Assembly in vitro of bacteriophage P22 procapsids from purified coat and scaffolding subunits

J. Nol. Biol.

(1982) 156, 633-665

Assembly in Vitro of Bacteriophage P22 Procapsids from Purified Coat and Scaffolding Subunitst MARGARET T. FULLERS AND JONATHAX KING

Massachusetts (Received

Institute

Department of Biology of Technology, Cambridge,

9 April

1981, and in revised form 19 October 1981)

Mass. 02139, C’.S.A

The coat and scaffolding proteius of bacteriophage P22 procapsids have been purified in soluble form. By incubating both purified proteins with a mutantinfected cell extract, lacking procapsids, but competent for DNA packaging in Gtro (Poteete et al., 1979), we were able to obtain assembly of biologically active procapsids in vitro. The active species for complementation in vitro in both protein preparations copurified with the soluble subunits, indicating that, these subunits represent precursors in procapsid polymerization. When t,he purified coat and scaffolding subunits were mixed directly. the! polymerized into double-shelled procapsid-like structures during dialysis from I.5 M-guanidine hydrochloride to buffer. When dialyzed separately under the same conditions, the scaffolding subunits did not polymerize but remained as soluble subunits, as did most of the coat subunits. No evidence was found for self-assembly of the scaffolding protein in the absence of t)he coat protein. The uuassembled coat subunits sedimented at 3.9 S and the unassembled scaffolding subunits sedimented at 2.4 S iu sucrose gradients. The Stokes’ radius. determined by gel filtration, was 25 A for the coat subunits and 34 A for the scaffolding subunits. These results indicate that the scaffolding subunit,s are relatively slender elongated molecules, whereas the coat subunits are more globular. The experiments suggest that the procapsid is built by copolymerization of the two protein species. Their interaction on the growing surface of the shell structure. and not in solution, appears to regulate successive binding interartions.

1. Introduction The protein shells of viruses are members of a large class of biological structures whose accurate assembly depends on the control of protein polymerization reactions (Klug, 1972: Kellenberger, 1972; Caspar, 1976: King, 1980; Wood. 1979). The capsids of double-stranded DNA phages are initially built as double-shelled structures. the outer shell composed of coat subunits, and the inner shell of scaffolding or assembly core protein (Showe & Black, 1973 ; King 8r Casjens. IQ74 : Rhowe Br Kellenberger, 1976; Paulson & Laemmli, 1977: Van Driel & Couture. 1978a ; Earnshaw et al., 1979). They also contain a set of minor proteins. some of t A preliminary version of’ some of these experiments was reported in the Proc.redings of‘ the, Riophysical Discussions. held at Airlie, Virginia. May 1980. 1 Present address: Department of Biology. University of Indiana, Bloomington. Intl. 47401. I1.S.A. ow2%2X36/82/110833-33

$03.00/O

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‘(“’ 1982 Academic Press Inca.(London) Ltdl.

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M. T. FULLER

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J. KING

which form the special vertex that initiates shell assembly, and through which the DNA will be packaged and later ejected (Murialdo & Becker, 197%). Though a great deal is known of the topology of subunit packing in virus capsids (Caspar & Klug, 1962; Crowther & Klug, 1975; Kellenberger, 1980), much less is understood of the detailed pathway by which coat and scaffolding subunits initiate, propagate and close the precursor shell. In the course of the DNA packaging reactions, procapsid shells undergo a major transformation: the outer shells expand, with the coat’ subunits altering their bonding and, in some phages, undergoing proteolytic cleavage (Laemmli et al., 1976; Steven et aE., 1976). The scaffolding proteins are either cleaved as in phage T4 (Laemmli, 1970), or exit and recycle as in phage P22 (King & Casjens, 1974). As a result of these processes, the coat subunits found in the mature capsids represent a state quite different from that found in the procapsid, or in the unassembled precursor subunits (Kellenberger, 1980; King, 1980). Thus, studying the control of the shell polymerization process requires subunits derived either from the procapsids themselves, or from earlier precursors. In the control of the polymerization of bacterial flagellin (Uratani et al., 1972) and of the structural proteins of the T4 tail (Kikuchi & King, 1975), the key feature appears to be the synthesis of the subunits in a non-reactive form and their conversion to a reactive state only on the polymerization sites of the growing structure. These essentially irreversible processes, which depend on the switching of subunits from inactive to active conformation, have been termed self-controlled or self-regulated assembly processes (Caspar, 1976; King, 1980) as opposed to the characteristic of condensation reversible monomer-polymer associations polymerization (Oosawa & Asakura, 1975). Though both models involve specific initiation steps, in the former case subunits are not in equilibrium in solution between their reactive and unreactive states. Previous work on phage assembly has suggested the applicability of the self-regulated models (Casjens & King, 1975: Showe & Kellenberger, 1976; Wood, 1979; Wood & King, 1979; King, 1980). However, resolution of these questions with respect to capsids requires experimental systems in which shell assembly can be studied directly. The assembly of the procapsid from its precursor subunits or related forms has been studied in phage lambda and phage T4. In the case of T4, Van Driel &. Couture (1978a) isolated coat and scaffolding subunits as well as the major vertex protein gp20, by dissociation of procapsids. These subunits reassembled into a variety of structures when returned to physiological conditions. All three proteins were required for formation of closed prolate shells. Unfortunately, DNA could not be packaged into T4 procapsids in vitro. Thus, it was not possible to determine which of the in vitro-assembled structures were biologically important precursors. Murialdo & Becker (1977) assembled biologically active procapsids of bacteriophage lambda in vitro by complementation between extracts of mutant infected cells. In particular, when E- extracts, which lack the major coat protein gpE but contain the lambda scaffolding protein gpNu3, were mixed with Nu3extracts, which lack the scaffolding protein gpNu3 but contain the coat protein gpE. viable procapsids were formed that were able to package DNA and mature to infectious phage. Murialdo & Becker (19786) exploited the genetics of lambda to

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map out the sequence of gene product interactions in lambda early capsid assembly. Their experiments revealed that a number of interactions must occur between phage and host proteins for the scaffolding protein to be competent for capsid assembly in vitro. However, purified quantities of lambda gpE and gpNu3 have not been available for direct study of the capsid assembly reaction. The procapsid of bacteriophage P22 is an isometric structure of radius 256 A (Earnshaw et al., 1976). The outer shell is composed of about 420 molecules of the viral coat protein, the product of gene 5 (gp5), while the inner shell is composed of about 200 molecules of the 42,000&f, scaffolding protein, the product of gene X (Botstein et al., 1973; King & Casjens, 1974; Earnshaw et al., 1976: Casjens, 1979). If the coat protein is removed by mutation, the scaffolding protein probably remains as soluble subunits (Fuller & King, 1981). These subunits autogenously repress their own synthesis, so that the scaffolding subunits are present at low levels in 5--infected cells (King et al., 1978). If the scaffolding protein is removed by mutation, the coat subunits do not assemble efficiently, but aberrant aggregates of coat protein do accumulate (King et al., 1973; Lenk et al., 1975; Earnshaw $ King, 1978). Thus, the scaffolding protein appears to regulate both the rate and accuracy of coat protein polymerization. Four minor proteins are found in P22 procapsids, the products of genes I, 7. Ifi and 80. The latter three are not needed for capsid assembly; if removed by, mutation, morphologically normal phage are produced, but these part’icles are now infectious, due to the inability to inject their DNA (Botstein et al.. 1973: Hoffman 8: Levine, 1975; Bryant, 1978). If the gene 1 protein is removed by mutation. procapsids are formed (lacking gpl), but these are unable to package DSA (Botstein et al., 1973; Poteete & King, 1977). The genetic analysis of particle morphogenesis indicates that only two viral proteins, the products of genes 5 and 8, are required to assemble closed, accurately dimensioned double shells. However, the four minor proteins must be incorporat,ed if these shells are to be biologically active. P22 procapsids are distinguished by their stability and relative ease of isolation (Earnshaw et al., 1976; Fuller & King, 1981). Starting with purified procapsids. we have isolated soluble forms of the scaffolding and coat subunits by gentle dissociation with guanidine hydrochloride (Fuller & King, 1981). This mimics the release of the scaffolding protein, which occurs in vivo ; the scaffolding subunits exit through the coat protein shell, which remains intact. These shells were further dissociated with Gu. HClt to yield soluble forms of the coat subunits. As with lambda, P22 procapsids can be filled with DNA and converted into viable phage in vitro in extracts of mutant infected cells (Poteete et al., 1979; Poteete & Botstein, 1979). The DNA donor extract used in this reaction was made from cells infected with phage carrying two amber mutants in the coat protein gene. so that no coat protein and thus no pre-assembled procapsids are present. The extract does contain the four minor procapsid proteins gpl, gp7, gp16 and gp20. We used such extracts as react,ion mixtures for the assembly of biologically active procapsids from purified subunits. t Abbreviations product.

used: Gu. HCl, guanidine

hydrochloride;

SDS. sodium dodecyl sulfate:

gp. gene

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In this paper we report the assembly of P22 procapsids in vitro, both in crude extracts and with purified proteins alone. We use these assays to define the subunit species that are precursors in shell assembly, and to delineate the sequence of subunit interactions during their polymerization into closed shells. The results suggest that once the shell is initiated, the coat and scaffolding subunits do not interact in solution but only on the rim of the growing shell.

