Prohead II (confirmed below). The more abundant slower band migrates at the position of wild-type Head II, but the amount of assembly and processing of gp5 to gp5* in the mutant appeared insufficient to produce that much Head II, since the bulk of the radiolabel is in the 42 kDa form, suggesting that Prohead I and capsomers should be the major forms found in the lysate. Indeed, upon further examination, the band at the wild-type head position was found actually to be E219K mutant Prohead I, which we determined by combining agarose gel and velocity sedimentation analysis as described in the next section. The combined analysis revealed that E219K Prohead I and Prohead II have very different mobilities in agarose gels, in contrast to wild-type Prohead I and Prohead II, which have nearly the same mobility as each other, and as E219K Prohead II. The most unexpected result in these initial characterizations is shown in Figure 2(c). This is a non-denaturing PAGE gel, which separates wildtype gp5 hexamers and pentamers cleanly. In this gel the E219K mutant shows only a single band, running nearly identically to the wild-type hexamer, in contrast to the two capsomer bands that are normally found in such gels for any HK97 lysates, as can be seen for the wild-type or ProteaseK control lanes in the same gel. We characterize these mutant capsomers in more detail below.

E219K mutant Prohead I and Prohead II assembled in vivo have both hexons and pentons The discovery that the E219K mutant displayed only one type of capsomer by native gel analysis showed that capsomers of E219K protein have unusual properties and further raised the possibility that the E219K Prohead I-like particles we observed might actually be proheads that had assembled without pentons. This was unexpected because all HK97 capsids or procapsids examined so far have pentons, clearly visible in 3D reconstructions of HK97 particles.25,31,33 Also, as noted above, assembly of Prohead I appears to require both hexamers and pentamers.34 To test whether the particles in E219K lysates were Prohead I and Prohead II and also whether they contained pentamers, we examined their sedimentation velocity in glycerol gradients. If the E219K mutant made proheads without pentamers they should sediment more slowly than their wild-type counterparts, because they should have only 86% (360/420) of the mass of wild-type particles. A lysate of cells induced to produce E219K proheads was sedimented in glycerol gradients in parallel with wildtype Prohead I and Prohead II, shown in Figure 3. An agarose gel of the E219K gradient fractions (Figure 3(a)) shows two peaks resolved as bands with different gel mobilities that co-sediment with wild-type Prohead I and Prohead II, suggesting that both mutant particles have the same masses as the corresponding wild-type particles and so indeed have pentamers. An SDS polyacrylamide gel of the same region of the E219K lysate gradient (Figure 3(b)) shows that sizes of the proteins in the two peaks correspond to those expected for Prohead I (42 kDa) and Prohead II (31 kDa). These results also demonstrate that the slow migrating

HK97 “Whiffleball” Capsids without Pentons

171 linking (not shown). This allowed us to carry out an additional test for the presence of pentons. E219K Prohead II was purified, converted to Head II in vitro and tested for the presence of covalent circular protein hexamers. As described in detail elsewhere,30,31 the peculiar geometry of chainmail cross-linking dictates that every such hexameric covalent circle contains one subunit derived from a pentamer of gp5*. The assay, done in parallel with wild-type Head II, uses partial proteolysis to release oligomers from large chainmail complexes in Head II and showed that E219K Head II clearly had circular hexameric gp5* oligomers and therefore contained pentons (data not shown). This result is corroborated by the structural studies of E219K Prohead I described below. Capsomers from E219K Prohead I are hexamers

Figure 3. Sedimentation properties of E219K proheads. Wild-type Prohead I (PI), Prohead II (PII) and E219K Proheads were expressed from plasmid pVK, pV0 and pV0-E219K, respectively in host BL21(DE3)pLysS. Cells were lysed and cell debris removed by centrifugation. Crude lysates were loaded onto 10–30% glycerol gradients and centrifuged at 35,000 rpm in a Beckman SW41 rotor for 100 minutes at 4 8C. Fractions were collected by drop from the bottom of each tube. (a) Equal volumes of the fractions were subjected to electrophoresis on a 0.8% agarose gel. (b) Fractions corresponding to proheads from the E219K lysate (fractions 4–10) were precipitated with TCA, resuspended in SDS sample buffer, and run in a 12% SDS-polyacrylamide gel. (c) Fractions from the E219K lysate gradient were electrophoresed in a 7.5% native polyacrylamide gel.

E219K prohead band in agarose gels is correctly identified as Prohead I and the faster band as Prohead II. E219K Prohead I migrates much slower than wild-type Prohead I and E219K Prohead II, but the reason remains unclear. A native polyacrylamide gel confirmed the existence of the one hexamer-like capsomer band derived from fractions containing E219K Prohead I (Figure 3(c)); this band appears only in fractions that contain E219K Prohead I, since Prohead II is too big to enter the gel and, unlike Prohead I does not partially dissociate into capsomers under these conditions. E219K Proheads were also found to have the ability to continue through the head maturation pathway in vitro and catalyze intersubunit cross-

In order to further characterize the E219K capsomers we added a protease (gp4) knock-out mutation to the E219K expression plasmid so that it exclusively makes Prohead I. E219K Prohead I was purified and samples were dissociated into capsomers using 30% (w/v) glucose in 20 mM Tris–HCl (pH w9.5), conditions that effectively dissociate wild-type Prohead I. Wild-type and mutant particles were treated and both were successfully dissociated into capsomers as shown by the conversion of a fast migrating sharp prohead band to a slower migrating diffuse capsomer band or spot in an agarose gel (Figure 4(a)). All clues to the identity of the E219K capsomer band suggested that it is composed of hexamers, but migration of native protein complexes in gels is sensitive to both size and surface charge. Since the E219K protein nominally has a C2 charge difference from wild-type gp5, we could not rule out the possibility that the E219K capsomer band contains pentamers that migrate like wild-type hexamers because of altered surface charge properties. In order to discover which oligomer was present, we used Ferguson plot analysis, an electrophoretic method that can distinguish between the effects of size and charge on electrophoretic mobility of proteins.35,36 In this technique, the log of the relative migration (Rf) of individual protein complexes is plotted against the gel concentration (T); the data are fit to a line for each protein complex and the slopes and intercepts are compared. The gel data and Ferguson plot for wildtype and E219K capsomers are shown in Figure 4(b) and (c). If two species have identical charge density but different size then the relative migration will depend strictly on size and the plots will have different slopes and intersect at TZ0%. This is true for the wild-type hexamer and pentamer data in Figure 4: the lines do intersect near zero. If two species have identical size but different charge then increasing the gel concentration does not affect the relative migration of the two species and the plot gives parallel lines. This second condition is true for wild-type hexamers and E219K capsomers showing