2. Materials and Methods (a) Strains All bacterial strains are derivatives of SaZmoneEla typhimurium LT2 from the collection of D. Botstcin. DNA donor extracts were prepared by induction of the non-permissive lysogen DB7130 (DB7OOO(P22 immL/c2ts52/5-amN8/5-amN13/I3-amH715)) described by Poteete et al. (1979). Phage produced by encapsulation in vitro were titred on DB7002 (ZeuA am414/supD) or on the lysogen DB7002(P22int3/&6). When plated on the lysogen, only phage with the immL genotype of the DNA donor produce plaques. The phage and bacterial strains employed in preparation of coat and scaffolding protein are described by Fuller & King (1981). Since all of these phage carry the clear plaque mutation cl-7 and the 13- allele HlOl, which is poorly suppressed by DB7002, phage present as background in protein or particle preparations produce small, clear plaques on DB7002. These phage can be easily distinguished from the large turbid plaques produced at 30°C on DB7002 by phage of the DNA donor genotype. (b) Media and chemicals Super broth is described by Fuller & King (1981). Buffer B is 50 mM-Tris .HCl (pH 7.6), 25 mM-NaCl, 2 mm-EDTA, 3 mM-2-mercaptoethanol, 1% (v/v) glycerol. Dilution fluid con tains 1 g of Tryptone (Difco), 7 g of NaCl per litre. TSM/3 contains 10 miw-Tris.HCl (pH 7.4), 60 mM-spermidine .HCl, 200 mi%MgCl,, 30 mM-2-mercaptoethanol. ATP stock solution was made to 150 mM in double distilled water, neutralized with NaOH, and stored frozen in 05 ml portions. Both spermidine HCl and ATP (disodium salt) were obtained from Sigma Chemical Co. Egg-white lysozyme was obtained from Calbiochem. Fresh solutions of 3 mg lysozyme/ml in 250 mM-Tris. HCl (pH 7.6) 20 mM-EDTA were made for each experiment. EDTA was stored as a stock solution at 05 M in double distilled water brought to pH 9.4 with NaOH. (c) Preparation of DNA donor extracts DNA donor extracts were prepared by a slight modification of the procedure described by Poteete et al. (1979). The DNA donor lysogen was grown to 5 x 10s cells/ml at 28°C with vigorous aeration in super broth. Production of phage components was induced by shifting the culture to 39°C for 90 min. Cells were harvested by centrifugation for 10 min at 8000 revs/mm in a Sorvall SS-34 rotor, resuspended in 002 vol. 50 m&r-Tris.HCl (pH 7.4), 10% (w/v) sucrose, and stored frozen in liquid nitrogen in 1 ml portions. Fresh DNA donor extracts were prepared for each experiment. A 1 ml portion of frozen cell suspension was quickly thawed at 35”C, but with care taken that the contents stayed cold. Cells were incubated on ice with 61 ml of a solution of 3 mg lysozyme/ml, 25 mMTris. HCl (pH 7.4), 20 mm-EDTA for 30 min. After lysis, 91 ml TSMjl and 625 ml of 150 mMATP were added per original 1 ml portion, the extract was drawn through a 20-gauge needle once or twice to reduce viscosity and immediately distributed to tubes on ice and mixed with the other components of the reaction. (d) Protein donors Protein donors were either purified P22 procapsids formed in wivo in cells infected with mutants blocked in DNA packaging (l-/23or Z-/13- infections) or soluble coat and

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scaffolding protein isolated from procapsids as described by Fuller & King (1981). Samples containing Gu. HCl were dialyzed against buffer B at 4°C before mixing with the complementing protein and extract. When Gu . HCl was removed from coat protein samples by passage over a desalting column, the protein was significantly less active for complementation. Proteins were mixed on ice immediately before addition of the DNA donor extract. The complementation activity of the soluble protein preparations in titro was not st,able for more than a period of a few weeks at 4°C. Samples of the scaffolding protein frozen in liquid nitrogen remained active for at least a year. (e) Complementation in vitro Complementation in vitro mixtures were commonly made with 050 ml of DNA donor extract mixed with @50 ml of P22 procapsids or 25 ~1 each of coat protein and scaffolding protein preparations or buffer B. For experiments testing activity across columns, 25 ~1 of each column fraction was mixed with 25 ~1 of the supplementary protein, or buffer B, on ice. After addition of DNA donor extract, the mixture was vortex mixed and incubated at 23°C’ overnight in loosely covered glass tubes (13 mm x 100 mm). Reactions were terminated by IO-fold dilution with chilled dilution fluid. im,mL phage were titred on DB7002 (P22 int3/sie6) at 30°C. The procapsids purified from 2- infected cells as described by Fuller & King (1981) complemented the DNA donor extract. At 10” procapsids/ml or less, the production of phage in the undiluted DNA donor extract was linear with respect to procapsid concentration, and the efficiency of conversion of procapsids to phage was 0.1% to 2%. This value is consistent with the 01~~ efficiency of DNA packaging and maturation in vitro for procapsids isolated from crude extracts by sucrose gradient centrifugation described by Poteete et al. (1979). In the complementation in vitro experiments described here, the concentrations of procapsids assembled in vitro were well below this range, and so the infectious phage produced in the complement&ion reaction should be proportional to the amount of active procapsids assembled in vitro. At the low procapsid concentrations produced, the complementation reaction was not sensitive to small variations in the DNA donor concentration. At procapsid concentrations above 1013/ml, production of phage was no longer linear with respect to procapsid concentration, and procapsids appeared to be in excess. The same sample of purified procapsids (lO”/ml) was used in the experiments described throughout this paper. (f) Assembly in vitro Soluble coat and scaffolding protein were obtained by dissociation of purified P22 procapsids with Gu. HCl as described by Fuller & King (1981). Procapsids were first treated with @5 M-Gu . HCl to induce release of the scaffolding protein and the minor proteins gp16 and gp20 from within the coat protein shell. The released proteins were separated from the intact coat protein shells by molecular sieve chromatography and further purified by iorlexchange chromatography on DEAE-cellulose. The intact shells, containing the coat protein and t,he minor protein gpl , were dissociated with 3 M-GU . HCl, and the disassembled coat protein was separated from remaining intact structures by column chromatography. Coat and scaffolding proteins were mixed for reassembly in the following manner: 300 ~1 of coat protjein in 2 M-GU . HCl in buffer B was mixed with 100 ~1 of soluble scaffolding protein in buffer B containing 10 to 100 mM extra NaCl (a result of the ion-exchange purification step). The concentration of Gu . HCl in all reaction mixtures was 1.5 M. The mixtures were dialyzed for 12 to 18 h at 23°C or at 4°C against 100 to 500 ml of buffer B in 2 changes. Mixtures missing one of the proteins were made with the respective buffer. The ratio of coat to scaffolding protein varied from experiment to experiment. Most often, the assembly reaction mixture contained scaffolding protein in slight excess over the coat protein as determined by Coomassie blue staining of proteins banded by sodium dodecyl sulfat,e/polyacrylamide gel electrophoresis. If very concentrated preparations of coat protein

638

M. T. FULLER

AND J. KING

were dialyzed at 23”C, some of the protein precipitated. When the same preparation dialyzed to remove the Gu . HCl at 4”C, no precipitate formed.

was

(g) Sucrose gradient sedimentation Assembly mixtures in vitro were layered on top of 5-ml, 5% to 20% (w/w) sucrose gradients with a @3 ml 600/b (w/w) sucrose cushion. To display procapsid-sized structures 2/3 of the way down the centrifuge tube, gradients were centrifuged for 40 min at 35,000 revs/min at 20°C or for 70 min at 35,000 revs/min at 4°C in a Beckman SW50.1 rotor. To analyze small molecular weight components, gradients were centrifuged for 20 h at 45,000 revs/min at 4°C in a Beckman 50.1 rotor: 10 ~1 of 10 mg hemoglobin/ml and 10 ~1 of 10 mg lysozyme/ml were added to 100 ~1 of sample as sedimentation markers for the longer centrifugation. Gradients were collected in constant-volume fractions from a pinhole at the bottom of the tube. The protein content of gradient fractions was analyzed by SDS/polyacrylamide gel electrophoresis. The protein content of Coomassie blue-stained gels was quantitated with a Joyce-Loebl scanning microdensitometer.

(h) Measurement of Stokes’ radius The Stokes’ radius of the scaffolding protein was measured by molecular exclusion chromatography on a Sephacryl S-200 column (Pharmacia Fine Chemicals) with marker proteins. The column was poured as specified by Pharmacia. The Sephacryl S-200 suspension was mixed with buffer B at 4°C and decanted several times to remove the fines. The suspension was degassed and poured at once into a glass column (0.9 cm x 40 cm). The column was packed at a flow-rate of 31 ml/h, maintained with a peristaltic pump. The final height of the packed Sephacryl was 38 cm, and the final bed volume was 35.5 ml. The column was run at a flow-rate of 4 ml/h under force of gravity, with buffer B at 4°C. Stock solutions were made of the marker proteins egg-white lysozyme (Calbiochem), bovine hemoglobin (Sigma) and yeast alcohol dehydrogenase (Sigma) at 10 mg/ml in buffer B. The stock solution of catalase was a 50-fold dilution of a 25 mg/ml crystalline suspension with buffer B. Bovine serum albumin was 10 mg/ml in O%% (w/v) saline. The P22 tail protein, gp9 was a generous gift from D. Goldenberg and D. Smith. A stock of wild-type P22 phage was diluted in buffer B to 1.5 x lo9 phage/ml for the void volume marker. 14Clabeled amino acids were purchased from New England Nuclear at 100 &X/ml in 0.1 M-HCl. A sample of purified scaffolding protein was mixed with 10 to 25 ~1 of each of the marker protein solutions on ice. Then 10 ~1 of the P22 phage solution and 2 ~1 of 14C-labeled amino acids were added to mark the void and exclusion limits of the column. The total sample volume was O-30 to 043 ml. Glycerol (1 drop) was added to increase the density and the sample was layered under the buffer on top of the running column. Fractions (25 drops, about @6 ml) were collected with a Gilson Micro-fraction collector. The same column was used for all experiments. The protein content of fractions collected from the column was monitored by measuring absorbance at 280 nm. The hemoglobin peak was easily distinguished by its brown color. The distribution of individual proteins in the effluent fractions was determined by SDS/polyacrylamide gel electrophoresis. The void volume was determined by titre of P22 phage. A sample of each of the final column fractions was counted in a scintillation counter to detect 14C-labeled amino acids. The elution behavior of the P22 tail protein gp9 was measured by spot complementation assay on agar plates (Berget & Poteete, 1980). This assay is based on the ability of the tail protein to assemble onto 9- phage particles embedded in soft agar in a growing bacterial lawn. The assembly event allows the 9- particles to infect the permissive bacteria and grow, producing a cleared spot in the lawn where the tail protein was added. The production of a cleared spot requires a threshold level of gp9. Serial dilutions of consecutive column fractions were tested for the threshold level of tail protein.

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3. Results (a) Complementation of the 5- DNA donor extract with purified coat and scaffolding proteina To determine whether the subunits purified from procapsids were related to precursor states in the capsid polymerization pathway, we attempted to find conditions in which biologically active procapsids could be assembled in vitro. Poteete et al. (1979) demonstrated in vitro DNA packaging and capsid maturation when a crude infected cell extract, containing viral Dh’A and viral proteins but lacking the capsid protein: was supplemented with P22 procapsids that had been the DNA packaging extract with purified. assembled in vivo. We supplemented unassembled subunits of the P22 coat and scaffolding proteins. If these subunits were able to assemble with the minor proteins into complete procapsids. subsequent DNA and capsid maturation would result in the formation of infectious the only phage scored are those with the phage part,icles. In these experiments, c + immL genotype of the DNA donor extract. Viral coat and scaffolding proteins were isolated from P22 procapsids after dissociation with low concentrations of Gu. HCl as described by Fuller & King

IIP5

-

clP8

Fro. 1. SDS/polyacrylamide gel electrophoresis of P22 procapsids, purified coat protein. and purified scaffolding protein. P22 coat protein, scaffolding protein and procapsids were purified a8 described by Fuller & King (1981). Coat and scaffolding subunits were derived from dissociation of I - procapsids by 3 M-GU HCI. Scaffolding protein wair further purified by ion-exchange chromatography. The procapsids shown, which were used in the in vilro complementatlon experiments. were isolated from g--infected cells. Samples were dialyzed against buffer B before mixing with SDS sample buffer. The procapsid minor proteins of lower molecular weight (gp4. gp7 and gp26) are not detectable in these stained gels.

640

M. T. FULLER

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(1981). Figure 1 shows SDS/polyacrylamide gel electrophoresis of preparations of P22 procapsids and purified coat and scaffolding protein typical of those tested in these experiments. To test the ability of the purified, soluble subunits of coat and scaffolding protein to assemble into active procapsids in vitro, we mixed the proteins with the DNA donor extract as follows. A freshly prepared coat protein sample (25 ~1; 69 mg/ml) was mixed on ice with an equal volume of a freshly thawed sample of purified scaffolding protein (O-6 mg/ml). After addition of 50 ~1 of freshly prepared DNA donor extract, the reaction mixture was vortex mixed and immediately shifted to 23”C, incubated overnight and assayed for the formation of infectious phage carrying the genotype of the DNA donor. We explored different methods of lysis of the DNA donor cells, as shown in Table 1. Addition of purified procapsids to the 5- extract resulted in an increase of viable phage of lo5 above the background level produced when buffer alone was added to the extract (as shown in Table 1). Measurements of background levels of phage were at the limit of detectability, and were subject to large statistical fluctuation. (Phage yields of 5 x lo2 represent 5 plaques/plate; phage yields of lo4 or more represent .more than 100 plaques/plate.) The efficiency of DNA packaging and subsequent assembly reactions was not sensitive to the method of preparing the lysate when intact procapsids were added to the crude extract. Initial experiments in which the DNA donor extract was prepared with chloroform and incubated at 35”C, as in the experiments described by Poteete et al. (1979), failed to reveal assembly of active procapsids in vitro. However, when the complementation reaction was carried out at 23°C instead of at 35°C: and a different method of lysis was employed, an increase of infectious virus was observed in vitro. Packaging of DNA into intact procapsids was not as temperature dependent; incubation of procapsids mixed with DNA donor produced only twofold more phage at 23°C than at 35°C.

TABLE

1

Complementation in vitro of DNA donor extracts prepared by different lysis procedures

Protein donort Procapsids i?P5+ gP6 Buffer B

LYS/EDTA

DNA donor extractt LYS/EDTA/SON

6.9 x 10s 1.5 x lo6 1 x lo3

7.2 x 10s 36 x lo5 5 x 10s

CHCl,

50 mM-Tris

5.9 x 10s 6 x lo3 1 x103


Coat protein was dialyzed at 4°C to remove Gu . HCl : 50 ~1 of DNA donor, 10 ~1 of 150 miv-ATP and 50 ~1 of protein donor were incubated overnight at 23°C. diluted and titred on DB7002. t DNA donor extracts were prepared with lysozyme and EDTA (LYQEDTA), lysozyme, EDTA and sonication (LYS/EDTA/SON), or with chloroform (CHCI,) as described in Materials and Methods. $ Protein donors were 2- procapsids at 10’2/ml or soluble coat (gp5) and scaffolding (gp8) protein prepared as described in Materials and Methods.