172

HK97 “Whiffleball” Capsids without Pentons

obvious precipitate when we do dissociation reactions, even on a large scale. We do not believe that the mutant protein converts to monomers because HK97 gp5 appears to be an obligate oligomer.34 Soluble free monomers of HK97 gp5 are not stable, except under denaturing conditions; denatured monomers that bind to the GroEL chaperonin assemble into hexamers and pentamers when released.34 Wild-type hexamers and pentamers also have the unusual property of being interconvertible under special solvent conditions (in magic pentamer buffer or magic hexamer buffer).34 We took advantage of these special hexamer and pentamer promoting buffer conditions to ask if we could force the E219K hexamers to convert to pentamers. We put the E219K capsomers into the magic hexamer or pentamer buffers and compared the results with those obtained for a wild-type capsomer mixture (Figure 5(a)). Both wild-type and E219K capsomers showed essentially a single band at the positions of wild-type hexamers in magic hexamer buffer (compare lanes 2 and 7). When both

Figure 4. E219K capsomers from Prohead I are exclusively hexamers. (a) Wild-type and E219K Prohead I were prepared using plasmids pVK and pVK-E219K, respectively as described in Materials and Methods. Prohead I samples were incubated in 35% glucose, 15 mM Tris–HCl (pH 9.5) at room-temperature overnight to convert Prohead I into capsomers and then dialyzed against 10 mM Tris–HCl (pH 8.0) overnight. Samples of wild-type and E219K before (PI) and after (Cap) treatment were run in a 0.8% agarose gel. (b) Gel data for Ferguson plot analysis. Purified E219K and wild-type capsomers were subjected to native PAGE at a series of gel concentrations as indicated. Electrophoresis was performed at w90 V until the dye front just reached the bottom edge. (c) Ferguson plots of E219K capsomers, wild-type hexamer and pentamer. Relative mobilities were measured from (b) using the distances between the bands and the upper edge of the separating gel, divided by those between the front edges and the upper edges, respectively. Data fit to straight lines on a semilogarithmic graph.

that the E219K capsomers are indeed hexamers. There is a very small but consistent difference in the Rf of these two species, which implies a small difference in effective charge. We do not know the fate of pentamers that were present in the in vivo assembled E219K Prohead I following dissociation. The pentamers could convert into hexamers, a real possibility, since wild-type gp5 can do this (see below), or even precipitate, although we have not observed an

Figure 5. E219K hexamers cannot convert to stable, soluble pentamers. (a) E219K hexamers failed to convert to pentamers using solvent conditions that strongly favor pentamers for wild-type capsomers, but remained stable under hexamer-promoting conditions. Wild-type and E219K capsomers (0.5 mg/ml final) were treated with 0.5 M NaSCN in 5 mM Tris–HCl (pH 7.5), “magic hexamer buffer”, at room-temperature overnight for pentamer-to-hexamer conversion (5/6); or with 0.2 M GuHCl and 1% DMF, “magic pentamer buffer”, at the indicated pH (20 mM NaOAc, pH 5.2, and 20 mM Tris– HCl, pH 6.8 or 7.5) for hexamer-to-pentamer conversion (6/5)34. The reactions were resolved on 7.5% native PAGE. 5-mer, pentamer; 6-mer, hexamer. (b) E219K capsomers precipitate under pentamer-promoting conditions. Samples 2–5 and 7–10 from (a) were centrifuged at 16,000 g for ten minutes. The supernatant was removed, the pellet suspended in water, and both were TCA precipitated, dissolved in SDS sample buffer, denatured and run in SDS polyacrylamide gels. The gp5 (42 kDa) regions of the gels are shown.

HK97 “Whiffleball” Capsids without Pentons

173

types of capsomers were put under magic pentamer buffer conditions at pH 7.5, 6.8 and 5.2, the wildtype capsomers converted to pentamers with increasing efficiency as the pH was lowered, but the E219K capsomers never converted to a band that migrated faster, as would be expected if they could convert to pentamers. Instead, the E219K protein remained as hexamers, but became less soluble after treatment in magic pentamer buffer: a fraction precipitated when the pH was near neutral, and all of the E219K protein precipitated at pH 5.2 (Figure 5(b)). The nature of this precipitate has not been investigated further. Wild-type pentamers remained mostly soluble under all conditions tested in the experiment. E219K capsomers re-assemble into Whiffleballs: proheads without pentons Wild-type capsomers reassemble into Prohead I in reactions containing Mg2C or Ca2C and w6% polyethylene glycol,34 but the E219K capsomers did not assemble efficiently under similar conditions. However, purified E219K hexamers did reassemble efficiently into Prohead I-like particles when they were concentrated by precipitation with ammonium sulfate and resuspended in phosphate buffer (Figure 6(a)). (Wild-type capsomers assemble under these conditions, but less efficiently (data not shown).) Reassembly of E219K capsomers was found to be highly concentration-dependent (data not shown). The reassembled particles co-migrate with E219K Prohead I in the agarose gel, significantly slower than wild-type Prohead I. Since we presumed that no pentamers were present, we suspected that these reassembled particles might lack pentons, so we compared their sedimentation properties to those of E219K Prohead I particles with pentons and wild-type Prohead I and Prohead II as shown in Figure 6(b) and (c). The reassembled particles sedimented significantly slower than wildtype and E219K Prohead I, to a position in the gradient midway between Prohead I and Prohead II. This is the position expected for particles with uncleaved (42 kDa) subunits but lacking pentons, since they are predicted to have a mass very nearly midway between that of Prohead I and Prohead II. Negative stain electron microscopy showed that the reassembled particles resembled wild-type and E219K Prohead I, but had more stain penetration and appeared to have gaps in the shell outline as would be expected for penton-less Prohead I (data not shown). We named the particles “Whiffleballs” because they are conceptually similar to the plastic Wiffleballw baseball toy that also has distinctive holes. Cryo-EM reveals that Whiffleballs lack pentons but are otherwise indistinguishable from Prohead I E219K Prohead I and the reassembled particles were examined by cryo-electron microscopy