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Complementation

in

PH (1012/ml) gP5 + gP8 gP5 a8 Buffer B VA ratio of yields : 5+8/I’H Yield above background

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2

vitro of DNA donor extract by puri$ed and scaffolding

Protein donor

IN

Expt I

coat

protein Expt II

Expt III

1.3 x lo9 2.2 x lo5 5x lo3 3x102 5x lo2

3.4 x 5.4 x 2x 3x 4x

2.6 x 2.3 x 2x 2x 1x

0.02 400-fold

0.02 100.fold

lo8 lo4 103 103 lo2

lo8 lo4 lo3 lo3 lo3

0.01 20-fold

DNA donor extracts were prepared by lysis with lysozyme and EDTA as described in Materials and Methods. Reactions were incubated overnight at 23°C. The coat protein subunits were purified from freshly dissociated 2- procapsids, and dialyzed at 4°C to remove Gu. HCl. Scaffolding protein was purified on DEAE-cellulose from J-/20- procapsids, stored frozen in liquid nitrogen. and thawed on day 1. Experiment I took place on day 1, experiment II on day 2, and experiment III on day 12. The same protein preparations were used for each experiment, and stored at 4°C during the intervening time. A fresh portion of Dh’A donor extract from the same batch was thawed for each experiment.

As shown in line 2 of Table 1, addition of coat and scaffolding protein to the DXA donor extract resulted in 100 to lOO@fold increase in viable phage over background in those extracts prepared without chloroform. In subsequent experiment’s, extracts were prepared with lysozyme and EDTA, and reactions were incubated at 23°C. The possibility remained that the phage produced when the coat and scaffolding protein were added to the DNA extract were the result of packaging of DNA into a small number of procapsids present as contaminants in either the coat or t,he scaffolding protein preparations. The experiments shown in Table 2 demonstrate that addition of both the coat and the scaffolding protein preparations is required for assembly of active phage; the phage produced could not have been the result of previously assembled procapsids present in either preparation. Table 2 shows the results of three separate complementation experiment)s performed over a 12-day period using the same protein samples. Between experiments, the protein and procapsid samples were stored at 4°C in buffer B. Portions of a single preparation of DNA donor cells were freshly thawed for each experiment. The first line of Table 2 shows the results of complementation of the DNA donor extract with a standard sample of purified 2- procapsids (10’2/ml). Roughly lo6 phage were produced above background. The variation in complementation activity from day to day was typical, and probably reflects variation in the activity of the DNA donor extract preparations. When the scaffolding protein alone was added to the extract, only background levels of phage were detected. Addition of the coat protein alone to the extract in experiment 1 may have produced more than the background level of phage : in most

M. T. FULLER

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experiments, coat protein added to the extract alone did not yield an increase over background. When both the coat and the scaffolding protein were added to the DNA donor extract, phage were produced 20 to 400-fold above the background level. This indicates that the soluble subunits of the coat and scaffolding proteins added to the extract assembled into active procapsids in vitro. The minor proteins, gpl, gp7, gp16 and gp20, which must be incorporated into procapsids if infectious phage are to be produced, were presumably provided by the DNA donor extract. Although fewer phage were produced by complementation with coat and scaffolding proteins in experiment III, the yield of phage from the control procapsid complementation was also less than in the previous experiments. Some of the decrease in yield may have been due to a less active preparation of DNA donor extract.

(a)

14 t

142 2 ‘0 x 2 _”

(b’

IO-

6-

0.1 p!$++A&----q I I I IO 30

60

----I 90

I 120

\\r* ‘*

Time (mm) FIG. 2. Coat protein was dialyzed against buffer B at 4°C to remove Gu HCl. Procapsid and protein samples were the same as those used in the experiment presented in Table 2. DNA donor extract (50 ~1) prepared with lysosyme and EDTA was mixed with 50 ~1 of protein donor and 10 ~1 of 150 mwATP, and incubated at 23°C for the indicated time. Reactions were terminated by dilution with cold dilution fluid, and titred on DB7002. (a) Complementation in vitro by 2- procapsids; (h) Complementation in vitro by soluble coat (5) and scaffolding (8) proteins. Complementing species were: (A) procapsids; ( x ) buffer B ; (a) coat and scaffolding protein mixed ; (0) coat protein alone ; ( - - - - ) scaffolding protein alone. p.f.u., plaque-forming units.

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of phage production

t&t:1

in vitro

The time-course of phage production by complementation in vitro of the DS.4 donor extract with procapsids and with soluble proteins are compared in thr experiment shown in Figure 2. Addition of intact procapsids to the crude extract at This stopped b> 23°C was followed by a rapid increase in viable phage production. approximately 60 minutes after the start of the reaction. The production of phage by complementation in U&O with soluble coat and scaffolding protein at 23°C did not begin until at least 60 minutes af%er the start of the incubation. By 120 minutes, phage production had begun to plateau. The longer time required before onset of phage production upon complementjatSion with solul)le protein presumably reflects t’he time necessary for assembly of activcl procapsids in vitro. Once again. phage production was dependent on t)he addition of both coat) and scaffolding prot)ein to the extracts. Since phage production was 100.fold higher when both proteins were added to the reaction than when either was added alone. the production of phage in the extracts camlot be due t,o the packaging of DEu‘A into procapsids already formed and present as contaminants of eit,her of the protein preparations. Similarly. the dependence on both proteins rules out events due t’o viable phage in the protein preparation infectSing the surviving bacteria in the tJxtract. active coat and scaffolding (c) Assay of biologically during pu,ri$cation from 1’22 procapaids

protein

Altjhough the product,ion of phage by assembly in vitro of active procapsids from purified coat and scaffolding protein in the DK’A donor extract was significant,ly over background. phage production in this syst,em was still very inefficient. In order to determine if the active material in t,he protein preparation had the properties of the purified coat and scaffolding protein, we followed the behavior of t’he biological act,ivity during the purification of soluble coat’ and scaffolding subunits. Since the production of phage in vitro depends on the addition of both the (aoat and scaffolding protein to the 5- DNA donor extract,, t*o measure the actirit> of the scaffolding prot,ein in a set of column fractions, we supplemented each c~olumn fract’ion with a known amount of a standard coat protein preparation before adding the complementation extract. The reverse was true for the measurement of coat protein activity : each fraction assayed was supplemented with a constant amount of a standard scaffolding protein preparat,ion. The standard coat and scaffolding protein preparations were the same as t#hose described in Table 2. P22 procapsids purified from I- infected cells were treated with 0.5 M-GII . HCl t’o release the scaffolding protein, and the minor proteins gp16 and gp20 from the structure (Fuller & King, 1981). The released proteins were then separated from the int’act coat protein shells on a Biogel A-5m column run in 0.5 M-GLI. HCl. Figure 3 shows the elution profile of protein from the column. The coat protein remained in intact shells and eluted in the void volume of t,he column. The scaffolding prot,ein and the minor proteins gp16 and gp20 were included in the column. The scaffolding

M. T. FULLER

644 (a

ASD

J. KIXG

1

I-O

-

Void

7

!.O

0 ;

f , 2 d

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I.0

0.5

30

20 Froctlon

number

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number

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< F.

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protein migrated through the column as a single peak ; the minor proteins migrated independently of the scaffolding protein but with an overlapping distribution, as shown by SDS/polyacrylamide gel electrophoresis of the column fractions (Fig. 3(b)). Following dialysis to remove the Gu .HCl. samples from individual column fractions were supplemented with an equal volume of the standard coat protein preparation and kept on ice. After addition of the DNA donor extract, the reactions were mixed and incubated overnight at 23”C, and titred for immL phage produced by DNA packaging in vitro. The scaffolding protein in the column fractions was able to complement the DNA donor extract, but only when the exogenous coat protein was added to the reaction. When the column fractions were supplemented with buffer instead of with coat protein, no phage were produced The complementation activity in vitro was found in a single peak, coincident with the trailing half of the peak of protein included in the column. In the presence of a c,onstant’ amount of supplementary coat protein, the complementation activity was roughly proportional to the concentration of the scaffolding protein for the trailing half of the activity peak. The leading fractions of the scaffolding protein peak had greatly reduced specific activity. We do not know the reason for this decrease. Column fractions containing scaffolding protein from the Biogel ,4-5m column (Fig. 3) were pooled, dialyzed, and passed through a DEAE-cellulose ion-exchange column. The bulk of the minor proteins gp16 and gp20 did not bind to the column. The scaffolding protein did bind, however. and was eluted with a gradient of IVaCl (Fuller & King, 1981). Figure 4 shows the activity profile of the scaffolding protein eluted from the DEAE-cellulose column. The peak of complementation activity in oifro coincided with the peak of protein concentration measured by optical density. Again, the scaffolding protein had no complementation activity in vitro unless supplemented by the addition of coat protein to the reaction. Similar results were found with scaffolding protein purified from 2- procapsids. In both this and t,he previous experiment, the complementation activity of the supplementary coat protein was very close to background when it was added to column fractions containing little or no scaffolding protein. FIN. 3. Complementation activity in vi&o of scaffolding protein released from l- procapsids after gel filtration. Scaffolding protein was released from l- procapsids by treatment with 05 M-GU . HCI for 30 min at 37°C. The treated sample was passed over a Biogel A-5m gel filtration column in buffer B with 05 M-GII HCI. The scaffolding protein eluted from the column in fractions 24 to 34. (a) (‘omplementation activity of column fractions. Protein concentration was monitored by measuring absorbance at 280 nm. Fractions were dialyzed individually at 4°C against buffer B to remove the Gu . HCI : 25 ~1 of each column fraction was mixed on ice with 25 ~1 of buffer B or 25 ~1 of a st,andard coat protein preparation (1% mg/ml). The coat protein standard was the same sample used in the experiment shown in Table 2. After addition of 50 ~1 of fresh DNA donor extract prepared with lysozyme and EDTA as described in Materials and Methods, the mixtures were vortex mixed and incubated overnight at 23°C. Samples were then diluted with cold dilution fluid and tit& for phage with the immL genotype on DB7002(P22 int-l&e-). .r\bsorbance at 280 nm (0); phage produced by complementation in vitro when mixtures were supplemented with coat protein ( x ); phage produced by complementation in m&o when mixtures were supplemented with buffer B (A). p.f.u., plaque-forming units. (b) Protein content of column fractions assayed by SDS/polyacrylamide gel electrophoresis. After dialysis to remove Gu HCI, column fractions were mixed with sample buffer, incubated at 100°C for 2 min and electrophoresed through a polyacrylamide slab gel in the buffer system of Laemmli (1970) and King PELaemmli (1971). Proteins were stamed with Coomassie blue,

M. T. FULLER

15

30

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40

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50

Fraction number FIG. 4. Complementation activity in vitro of scaffolding protein purified on DEAE-cellulose. Fractions containing scaffolding protein, described in the legend to Fig. 3, were pooled, dialyzed to remove the Gu. HCI, and layered on a 20-ml DEAE-cellulose column run in buffer B at 4°C. Scaffolding protein was eluted from the column with a 0.0 to 0.5 M gradient of NaCI. Ionic strength was monitored by measuring conductivity. The scaffolding protein peak elukd at 85 mM-NaCl (total molarity). Column fractions were supplemented with coat protein or with buffer B, mixed with freshly thawed DNA donor extract, incubated and titred exactly as described for Fig. 3. Absorbance at 280 nm (0); conductivity (M-N&I) (0); phage produced by complementation in titro when mixtures were supplemented with coat protein ( x ); phage produced by complementation in vitro when mixtures were supplemented with buffer B (A). p.f.u., plaque-forming units.

Soluble coat, protein was obtained by dissociation of the empty coat protein shells remaining after release of the scaffolding protein from 1>22procapsids. Shells were treated with 3 M-GU . HCf and passed over a Biogel A-5m gel filtration column eluted with 2 M-GU . HCl to separate the soluble protein from undissociated structures (Fuller & King, 1981). Figure 5 shows the distribution of the complementation activity in vitro of the soluble coat protein in the Biogel A-5m column fractions following dissociation of I - shehs with 3 M-GU . HCI. Column fractions were dialyzed against buffer B at 4°C to remove the Gu. HCI, and then mixed with an equal volume of either buffer or the standard supplement,ary scaffolding protein preparation. After addition of DNA donor extract, the reaction mixtures were incubated overnight at 23°C and titred as described above. The peak

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complementation activity in vitro coincided with the peak of absorbance for the included coat protein. Although supplementary scaffolding protein enhanced complementation in this experiment. the coat protein contained in the column fractions was able to complement, the extract without the addition of supplementary scaffolding protein. This activity was probably not due to residual scaffolding protein contaminating the column fractions. Analysis by SDS/polyacrylamide gel electrophoresis (Fig. 4(b)) showed only traces of scaffolding protein. The complementation activity of the coat protein alone in this experiment may have been due to the endogenous scaffolding protein in the DNA donor extract. This effect was probably not seen in the previous experiments because the concentration of supplementary coat prot,ein in those experiments was lower than the coat protein concentration in the pea,k fractions of this column. These experiments show that the ability to stimulate phage formation in the extract was associated with the coat and scaffolding subunits themselves. and not with procapsids. phage particles or small molecular weight factors. (d) Assembly

in vitro of procapsid-like

particles

from,

purified

proteins

The low efficiency of conversion of subunits to mature particles in the crude extracts limited our ability to study the assembly reactions directly. We therefore attempted to find conditions in which the purified proteins would assemble int,o shell structures in the absence of infected-cell extracts. The successful protocol was essentially the reverse of the dissociation process used to prepare the proteins. Purified coat and scaffolding protein were mixed in 1.5 M-GU . HCl, which was then removed by dialysis overnight against buffer B. and the reaction mixtures were analyzed for assembly by electron microscopy and by sucrose gradient centrifugation. Figure 6(a) shows an electron micrograph of a negatively stained sample of the coat and scaffolding protein reaction mixture after dialysis at 23”t overnight. Procapsid-like structures are plentiful. Some aberrant) structures arc present, but, about 80% of the particles in the field show the external morphology and internal density characteristic of procapsids from infected cells (Botstein et al., 1973 ; Earnshaw B King, 1978). When dialysis took place at 4°C instead of at 23”(‘, very few procapsid-like structures were observed (Fig. 6(b)). To analyze the products of the incubation more quantitatively. the dialyzed samples were fractionated by sucrose gradient centrifugation and the protein content of each gradient fraction was determined by SDS/polyacrylamide gel electrophoresis. Figure 7 shows the sedimentation behavior of the purified proteins after dialysis at 23°C. The top gel (5) shows the gradient profile of coat dialyzed alone. All of the coat protein was found at the top of the gradient. The third gel (8) shows the sedimentation behavior of scaffolding protein dialyzed alone. All of the scaffolding protein was also found at the top of the gradient. III both cases, no higher molecular weight structure was detectable in the sucrose gradient. When the coat and scaffolding protein were mixed and dialyzed, (5+8), a significant portion of the protein formed a rapidly sedimenting complex, srdimrntinp similarly Taoprocapsids and with a similar ratio of gp5 t)o gp8. Roughly

648

M. T. FITLLER

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(0) 2.

I4

Ammo

/

20 Froctlon

acid

30 number

(b)

-4P5

20 Fraction

Frc:. 5.