Figure 6. E219K hexamers reassemble into particles that sediment between Prohead I and Prohead II. (a) Purified E219K hexamers were forced to reassemble by concentrating the protein using ammonium sulfate precipitation. The pelleted hexamers were resuspended in PBS to a high concentration (w35 mg/ml) followed by dialysis against the same buffer. The reassembled product (labeled WB) was compared to the starting material, capsomers (Caps), E219K Prohead I (PI) and wild-type Prohead I on a 0.8% agarose gel. (b) Reassembled E219K “Prohead I” made in vitro (upper panel) or E219K Prohead I made in vivo (lower panel) were added to a mixture of wild-type Prohead I and Prohead II (PII), and sedimented into a 10–30% glycerol gradient at 40,000 rpm for two hours at 4 8C in a Beckman SW41 rotor. Fractions were collected by drop from the bottoms of the tubes, fractions 3–15 were separated on a 0.8% agarose gel, fixed and stained with Coomassie Blue. (c) The stained gel was scanned with Zeineh Scanning Densitometer SLR-2D/1D (Biomed Instruments, Chicago, IL), bands were quantified with NIH Image, and the integated band density was plotted.

(cryo-EM) (Figure 7). Both are of the same size and shape and share the same visual characteristics as wild-type Prohead I.25 However, on closer inspection it became apparent from the presence

174

Figure 7. Cryo-EM reveals that reassembled E219K hexons make Whiffleballs. Cryo-electron micrographs revealed the structures of E219K Prohead I (a), and the reassembled Whiffleball particles (b). Both resemble images of wild-type Prohead I25. Note the peripheral notches (arrows) on reassembled particles in (b). There are also small low-density patches visible on reassembled particles with favorable orientation (compare the two particles indicated with a “5” in the Figure (each has an orientation centered close to a 5-fold particle axis). Both of these features indicate that there are lesions in the Whiffleball surface lattice (confirmed in the cryo-EM reconstruction in Figure 8(d)–(f)) showing 12 vacant vertices). The density maps from Figure 8 were reprojected in appropriate directions, scaled and shown in insets in a and b. The reprojections of Whiffleballs reproduced the notches and low-density patches discernible on the original noisy images (the leftmost reprojections ˚. are oriented on the 5-fold axis). The bar represents 100 A

of peripheral notches (arrows, Figure 7(b)) and small low-density patches (particle labeled 5 in Figure 7(b)) on the projected particles that there are lesions in the Whiffleball surface lattice. This impression was confirmed in a cryo-EM ˚ resolution (Figure 8(d)–(f)) reconstruction at 14 A which showed that its 12 vertices are vacant, i.e. Whiffleballs do indeed lack pentons. When this density map was reprojected in appropriate directions, it reproduced, more clearly, the notches and

HK97 “Whiffleball” Capsids without Pentons

low-density patches that were already discernible on the noisy original images (Figure 7 inset). The three-dimensional structure of the E219K Prohead I was also reconstructed to the same resolution (Figure 8(a)–(c)). This capsid is indistinguishable from wild-type Prohead I as ˚ ˚ resolution25 as well as at 14 A visualized at 25 A and beyond (J.F. Conway et al., unpublished results). The total absence of density at the Whiffleball vertex sites (Figure 8(d)–(f)) indicates that these reassembled particles are completely lacking of pentons. The particle consists of 20 triangular clusters, each of three hexons, and it is held together by pair-wise interactions between the two hexons on the sides of each of two interacting triangles. Everywhere except at the vertices, the Whiffleball resembles the E219K Prohead I closely (cf. Figure 8(d)–(f) and Figure 8(a)–(c)). Nevertheless, to probe for possible subtle differences in the placement and/or the individual structures of the hexons, a difference map was calculated (Figure 8 (g) and (h)). Its noise level was low, albeit, as expected, somewhat higher than those of the two contributing maps (cf. Figure 8 (b), (e) and (h)). We conclude that in Whiffleball assembly, the hexon lattice reproduces that of Prohead I with high fidelity, despite the absence of pentons. The difference map allowed us to define the molecular boundary of the pentons in Prohead I (Figure 8 (g) and (h)). The delta domains, i.e. the amino-terminal extensions of the gp5 subunits, contribute clearly defined structures on the undersides of the capsomers in both maps (arrows in Figure 8(b), (c), (e), (f), (h) and (i)). In Prohead I, they are present under the pentons as well as under the hexons (Figure 8(b) and (c)). Thus there is no reason to suppose that the mutation of residue 219 from E to K results in a major structural change in the delta domain (residues 2–102), which has been likened to the scaffolding proteins of other bacteriophages10,25 and is thought to play an important role in assembly. Whiffleballs can be “capped” by adding wildtype capsomers Wild-type HK97 Proheads normally assemble from a mixture of hexamers and pentamers, but the exact sequence of assembly events is not known, so it was not clear if wild-type pentamers could enter and fill the holes in Whiffleballs where pentons normally sit. We found however that wild-type pentamers will bind to Whiffleballs, even when added as a mixture of hexamers and pentamers. This reaction causes an electrophoretic mobility shift from the slow positions found for Whiffleballs (and E219K Prohead I) to the faster position of wild-type Prohead I as demonstrated in the time-course of a “capping” reaction presented in Figure 9. The source of pentamers for the reaction shown was a 0.3 mg/ml mixture of wild-type capsomers containing an excess of hexamers;

HK97 “Whiffleball” Capsids without Pentons

175

˚ resolution Figure 8. Cryo-EM reconstructions reveal penton-less E219K Whiffleballs. Cryo-EM reconstructions at 14 A of E219K Prohead I (a)– (c), Whiffleballs reassembled from E219K hexamers (d)–(f), and views derived from a difference map between Whiffleballs and E219K Prohead I (g)–(i). The Whiffleballs have holes where the pentons should sit. All panels show views along a 2-fold symmetry axis. (a) (d) and (g) show outside views, and (b), (e), and (h) show central sections of the reconstructions and the difference map in which the highest densities are in black and the lowest densities are in white. Inside views are shown in (c) and (f), and (i) shows a view of the Whiffleball particle with the difference map included, colored blue. The difference map clearly shows the boundaries of the pentons present in E219K Prohead I but ˚. missing in the Whiffleball. The bar represents 100 A

Whiffleballs were at 2 mg/ml. The reaction proceeded more than half-way to completion by ten minutes, even at this modest concentration of capsomers. The reason for the change of mobility of Whiffleballs when wild-type pentamers are added is not clear, but could involve shielding of residues that are exposed in both Whiffleballs and E219K Prohead I and hidden only when a wildtype, but not a mutant pentamer binds. We looked in more detail at the products of the capping reaction by using a [35S]-methionine labeled

capsomer preparation as a radioactive tracer. As controls in the same experiment we looked for incorporation of 35S-labeled capsomers into E219K Prohead I and wild-type Prohead I. Figure 10(a) shows a stained agarose gel of the capsomer incorporation experiment and Figure 10(b) shows the corresponding autoradiograph. The radiolabeled protein was at a low concentration, so two reactions were performed with each sample: one with radio-labeled capsomers alone (Rxn 2, 5, and 7), and one supplemented with a higher

176

HK97 “Whiffleball” Capsids without Pentons

towards that of wild-type Prohead I. Adding excess unlabelled capsomers caused more of the E219K Prohead I to migrate slightly faster, but diluted the specific radioactivity of the incorporated label (Rxn 6). The faster migration of capsomer-labeled E219K Prohead I particles suggests that the mobility shifts are due to exchange of wild-type labeled pentamers with the E219K Prohead I. In contrast, incubation of wild-type Prohead I with radio-labeled capsomers showed no evidence of capsomer exchange at high (Rxn 7) or low (Rxn 8) capsomer concentrations. The clear differences between E219K and wild-type particles in capsomer exchange behavior suggests that E219K particles have weakly bound pentons. In order to show whether it was pentamers or hexamers that were incorporated into Whiffleballs or E219K Prohead I, two of the radio-labeled incorporation reaction products (Rxn 2 and 5) were centrifuged at high speed to pellet the proheads and separate the incorporated label in the pellet from the unincorporated label in the supernatant. The supernatant and pellet fractions were run in a native polyacrylamide gel to separate hexamers and pentamers (Figure 10(c)) and the radio-labeled hexamer and pentamer bands were quantified (Figure 10(d)). The results clearly show that pentamers were preferentially, if not exclusively incorporated into Whiffleballs and into E219K Prohead I. Figure 9. Wild-type capsomers change the electrophoretic mobility of Whiffleballs. (a) Agarose gel of capping reactions. The final concentration of capsomers was 0.3 mg/ml and Whiffleballs at 2 mg/ml; all components were in PBS. Samples were mixed in reverse and staggered order, longest time point first, so that all reactions could end at the same time and immediately be loaded onto the gel. (b) Relative mobility of the gel bands shown in (a).

concentration of unlabeled capsomers (Rxn 3, 6, and 8) to drive the reaction towards completion. Note also that the radio-labeled capsomer preparation contained a small amount of undissociated Prohead I (Rxn 9), which appears as a background band in Rxn 2, 3, 5, 6, 7, 8 and 9 in Figure 10(b). When Whiffleballs were incubated with trace amounts of labeled capsomers the particles clearly became labeled, but their mobility changed only slightly (Rxn 2). When excess capsomers were added to Whiffleball the reaction was driven towards completion and the Proheads increased their mobility to nearly that of wild-type Prohead I (Rxn 3), but some particles had intermediate mobilities. When E219K Prohead I was incubated with trace radio-labeled capsomers, some of the Prohead I became labeled, but the label appeared to be concentrated in the higher mobility material (Rxn 5: compare Figure 10(b) with (a)), suggesting that labeled capsomers were rapidly altering a subset of particles and changing their mobility substantially

Discussion The E219K mutant of the HK97 capsid protein gp5 is partially defective for making infectious phage virions, as judged by a reduced efficiency in complementing a phage with an amber mutation in gene 5 (data not shown). However, the mutant protein is able to support the formation of infections virions at reduced efficiency. Furthermore, the mutant protein appears able to carry out all of the experimentally defined biochemical and morphological steps of capsid assembly and maturation. Thus if the mutant protein is expressed by itself in cells, it assembles into Prohead I structures that are morphologically indistinguishable from the wild-type Prohead I, and if the HK97 maturation protease gp4 is expressed together with the mutant gp5, apparently normal Prohead II shells form. These structures can be matured in vitro, including both the shell expansion and the subunit cross-linking steps, to yield Head II shells that are essentially identical with the corresponding wild-type structures. Because of these extensive functional similarities between the wild-type and mutant proteins, we are confident that the fundamental mechanisms of shell assembly are the same for both, and therefore that it is very likely that the differences the wild-type and mutant proteins display are informative about the properties of their shared capsid assembly and maturation pathway rather than delineating fundamentally different processes.

177

HK97 “Whiffleball” Capsids without Pentons

Unique properties of E219K pentamers

Figure 10. Wild-type pentamers add to Whiffleballs and exchange with E219K pentons. E219K WhiffleBalls, E219K Prohead I, and wild-type Prohead I (all at a final concentration of 1 mg/ml), were mixed with radiolabeled wild-type capsomers (0.1 mg/ml final) and cold wild-type capsomers (0.5 mg/ml final) in PBS, and incubated at room-temperature overnight. Aliquots were electrophoresed on a 0.8% agarose gel. The gel was fixed and stained with Coomassie Blue to visualize total protein in the reactions (a), and autoradiographed to visualized radioactive materials (b). Since radioactive materials co-migrated with proheads in reactions (Rxn) 2, 3, 5 and 6 (a) and (b), proheads in reactions 2 and 5 were pelleted at 65,000 rpm in a Beckman TLA100 rotor for 15 minutes at 4 8C and resuspended in PBS. Both pellets and