number

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30 to 40% of the total protein present in the reaction mixture sediment& as procapsid-sized material. The bottom gel (PH) shows a parallel sucrose gradient, upon which was layered reference procapsids made in vivo. (Due to overloading of the gradient, some of the procapsid material sedimented to the bottom of the gradient.) The higher molecular weight structures formed in vitro in the mixture of coat and scaffolding protein (5 + 8) was found in the same gradient fraction as the peak of the distribution of reference in vivo procapsids in the parallel gradient (PH). The ratio of coat to scaffolding protein in the in vitro structures was similar to that in the reference procapsids, even though scaffolding protein wa.~ in excess in the original reaction mixture. When the Gu. HCl was removed by dialysis at 4°C instead of 23”C, no higher molecular weight aggregates were detected by similar sucrose density gradient analysis. The protein sedimented at the top of the gradient in all three cases: coat protein dialyzed alone, scaffolding protein alone, and the mixture of coat and scaffolding proteins. (e) Assembly of aberrant particles from more concentrated coat protein preparations In the experiments described above, the scaffolding protein was in excess over the coat protein in the assembly mixtures. However, roughly twice as many coat protein as scaffolding protein subunits are contained in a procapsid. Coat protein preparations that were five- to ten-fold more concentrated than those described above were mixed with the same scaffolding protein samples previously employed so bhat the coat protein was in excess in the reaction mixtures. Under these conditions, some of the coat protein precipitated upon dialysis from 15 M-GU . H(‘1 at 23°C. Precipitates were not formed in reaction mixtures dialyzed at 4°C. Figure 8 shows electron micrographs of the reaction mixtures after dialysis at 23°C. As in the previous experiments, the mixture of coat and scaffolding protein dialyzed at 23°C contained many particles that resembled P22 procapsids (Fig. 8(b)). Scaffolding protein dialyzed alone at 23°C (Fig. 8(c)) contained only a FIG. 5. Complementation activity in w&o of coat protein dissociated from l- shells with 3 M-GU HVl. I - procapsids were treated with @5 M-Gu . HCI to release the scaffolding protein and the minor proteins gp16 and gp20 as described for Fig. 3. Intact coat protein shells from the void volume of the column shown in Fig. 3 were pooled and concentrated with an Amicon ultrafiltration device with filter PM30. The l- shells were then dissociated with 3 M-GU HCl for 30 min at 37°C and loaded on a Biogel A-5m gel filtration column equilibrated with 2 M-GU . HCI in buffer B. (a) Complement&ion activity of column fractions. The column &ant was monitored for absorbance at 280 nm. Column fractions were individually dialyzed against buffer B at 4°C to remove the Gu HCl 25 ~1 of each fraction was then mixed with 25 ~1 of a standard scaffolding protein preparation or with 25 $ of buffer B. After addition of 50 ~1 of fresh DNA donor extract prepared with lysozyme and EDTA, the reaction mixtures were vortex mixed and incubated at 23°C overnight. Reactions were diluted with cold dilution fluid and titred on DB7002 (P22 d-/tie-) to detect phage with the immL genotype of the DNA donor extract. The scaffolding protein standard was the same preparation used in t,he experiments shown in Table 2. Absorbance at 280 nm (a); phage produced by complementation in vitro when the column fractions were supplemented with scaffolding protein ( x ) ; phage produced by complementation in vitro when the column fractions were supplemented with buffer B (A). p.f.u.. plaque-forming units. (b) Protein content of column fractions assayed by SDS/polyacrylamide gel electrophoresis. After dialysis to remove Gu HCI, column fractions were mixed with sample buffer. incubated at 100°C for 2 min and electrophoresed through an SDS/polyacrylamide slab gel.

650

M. T. FULLER

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few head-related structures, probably undissociated procapsids present as contaminants in the scaffolding protein preparation. The micrograph of the concentrated 2- coat protein dialyzed alone at 23°C (Fig. 8(a)) shows a number of head-related structures. However, as described below, these particles did not sediment as procapsids and they did not contain scaffolding protein. Again, when the coat and scaffolding proteins were dialyzed at 4”C, they remained primarily unassembled. Only a few head-related st,ructures were seen in samples of either the scaffolding protein alone or the coat protein alone. A feM procapsid-like particles were observed by electron microscopy in the mixture of coat and scaffolding protein dialyzed at 4”C, but the level of particle production remained below that detectable by sucrose gradient analysis. The relative amount of procapsid assembly in the reaction mixtures dialyzed at 23°C’ was analyzed by sucrose gradient centrifugation. The protein content of each gradient fraction was analyzed by SDS/polyacrylamide gel electrophoresis and is shown in Figure 9. As in the previous assembly experiment, particles with the same A value as P22 procapsids (fractions 7 to 9) were formed when coat and scaffolding protein were mixed together and dialyzed at room temperature (Fig. 9(b)). Although the concentration of the coat protein was fivefold higher than in the experiment shown in Figure 7, the yield of procapsids was not increased proport’ionately. Once again, a significant amount of the coat and scaffolding protein present in the mixture remained at the top of the gradient. When the concentrated coat protein alone was dialyzed at, 23°C. two fastsedimenting species of higher molecular weight structures were formed (Fig. 9(a)). One population sedimented slightly slower than procapsids. while the ot.hrr populat,ion sedimented slightly faster. These two populat,ions correspond to the aberrant aggregates of coat protein that form in cells infected with mutants defective in scaffolding protein synthesis (King et al., 1973: Earnshaw 6 King. 1978). The more rapidly sedimenting material included spiral and other aberrant’ aggregates (Earnshaw & King, 1978), while the more slowly sedimenting material included closed empty shells. Few structures with the same s value as procapsids were formed. When scaffolding protein was present in the mixture. all higher molecular weight particles formed had the characteristic procapsid Rvalue. and felr aberrant shells were detected in the sucrose gradient (Fig. 9(b)). As in the previous rxperiment, scaffolding protein dialyzed in the absence of coat protein remained unaggregated and sedimented at the top of the gradient (Fig. 9(d)). Figure 10 shows micrographs of negatively stained samples of the sucrose gradient fract,ions corresponding to the procapsid s value for reaction mixt,ures dialyzed at 23°C. For comparison, Figure 10(d) shows sucrose gradient’-purified procapsids, formed in viva in a 2-/13- infection. The procapsid-sized products of t.he ir/ vitro reaction (Fig. 10(a)) resemble the procapsids produced in ~qi~o Frc:. 6. Electron micrographs of procapsid-like particles formed in vitro. Reaction mixtures were made as follows: 300 ~1 of coat protein in 2 M-Gu.HCl in buffer B was mixed with 100 ~1 of scaffolding protein in buffer B and dialyzed overnight against buffer B at 23°C. Scaffolding protein was in slight molar excess over coat protein. A drop of the dialyzed reaction mixture was applied to a carbon-coated copper electron microscope grid. and stained with 2% (w/v) uranyl acetate (pH 45). The bar represents @l +v. (a) (‘oat and scaffolding protein dialyzed at 23°C; (b) coat and scaffolding protein dialyzed at 4°C’.

652

M. T. FULLER

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tJ. KING

PH 1 bottom

Sucrose gradient fraction number mc:.7.

top

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(Fig. 10(d)). F’g 1 ure 10(b) and (c) shows the gradient fractions corresponding to the procapsid s value for coat and scaffolding proteins dialyzed separately. Few, if any, procapsid-sized particles were observed in the gradient fraction corresponding to procapsid sedimentation when either one of the two major structural proteins was removed from the reaction mixture. Those observed are probably due to the few higher molecular weight structures contaminating the soluble protein preparations or, in the case of the coat protein, to the low level of aberrant structures formed in vitro by concentrated coat protein. (f) Assembly in vitro ofprocapsid-like

particles in the absence of the gene 1 protein

The preparation of coat protein used in the above assembly experiments was prepared from 2- P22 procapsids and could have contained very small amounts of the minor protein gpl. Since gpl is normally included in procapsids in only a few copies per particle, even this small amount might be significant. To determine if the gpl protein was a required initiator protein for assembly of procapsid-like structures in vitro, similar to gp20 of T4 (Van Driel & Couture, 197&b). we prepared coat protein from procapsids purified from cells infected with phage carrying an amber mutation in gene 1. This purification was carried out in parallel with the preparation of coat protein from 2- procapsids shown in Figure 9(c). gpl was thus genetically removed from the coat protein samples. The sample of coat protein from the I- infection was mixed with scaffolding protein and dialyzed at 23°C in parallel with the coat protein from 2- procapsids as described above. A single class of high molecular weight aggregates, corresponding to procapsids in s value and in ratio of coat to scaffolding protein, was found in similar yield when scaffolding protein was dialyzed with coat protein prepared from either I- or 2procapsids (Fig. 9(b) and (c)). Thus gpl was not required for the initiation of assembly of procapsid-like structures from purified coat and scaffolding protein itz vitro. (g) Characterization

of free subunits by sedimentation

Although no procapsid-sized aggregates were found at 4”C, small aggregates of coat and scaffolding protein may have formed, and these would sediment at the top of the gradients shown in Figure 7. To determine the assembly state of the small molecular weight material remaining after dialysis, we centrifuged samples from

Fu:. 7. In vitro assembly experiment; dialysis at 23°C. Assembly reaction mixtures were made as described in Materials and Methods: 300 ~1 of coat protein with A2s0 0475 in 2 M-Gu. HCI in buffer B was mixed with 100~1 of scaffolding protein in buffer B with 5OpM additional NaCi. Gu’HCI was removed by dialysis (overnight against 2 changes of 250 ml each of buffer B at 23°C). Reaction mixtures were centrifuged for 40 min at 35,000 revs/min at 20°C on 5% to 20% (w/v) sucrose gradients with @3 ml 60% sucrose cushions in a Beckman SW50.1 rotor. Gradient fractions were analyzed by SDS/polyacrylamide gel electrophoresis. The rightmost lane of each gel in the Figure shows the protein content of the top gradient fraction. The direction of sedimentation was from right to left. The leftmost lane in each panel shows a sample of P22 procapsids applied to the gel for markers. 5: coat protein mixed with buffer B. 5 + 8 : coat protein mixed with scaffolding protein. 5 : scaffolding protein in buffer B. PH, Sediment,ation on a parallel sucrose gradient of purified control P22 procapsids from a Z-/13- infection.

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’

I 3

1

I 5

1

I 7

’

I 9

Roction

1

I II

’

1 13

’

I 15

’

I 17

I Top

Scoffold

ON

number

Fro. 9. Assembly of procapsids from parallel I- and l+ preparations of coat protein. Reaction mixtures were analyzed by sucrose gradient centrifugation and SDS/polyacrylamide gel electrophoresis as described for Figs 7 and 8. Right lanes show samples loaded on gradients.

the reaction mixtures through sucrose gradients at 45,006 revs/mm for 20 hours at 4°C. Figure 11(a) shows the sedimentation behavior of the coat protein alone. The distribution of gp5 was relatively symmetric, and sedimented slightly more slowly than hemoglobin. The sedimentation coefficient calculated with reference to hemoglobin and lysozyme was 3.9 S. The sedimentation of the scaffolding protein is shown in Figure 11(b). It sedimented only slightly faster than lysozyme, at about 2.4 S. The suggestion of a shoulder on the leading edge of the scaffolding protein distribution was not observed in other experiments. Figure 11(c) shows the sedimentation profile of the mixture of coat and scaffolding protein after dialysis at 4°C. The distributions of the two proteins were overlapping, but independent. The coat and scaffolding protein in the mixture displayed the same sedimentation behavior as the purified coat and scaffolding protein centrifuged separately (Fig. 1 l(a) and (b)). We detected no small mixed multimeric aggregates of the coat with the scaffolding protein, coat with coat protein, or the scaffolding protein with itself. If we compare the molecular weight (67,000) and s value of the hemoglobin tetramer (4.3 S) with the molecular weight of the polypeptides of the coat (55,000) and scaffolding (42,000) proteins, determined by SDS/polyacrylamide gel FK. 8. Electron micrographs of mixtures of scaffolding protein with concentrated preparation of coat protein after dialysis at 23°C. Concentrated coat protein (A,,, 1.8) was mixed with scaffolding protein as described in the legend to Fig. 7, and dialyzed against buffer B at 23°C. Samples from the onfractionated reaction mixtures were applied to the grids and stained with uranyl acetate. The bar represents 0.1 pm. (a) Coat protein: (b) coat protein and scaffolding protein; (c) scaffolding protein

656

M. T. FULLER

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J. KING

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Fmctlan (a)

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I

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(cl

Fu:. 11. Sucrose gradient sedimentation of coat and scaffolding subunits. Samples (100 ~1) of the coat (C) protein (a), scaffolding (S) protein (b), and the mixture of both (c), all dialyzed from Gu. HCl at 4C. were mixed with 10 ~1 of 10 mg hemoglobin/ml (H) and 10 ~1 of 10 mg lysozyme/ml (L), layered on a prechilled 5% to 20% sucrose gradient, centrifuged for 20 h at 45,000 revs/min at 4°C in a Beckman SW50.1 rotor and collected in @2 ml fractions. Protein content of gradient fractions was analyzed by HDS/polyacrylamide gel electrophoreeis and microdensitometry as described in Materials and Methods.

electrophoresis (Botstein et al., 1973), we see that both the coat and scaffolding proteins probably sedimented as monomers (Martin & Ames, 1961). When the coat and scaffolding proteins were mixed and dialyzed at room temperature under assembly conditions, a significant amount of the coat and scaffolding proteins remained unassembled, as shown in Figure 7(b). This unassembled coat and scaffolding protein also sedimented independently as monomers upon longer centrifugation (data not shown). Thus, even under conditions that favored the assembly of procapsid-like particles, no intermediate complexes of the coat and scaffolding protein accumulated. Although we did not detect small multimeric complexes of the coat and scaffolding proteins in the reaction mixtures by sucrose gradient centrifugation, we occasionally saw small cyclic structures in negatively stained samples in the electron microscope (Fuller & King, 1981). These complexes appeared to be fivesided. The observation of these complexes in the electron microscope led us to reexamine whether complexes of the proteins existed in solution. To rule out the Fro. 10. Electron micrographs of sucrose gradient-purified procapsid-sized particles formed in vitro. After dialysis at 23”C, the reaction mixtures shown in Fig. 8 were centrifuged for 40 min at 35,000 revs/min at 20°C. Gradients were collected in consecutive @3 ml fractions from a pinhole slightly offcenter from the bottom of the tube. Procapsid-like structures, assembled when both coat and scaffolding protein were present in the reaction mixtures, were found in gradient fraction 7. The corresponding gradient fraction from each assembly mixture was applied to a carbon-coated electron microscope grid. wmhed with 5 drops of distilled water and stained with 2% uranyl acetate. The bar represents @l pm. (a) Coat protein plus scaffolding protein ; fraction 7 ; (b) coat protein alone ; fraction 7 ; (c) scaffolding protein alone; fraction 7 : (d) procapsids formed in viva in a Z-/13- infection.

658

M. T. FULLER

Stokes’

AND

J. KING

radius (8)

FIG. 12. Determination of Stokes’ radius of coat and scaffolding protein by molecular exclusion chromatography. The elution behavior of the purified coat and scaffolding proteins were measured with the same column of Sephacryl S-200. The column was first calibrated (0) with a mixture of the marker proteins lysozyme, hemoglobin (dimer), bovine serum albumin (BSA), yeast alcohol dehydrogenaae (YADH). catalase and P22 tail protein (gp9). In the second run (A) the scaffolding protein (gp8) was chromatographed with a mixture of lysozyme, hemoglobin, bovine serum albumin, yeast alcohol dehydrogenase and catalaee. In the third run ( x ) the purified coat protein was chromatographed with a mixture of hemoglobin, bovine serum albumin, yeast alcohol dehydrogenase and catalase. To all samples, P22 phage were added to mark the void volume of the column, and “C-labeled amino acids were added to mark the exclusion limit. The elution volume (V,) of each protein was plotted in terms of ( -log &)I’* W-SW the Stokes’ radius, where : K,, = V, - VJV, - V,,, where V, was the elution volume of P22 phage, and V, was the elution volume of 14C-labeled amino acids in the same column run. The data were fit to a straight line by a lea&-squares method. Stock solutions of the marker proteins are described in Materials and Methods. Data from 3 separate run8 are combined. Marker run: 10 rg of P22 phage, 10 pg of gp9, 50 pg of catalaee, 50 pg of hemoglobin, 100 pg of lysozyme, 100 pg of yeast alcohol dehydrogenase, 50 pg of bovine serum albumin, 2 pg of 14C!-labeled amino acids, 1 drop of glycerol. Scaffolding protein run: 300 pg of scaffolding protein (1.3 mg/ml), purified by DEAE-cellulose chromatography a8 described above, 10 fig of P22 phage, 25 rg of catalase, 25 rg of hemoglobin, 25 pg of lysozyme, 25 rg of yeast alcohol dehydrogenase, 2 pg of 14C-labe1ed amino acids, 1 drop of glycerol. Coat protein run : 250 pg of coat protein (2 mg/ml), purified by gel filtration in 2 M-GU . HCl and dialyzed against buffer B at 4”C, 10 pg of P22 phage, 50 pg of catalaee,50pg of yeast alcohol dehydrogenase, 50 rg of bovine serum albumin, 50 pg of hemoglobin, 2 pg of 14C-labeled amino acids, 1 drop of glycerol.

possibility that the sucrose itself was effecting the state of the subunits, we determined the sedimentation behavior of the coat and scaffolding proteins in gradients stabilized by ‘H,O. The two proteins sedimented similarly, with respect to hemoglobin and lysozyme markers, as they had in the sucrose gradients (data not shown). (h) Determination

of the Stokes’ radii of the coat and scaffolding by molecular exclusion chromatography

proteins

To explore the conformation of the coat and scaffolding protein molecules, we estimated the Stokes’ radii of the purified proteins by gel filtration on a column