Most of the differences we have seen between the wild-type and E219K mutant proteins relate to pentamers. For example, although mutant protein in the bacterial cytoplasm can evidently form pentamers that are successfully assembled into Prohead I, the protein remains as pentamers in the buffer conditions we have employed in vitro only as long as the pentamers are constrained in the Prohead I structure. When mutant Prohead I is disassembled in vitro the hexamers are stable but the pentamers disappear, possibly rearranging to hexamers upon dissociation. In earlier studies with wild-type capsomers we showed that hexamers and pentamers can be interconverted in vitro, with the direction of conversion depending on the composition of the solvent.34 In ordinary aqueous solvents, the wild-type protein converts from pentamer to hexamer, and when 1% dimethyl formamide is added to the solvent the capsomers convert in the opposite direction, from hexamers to pentamers. Conversion in either direction for wild-type protein requires a small amount of denaturant (e.g., 0.2 M Guanidine HCl or 0.5 M NaSCN). We interpreted these data to mean that the pentamers and hexamers are potentially in equilibrium with each other, with the equilibrium point set by the solvent properties, but that the equilibrium cannot be achieved in vitro without adding enough denaturant to lower the energy barrier of the transition. In this context, we suggest that for the E219K mutant protein, as for the wild-type, the hexamer is the energetically favored form in our in vitro buffers, but that the pentamer form is so disfavored energetically under these conditions that it does not require denaturant to breach the energy barrier to hexamer formation, provided the pentamer is released from the constraints of the prohead structure. If we put the mutant hexamers under the solvent conditions that promote conversion of the wild-type protein to pentamers, the protein precipitates, supporting the idea that the pentamerforming conformation of the mutant protein is disfavored in these conditions. The pentamers in the mutant Prohead I structure exhibit aberrant behavior in that context as well. When wild-type capsomers are mixed with mutant Prohead I, the mutant pentamers are selectively replaced with wild-type pentamers, but a similar exchange between added capsomers and wild-type Prohead I is not observed. This reinforces the view that the pentamer form of the mutant protein is not optimally functional, in that it is evidently not able to bind as tightly to the surrounding hexamers of the prohead as do wild-type pentamers. The fact that wild-type pentamers but not wild-type supernatants were electrophoresed in a 7.5% native PAGE and autoradiographed (c). The gel was also scanned (counted) and the bands quantified with an AMBIS Radioanalytic Imager (Ambis, Inc., San Diego, Calif.) (d).

178 hexamers are able to exchange into E219K Prohead I argues that the mutant pentamers are less tightly bound into the structure than the mutant hexamers. While we do not have direct evidence that the same is true for the wild-type proheads, there is evidence from several other icosahedral viruses that the pentamers are labile relative to the hexamers. For example, pentons have been shown to be specifically removable both from capsids that use a special protein to make the pentons (T44,5and Adenovirus6) and from capsids that use the same protein for both hexons and pentons (P2237 and Herpes Simplex Virus38). The differing mobilities in agarose gel electrophoresis of the various prohead forms examined here are also informative about the interaction between the pentamers and the rest of the prohead. All of these proheads have very nearly the same size and shape, so the differences in mobility must represent differences in charge. The relevant charge for this purpose is charge located at or near the surface of the particles, since a charged group in the protein can only affect the electrophoretic mobility of the particle if its counter ion can exchange freely with the bulk solvent. There are essentially two different mobilities among the proheads examined here: the wild-type Prohead I and Prohead II as well as the E219K Prohead II all have nearly the same mobility, and the E219K Prohead I and Whiffleballs have the same mobility as each other, w10% slower than that of the first group. The charge difference appears to derive from the pentamers or their immediate surroundings, since when wild-type pentamers are added to either Whiffleballs or mutant Prohead I, the mobility shifts to that of the wild-type proheads; furthermore it appears to shift in multiple steps, as if each added wild-type pentamer contributes an increment to the charge. We do not believe that the charge differences in the Whiffleballs and the mutant Prohead I can be attributed directly to the mutational change, even though it is nominally a two charge change in the direction that would retard the mobility on the agarose gel. The side-chain of glutamate 219 (Glu219) is within the main fold of the protein and pointing toward the center of the prohead shell, where it would have no access to the bulk solvent. Although the acidic group of Glu219 approaches the interior surface of the protein subunit, the structure suggests that it may not actually reach the surface and therefore may not be in full contact with the interior solvent, and we expect the same would be true of the basic amino group of the lysine that substitutes for Glu219 in the mutant. This view is supported by the observation that the electrophoretic mobility of the mutant hexamers is only slightly different from that of the wild-type hexamers. We suggest instead that the most plausible explanation for the prohead mobility differences is that there are charged groups in the interface between the pentamer and the surrounding hexamers that are buried when the wild-type

HK97 “Whiffleball” Capsids without Pentons

pentamers are in the prohead but are exposed when the mutant pentamers are there, implying a slightly different positioning for the mutant pentamers. In this view, the changed mobility of the mutant Prohead II to match the mobility of the wild-type proheads would reflect burial of those charges as a result of tightening the interactions between capsomers during the transition from Prohead I to Prohead II. Other scenarios are possible to explain the observed mobility differences among the proheads, but they appear more complex. Conformational switch model and HK97 assembly with and without pentons The most dramatic phenotype of the E219K mutant, its ability to assemble into penton-less Whiffleballs, presents what appears initially to be a contradiction with earlier results. We showed previously with wild-type protein that in vitro assembly of Prohead I from capsomers requires both pentamers and hexamers, with the most efficient assembly occurring when the pentamer to hexamer ratio was the same in the assembly reaction as in the assembled TZ7 structure. The efficient assembly documented here of mutant hexamers into Whiffleballs, in the absence of pentamers and to the correct TZ7 size, appears to call into question the role of pentamers in the assembly pathway. We believe, however, that the two sets of experiments can be reconciled in a way that is informative about the probable pathway of capsid assembly for HK97 and about how that pathway is regulated. Our hypothesis requires one new assumption about the mechanism of assembly, namely that an initial interaction between pentamers and hexamers changes the hexamers in such a way that they become competent for the subsequent steps of assembly. This change might most plausibly be a conformational change in the hexamer, such as a transition from a hypothetical 6-fold symmetric “assembly naive” hexamer into the strongly skewed, 2-fold symmetric hexamer found in the proheads. According to this view, if the hexamers retained the assembly competent form following prohead disassembly, they should be able to reassemble without again requiring interaction with a pentamer. We suggest that this may be what happens in the Whiffleball assembly experiment, where hexamers produced by dissociation of E219K Prohead I assembled into Whiffleballs in the complete absence of pentamers. In the earlier experiment in which wild-type Prohead I was disassembled and reassembled, pentamers were required for reassembly. This could mean that wildtype gp5 is less able than the mutant protein to retain the assembly competent conformation. Perhaps more importantly, in this experiment the capsomers were exposed to the special buffer conditions, including denaturant, that promote conversion between hexamers and pentamers, and this treatment may have erased the hexamer’s assembly competent conformation and therefore

179

HK97 “Whiffleball” Capsids without Pentons

required an additional interaction with pentamers before reassembly could go forward. These assumptions will require experimental testing, but they provide the prospect of understanding how different steps of capsid assembly can be constrained to follow the correct order, through a conformation switching mechanism. We note that such conformation switching assembly mechanisms have the potential to limit assembly to the correct TZ7 size.25 Structural lessons and implications Earlier work on HK97 prohead maturation indicates that there is a progression in the nature of the associations among hexamers and pentamers, from the loose and reversible association in Prohead I to the extensively intertwined and covalently bonded association in the mature Head. For example, Prohead I can dissociate spontaneously in ordinary buffers and will dissociate completely into hexamers and pentamers under rather mild conditions such as 2.0 M salt. In contrast, Prohead II, which differs from Prohead I only subtly in morphology beyond the loss of the delta domain, will disassemble only under strong denaturing conditions. The even more intertwined nature of the mature Head is perhaps best illustrated by the ˚ radius centered at a fact that a sphere of w17 A 3-fold symmetry axis encompasses parts of nine different gp5 subunits as well as three intersubunit cross-links. The work reported here with the E219K mutant illuminates some details of this progression. First, the fact that wild-type pentamers will add efficiently to Whiffleballs seems most easily reconciled with a view of Prohead I structure in which the individual capsomers are self-contained bricks that have not yet acquired the extensive intertwining of polypeptides across capsomer boundaries that characterize mature Heads and that might be expected to preclude the simple insertion of a pentamer bung into a Whiffleball bunghole. Second, the change in the electrophoretic mobility of the mutant proheads as they mature from Prohead I into Prohead II is suggestive of a tightening and consolidation of the contacts between capsomers; we suggest above that this change includes masking charged residues in the pentamer–hexamer interface. We are optimistic that as higher resolution structural information becomes available for one or both prohead forms, we will be able to identify the specific charged residues involved and infer the corresponding subtle structural changes.

Methods

Na2HPO4$7H2O, 2 g KH2PO4 per liter. Esherichia coli strains BL21(DE3) and BL21(DE3)pLysS were used as hosts for protein expression from T7-promoter containing plasmids.39 Plasmid pV0 (pT7-5Hd2.927) was used to produce Prohead II. Prohead I was made using pVB (pT7-5Hd2.9(fsBstB1)27), or pVK, a ProteaseK plasmid that was made by removing the DNA between two KasI sites in pV0 to inactivate the protease gene. The two plasmid derivatives pV0-E219K and pVK-E219K also contained the mutation E219K (gene 5 codon 219 changed from gaa to aaa; Yiyong Li PhD thesis, 2000, University of Pittsburgh). Growth media were supplemented with 50 mg/ml ampicillin and 25 mg/ml chloramphenicol, or ampicillin alone for BL21(DE3) cultures. RG glucose is an M9-derived minimal labeling medium lacking cysteine or methionine.40 Radio-labeling Selective radio-labeling was performed as described27,39. Briefly, BL21(DE3) cultures harboring pV0 or derivatives were grown at 37 8C in RG-glucose medium to about 4!108 cells/ml and isopropyl-b-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.4 mM to induce protein expression. After 20 minutes rifampicin was added to 0.2 mg/ml to shut down expression of host genes. After 30 minutes, [35S]-methionine was added to 10 mCi/ml and the culture was incubated for a further 15 minutes. Cells were harvested and lysed for analysis of native protein, or directly precipitated with TCA for analysis using SDS-PAGE. For native gel lysates 1 ml cells were collected by centrifugation, resuspended in 160 ml TKG containing 0.1 mg/ml each deoxyribonuclease I and ribonuclease A, mixed with 20 ml of 10 mg/ml lysozyme, left on ice for 15 minutes, supplemented with 20 ml of 1% Triton X-100, mixed and frozen in liquid nitrogen, thawed at room temperature to promote lysis, and stored at 4 8C. Prohead purification Proheads were purified by a combination of PEG precipitation, differential sedimentation, and velocity centrifugation in glycerol gradients using methods developed for Prohead II 26. Briefly, BL21(DE3)pLysS containing pVK (for wild-type Prohead I) or pVK-E219K (for E219K Prohead I) was grown to w5!108 cells/ml in LB and induced with IPTG. Cells were harvested by centrifugation, resuspended in lysis buffer and lysed by the addition of 0.2% Triton X100. Viscosity was reduced by treatment with deoxyribonuclease I at 20 mg/ml after MgSO4was added to 7.5 mM. The lysate was clarified by centrifugation, resuspended in TKG buffer and precipitated with 5% (w/v) PEG after adding 0.5 M potassium glutamate. The pellet was resuspended in TKG, repelleted in a 45Ti rotor (Beckman) for two hours at 35,000 rpm, resuspended, loaded onto 10–30% glycerol gradients in the same buffer and sedimented in the SW28 rotor (Beckman) at 27,000 rpm for 160 minutes. The prohead band was extracted with a syringe and the particles concentrated by sedimentation in a Ti45 rotor (Beckman) at 35,000 rpm for 1.5 hour. The pellet was resuspended in a small volume of TKG buffer.

Buffers, strains and media Dissociation of Prohead I TKG buffer is 20 mM Tris–HCl (pH 7.5), 100 mM potassium glutamate. Lysis buffer contained 50 mM Tris–HCl (pH 8.0), 5 mM EDTA. PBS, phosphate buffered saline, contained 80 g NaCl, 2 g KCl, 11.5 g

Prohead I was incubated in 30–40% glucose and 10–20 mM Tris–HCl (pH 9.5) at room temperature overnight, and the results were checked using agarose gels.

180 Generally, Prohead I was completely disassembled into capsomers. Purification of capsomers and reassembly in vitro Capsomers derived from Prohead I were purified by adsorption to a column of Poros HQ20 quaternary amine anion exchange media (ABI, Foster City CA) in 20 mM tris(hydroxymethyl)amino-methane hydrochloride (Tris– HCl)- bis-tris-propane buffer at pH 7.5 with 20 mM NaCl, and elution with a linear gradient of NaCl (to 0.5 M) at 13–33 ml/minute using a BioCAD Sprint chromatography system (ABI, Foster City CA). Fractions were collected by peak and resolved on native PAGE. Fractions containing homogeneous materials were pooled and the chromatographically purified E219K hexamers were reassembled by precipitating with 70% (w/v) (NH4)2SO4 at 4 8C and resuspending the pelleted protein in PBS to a high concentration (w35 mg/ml) followed by dialysis against PBS.

HK97 “Whiffleball” Capsids without Pentons

stock formulation containing 33.5% (w/v) acrylamide and 0.3% (w/v) methylene bis acrylamide. All samples were TCA precipitated to prevent unwanted spontaneous cross-linking of HK97 capsid protein.26 Precipitated protein was washed with acetone, dried under vacuum, resuspended in SDS sample buffer (0.063 M Tris–HCl (pH 6.8), 2% (w/v) SDS, 5% mercaptoethanol, 14% glycerol, and w0.025% bromphenol blue) and heated in boiling water for 2.5 minutes. Agarose gel analysis of HK97 head-related structures Non-denaturing agarose gel electrophoresis using TAMg buffer (40 mM Tris base, 20 mM acetic acid (pH 8.1), and 1 mM magnesium sulfate) was done as described previously.26 Samples were mixed with oneninth volume of dyes and glycerol (50% (v/v) glycerol, 0.025% (w/v) bromphenol blue, 0.025% (w/v) xylene cyanol XFF) before loading. Gels were fixed in 95% ethanol, dried, stained, destained and then dried onto filter paper for autoradiography.

Cryo-EM and image reconstruction Samples of E219K mutant particles were vitrified and then imaged at a nominal magnification of 38,000! and accelerating voltage of 120 kV on a CM200 FEG electron microscope (FEI, Mahwah, NJ) equipped with a Gatan 626 cryo-holder, as described.41 Focal pairs were collected with the first exposures being closer to focus, and digitized on a SCAI scanner (Z/I Imaging, Huntsville, ˚ at the AL) at a step size of 7 mm, corresponding to 1.8 A specimen. Image reconstruction, including contrast transfer function (CTF) correction, was performed as described.42 For the E219K Prohead I reconstruction, four focal pairs of micrographs yielded 1113 particles (each represented by two images), of which 746 (67%) were included in the final density map. For the reassembled E219K Whiffleball, the corresponding numbers were 1060 and 1581 particle images collected from six focal pairs of micrographs. Resolution was assessed in terms of the Fourier Shell Correlation coefficient43 with a threshold of 0.3: for both reconstruc˚ . Prior to difference tions this was estimated at 13–14 A mapping, the relative densities of the reconstructions were mutually calibrated by comparing central difference sections with varying contributions from each map and minimizing the difference. Analyses were performed on Macintosh G5 computers (Apple Computer, Cupertino CA, USA), and panels in Figure 8 were prepared on Linux workstations (Dell, Austin TX, USA) with Amira 3.1 software (Mercury Computer Systems/3D Viz group, San Diego CA, USA and Merignac, France). Capsomer interconversion Hexamers and pentamers of head protein were interconverted as described.34 Briefly, capsomers were treated with “magic pentamer buffer” (0.2 M Guanidine HCl and 1% (v/v) dimethyl formamide (DMF) at pH 7.5) at room temperature overnight to convert capsomers into pentamers, or with “magic hexamer buffer” (0.5 M NaSCN) to convert capsomers into hexamers. SDS polyacrylamide gel analysis The methods and conditions for SDS-polyacrylamide gel electrophoresis were modified from Laemmli,44 except that gels were prepared with a low-cross-linker

Non-denaturing polyacrylamide gel analysis Methods were modified from Davis,45 as described34 using 0.75 M Tris–HCl (pH 8.8) as the running gel buffer. Samples were diluted with 4! concentrated sample buffer before loading (final concentrations: 0.063 M Tris– HCl (pH 6.8), 10% glycerol, and w0.025% bromphenol blue).

Acknowledgements This work was supported by NIH grant R01 GM47795 to R.W.H. J.F.C. acknowledges support from the CNRS in the form of an ATIPE grant. We thank Brian Firek for much needed help in organizing the manuscript, Jun Xu for informative discussions, D. Humphrey for technical assistance and J. Young and C. Smith for their help with Whiffleball capping studies.

References 1. Ban, N. & McPherson, A. (1995). The structure of satellite panicum mosaic virus at 1.9 A resolution. Nature Struct. Biol. 2, 882–890. 2. Tsao, J., Chapman, M. S., Agbandje, M., Keller, W., Smith, K., Wu, H. et al. (1991). The three-dimensional structure of canine parvovirus and its functional implications. Science, 251, 1456–1464. 3. Caspar, D. L. & Klug, A. (1962). Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27, 1–24. 4. Muller-Salamin, L., Onorato, L. & Showe, M. K. (1977). Localization of minor protein components of the head of bacteriophage T4. J. Virol. 24, 121–134. 5. Black, L. W., Showe, M. K. & Steven, A. C. (1994). Morphogenesis of the T4 Head. In Molecular Biology of Bacteriophage T4 (Karam, J., ed.) 2nd edit., pp. 218–258, American Society of Microbiology, Washington, DC. 6. Prage, L., Pettersson, U., Hoglund, S., Lonberg-Holm,

181

HK97 “Whiffleball” Capsids without Pentons

7.

8. 9.

10. 11.

12. 13.

14. 15.

16.

17.

18. 19.

20.

21.

22.

23. 24.

K. & Philipson, L. (1970). Structural proteins of adenoviruses. IV. Sequential degradation of the adenovirus type 2 virion. Virology, 42, 341–358. Burnett, R. M. (1997). The structure of adenovirus. In Structural Biology of Viruses (Chiu, W., R.M, B. & Garcia, R. L., eds), pp. 209–238, Oxford University Press, New York. King, J. & Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature, 251, 112–119. Dokland, T., Bernal, R. A., Burch, A., Pletnev, S., Fane, B. A. & Rossmann, M. G. (1999). The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174. J. Mol. Biol. 288, 595–608. Fane, B. A. & Prevelige, P. E., Jr (2003). Mechanism of scaffolding-assisted viral assembly. Advan. Protein Chem. 64, 259–299. Laemmli, U. K., Molbert, E., Showe, M. & Kellenberger, E. (1970). Form-determining function of the genes required for the assembly of the head of bacteriophage T4. J. Mol. Biol. 49, 99–113. Serwer, P. (1979). Fibrous projections from the core of a bacteriophage T7 procapsid. J. Supramol. Struct. 11, 321–326. Steven, A. C. & Trus, B. L. (1986). The structure of bacteriophage T7. In Electron Microscopy of Proteins (Harris, J. R. & Horne, R. W., eds), vol. 5, pp. 1–35, Academic Press, London. Earnshaw, W. & King, J. (1978). Structure of phage P22 coat protein aggregates formed in the absence of the scaffolding protein. J. Mol. Biol. 126, 721–747. Howatson, A. F. & Kemp, C. L. (1975). The structure of tubular head forms of bacteriophage lambda; relation to the capsid structure of petit lambda and normal lambda heads. Virology, 67, 80–84. Georgopoulos, C., Casjens, S. & Tilly, K. (1983). Lambdoid phage head assembly. In The Bacteriophage Lambda II (Hendrix, R., Roberts, J., Stahl, F. & Weisberg, R., eds), pp. 279–304, Cold Sprig Harbor Laboratory, New York. Hagen, E. W., Reilly, B. E., Tosi, M. E. & Anderson, D. L. (1976). Analysis of gene function of bacteriophage phi 29 of Bacillus subtilis: identification of cistrons essential for viral assembly. J. Virol. 19, 501–517. Fuller, M. T. & King, J. (1982). Assembly in vitro of bacteriophage P22 procapsids from purified coat and scaffolding subunits. J. Mol. Biol. 156, 633–665. Prevelige, P. E., Jr, Thomas, D. & King, J. (1993). Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells. Biophys. J. 64, 824–835. Cerritelli, M. E. & Studier, F. W. (1996). Assembly of T7 capsids from independently expressed and purified head protein and scaffolding protein. J. Mol. Biol. 258, 286–298. Eiserling, F. A., Geiduschek, E. P., Epstein, R. H. & Metter, E. J. (1970). Capsid size and deoxyribonucleic acid length: the petite variant of bacteriophage T4. J. Virol. 6, 865–876. Mooney, D. T., Stockard, J., Parker, M. L. & Doermann, A. H. (1987). Genetic control of capsid length in bacteriophage T4: DNA sequence analysis of petite and petite/giant mutants. J. Virol. 61, 2828–2834. Katsura, I. (1983). Structure and inherent properties of the bacteriophage lambda head shell. IV. Small-head mutants. J. Mol. Biol. 171, 297–317. Katsura, I. (1980). Structure and inherent properties of

25.

26.

27. 28. 29.

30.

31.

32.

33.

34. 35. 36.

37.

38.

39.

40. 41.

the bacteriophage lambda head shell. II Isolation and initial characterization of prophage mutants defective in gene E. J. Mol. Biol. 142, 387–398. Conway, J. F., Duda, R. L., Cheng, N., Hendrix, R. W. & Steven, A. C. (1995). Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system. J. Mol. Biol. 253, 86–99. Duda, R. L., Hempel, J., Michel, H., Shabanowitz, J., Hunt, D. & Hendrix, R. W. (1995). Structural transitions during bacteriophage HK97 head assembly. J. Mol. Biol. 247, 618–635. Duda, R. L., Martincic, K. & Hendrix, R. W. (1995). Genetic basis of bacteriophage HK97 prohead assembly. J. Mol. Biol. 247, 636–647. Hendrix, R. W. & Duda, R. L. (1998). Bacteriophage HK97 head assembly: a protein ballet. Advan. Virus Res. 50, 235–288. Lata, R., Conway, J. F., Cheng, N., Duda, R. L., Hendrix, R. W., Wikoff, W. R. et al. (2000). Maturation dynamics of a viral capsid: visualization of transitional intermediate states. Cell, 100, 253–263. Wikoff, W. R., Liljas, L., Duda, R. L., Tsuruta, H., Hendrix, R. W. & Johnson, J. E. (2000). Topologically linked protein rings in the bacteriophage HK97 capsid. Science, 289, 2129–2133. Gan, L., Conway, J. F., Firek, B. A., Cheng, N., Hendrix, R. W., Steven, A. C. et al. (2004). Control of crosslinking by quaternary structure changes during bacteriophage HK97 maturation. Mol. Cell. 14, 559–569. Helgstrand, C., Wikoff, W. R., Duda, R. L., Hendrix, R. W., Johnson, J. E. & Liljas, L. (2003). The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44A resolution. J. Mol. Biol. 334, 885–899. Conway, J. F., Wikoff, W. R., Cheng, N., Duda, R. L., Hendrix, R. W., Johnson, J. E. & Steven, A. C. (2001). Virus maturation involving large subunit rotations and local refolding. Science, 292, 744–748. Xie, Z. & Hendrix, R. W. (1995). Assembly in vitro of bacteriophage HK97 proheads. J. Mol. Biol. 253, 74–85. Ferguson, K. A. (1964). Starch-gel electrophoresis– application to the classification of pituitary proteins and polypeptides. Metabolism, 13, 985–1002. Hedrick, J. L. & Smith, A. J. (1968). Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126, 155–164. Teschke, C. M., McGough, A. & Thuman-Commike, P. A. (2003). Penton release from P22 heat-expanded capsids suggests importance of stabilizing penton– hexon interactions during capsid maturation. Biophys. J. 84, 2585–2592. Newcomb, W. W., Trus, B. L., Booy, F. P., Steven, A. C., Wall, J. S. & Brown, J. C. (1993). Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes. J. Mol. Biol. 232, 499–511. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89. Georgopoulos, C. P., Hendrix, R. W., Casjens, S. R. & Kaiser, A. D. (1973). Host participation in bacteriophage lambda head assembly. J. Mol. Biol. 76, 45–60. Cheng, N., Conway, J. F., Watts, N. R., Hainfeld, J. F., Joshi, V., Powell, R. D. et al. (1999). Tetrairidium, a four-atom cluster, is readily visible as a density label in three-dimensional cryo-EM maps of proteins at 10-25 A resolution. J. Struct. Biol. 127, 169–176.

182

HK97 “Whiffleball” Capsids without Pentons

42. Conway, J. F. & Steven, A. C. (1999). Methods for reconstructing density maps of “single” particles from cryoelectron micrographs to subnanometer resolution. J. Struct. Biol. 128, 106–118. 43. Saxton, W. O. & Baumeister, W. (1982). The correlation averaging of a regularly arranged bacterial cell envelope protein. J. Microsc. 127, 127–138.

44. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. 45. Davis, B. J. (1964). Disc electrophoresis. Method and application to human serum proteins. Ann. New York Acad. Sci. 2, 121.

Edited by A. Klug (Received 17 December 2004; received in revised form 15 February 2005; accepted 23 February 2005)