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of Sephacryl S-200 (Siegel & Monty, 1966). Samples of the coat and scaffolding probein were separately mixed with marker proteins of known Stokes’ radius and chromatographed sequentially on the same column. Figure 12 presents data from three separate runs as ( -log K,,) li2 wersus Stokes’ radius (Siegel & Monty, 1966) t,o obtain a linear relationship between the elution volume of a given protein and its Stokes’ radius. The Stokes’ radii of the coat and scaffolding proteins were interpolated from the Stokes’ radii and elution volume of standards from the samcl column run. The Stokes’ radius of the coat protein was approximately 25 A and thfl Stokes’ radius of the scaffolding protein was approximately 34 A. Andrews (1964) found that hemoglobin was largely present as a dimer in dilut’r solutions comparable to those present in our experiments. The hemoglobin standard eluted from this column as expected for a molecule with the Stokes’ radius of the hemoglobin dimer. We rstimat,ed the axial ratios of the coat, and scaffolding proteins from t,heir calculated frictional ratios, flfO, according to Van Holde (1971), where f is the frict)ional coefficient, of the molecule calculated from its measured Stokes’ radius (a), and f0 is the frictional coefficient of an unhydrated sphere with t,he same molrcular weight. The calculation was based on the value of 0.725 cm3jg for the partial specific volume (V) of an average protein (Martin 8; Ames, 1961) and the monomer molecular weight for the two molecules, as determined by SDS/polyacrylamide gel electrophoresis (Botstein et al., 1973). f/‘,, = a/[(31jM4 ! LV)]“~ (Siegel & Monty, 1966). For a monomer of the coat protein, M, is 55,000. This gives a value for flfO of approximately 1, corresponding to a spherical molecule. For the scaffolding protein, flfO = 1.5 if we assume that the protein migrated in the column as a monomer of 42,OOOM,. This value offifo corresponds to a prolatr ellipsoid with an axial ratio of 9, or to an oblate ellipsoid with an axial ratio of 11.

4. Discussion The results reported above indicate that t,he coat and scaffolding subunits obtained from dissociation of procapsids can serve as precursors in the formation of infectious phage particles. Monomers appear to be the active form of both protein species. They show little or no tendency to aggregate by themselves. The simplest model consistent with our observations is as follows. Initiation of shell assembly requires the interaction of both species; once the shell is initiated, presumably at a specialized vertex, growth is through incorporation of alternating monomers: the incorporation of the monomers at the growing edge probably switches the subunits from the unreactive to the reactive state (Caspar, 1976 ; King. 1980). We have found no evidence to suggest that the scaffolding subunits first polymerize into an inner shell, which then serves as a t,emplate for coat prot,ein polymerization. A model of the polymerization pathway described above is shown in Figure 13(a). The coat protein lattice is drawn according to Casjens (1979). The scaffolding subunits have been placed at the 2-fold axes, which satisfies the stoichiometry of the subunits, and provides for an exit mechanism (Fuller & King, 1980.1981). As depicated in Figure 13(b), the scaffolding-scaffolding interactions occur at an inner

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5-fold complex

61

Coot

B!nds 5 scoffoldlng protein molecules

subunIt

And more scaffold

-

Scaffoldlnq

Coat,

protein

binds

Coat and scaffolding proiems bind sequenllolly to form shell 1

subunIt

(b)

Fro. 13. Procapsid assembly and organization. An assembly pathway consistent with our observations both in viva and m vitro. and incorporating findings from other double-stranded DNA phages (Murialdo & Becker, 197&z), is shown in (a). Though the gene I protein is not required for polymerization in vitro, it probably forms the initiating vertex together with other minor proteins in viva. Since the coat protein alone can polymerize, it has been shown as substituting for gpl in vitro. However, formation of this initiating B-fold vertex could proceed from the scaffolding protein or could require the interaction of both proteins, Once the shell is initiated, the coat and scaffolding subunits bind sequentially to sites on the growing shell, generating further reactive sites. The geometry of the subunit arrangement follows that reported by Casjens (1979), and the placement of the scaffolding subunits at local 2-fold axes satisfies the stoichiometry of the 2 proteins. Three-dimensional representation of the orientation of coat and scaffolding proteins shown in (b). The shape of the coat protein subunit is arbitrary. The long, slender shape of the scaffolding molecule is consistent with the axial ratio reported here, and with its ability to exit through the coat protein lattice (Griffin-Shea, 1977; Fuller & King, 1981). The location of the scaffolding molecules is consistent with the radial density profile of procapsids determined by X-ray scattering (Earnshaw et al.. 1976).

radius of the particle, which is not shown in the surface lattice of Figure 13(a). We presume that these sites are activated only after scaffolding subunits have interacted with the growing coat’ protein lattice: otherwise, it is difficult to understand why the scaffolding subunits do not self-aggregate. The copolymerization on the growing shell edge explains our failure to detect small mixed complexes of coat, and scaffolding subunits. This self-regulated assembly model differs from condensation polymerization in that the subunits are not in equilibrium between the two states in free solution (Oosawa & Asakura, 1975). Rather, the surface of the growing structure serves as a catalytic surface for inducing the conformational transition to the reactive conformation (Caspar, 1976; King, 1980). The guanidine-induced dissociation of the procapsid into free subunits can therefore be viewed as resulting from the switching of the subunits from the

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reactive (binding) form, back into the unreactive precursor state. We are attempting to obtain direct evidence of such subunit switching by laser Raman spectroscopic analysis of the free and organized forms of the subunits (Fish et ~1.. 1980). The question of the initiation of shell assembly is discussed in more detail l,f?lOW. (a) Complementation

in vitro

The complementation of the 5--infected cell extract was dependent, on the addition of both the coat and scaffolding protein, even though 5- extracts contain scaffolding subunits. However, because of the autogenous regulation of scaffolding protein synthesis (King et al., 1978), scaffolding synthesis is depressed to a very low level in 5--infected cells. The requirement for exogenous subunits probably reflects this low level accumulating in the cells. The 5- DNA donor extract also provided the gpl, gp7, gp16 and gp20 needed to assemble biologically active procapsids. We have no information as to whet,her these proteins were accumulating as free subunits, in a complex. or associated with the low level of scaffolding subunits present in the extracts. Poteete et al. (1979) 5- extracts attempted to assemble active procapsids in vitro by mixing directly with 8- extracts. They did not detect complementation above background in the mixtures. This may have been due to insufficient scaffolding protein accumulating in the 5- extract, as just described, or to the coat protein in the 8- extract accumulating primarily as aberrant aggregates (Earnshaw & King, 1978). Poteete et al. (1979) found that the efficiency of conversion of procapsids to liable phage in the DNA packaging extracts was about 1 in a 1000. The highest yields of viable phage obtained by complementing the extracts with coat, and scaffolding subunits was 10’ to lo6 phage per ml. Given bhe above factor, this must have represented t’he assembly of 10’ to 10’ complete procapsids per ml in t,he crude extracts. However. we know from the in vitro assembly experiments in the absence of extracts, that coat and scaffolding subunits can assemble into procapsid-like particles wit,hout the minor proteins. The yield of these particles was of the order of 10” per ml or higher. Such particles assembling in the crude ext’ract, without incorporation of a full complement of their minor proteins, could not have been converted to viable phage and would not have been detected. For both the coat and scaffolding proteins, the activity profile of the final step in the protein purification, as determined by complementation in vitro, corresponded to the absorbance profile at 280 nm. For the coat subunits, the biologically active polypeptides had the same size distribution as the bulk protein eluted from a Biogel A-5m column, run in the presence of 2 M-Gu. HCl. For the scaffolding protein. the active polypeptides shared the same affinit,y for DEAE-cellulose as the bulk of the protein. (b) Assembly

in vitro from pure

subunits

Both the coat and scaffolding proteins were required for efficient assembly of procapsid-like structures in the assembly reaction in z&o. Under the conditions of the assembly reaction, the scaffolding subunits did not form high molecular weight organized structures. This is consistent with the results obtained in vitro, where no organized structure of scaffolding protein has been detected in cells infected with

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mutants defective in the coat protein synthesis (Lenk et al., 1975 ; Fuller & King, 1981). These results indicate that the pathway for the assembly reaction in vitro does not involve prior formation of an inrler shell of gp8, followed by polymerization of gp5 subunits around it, as suggested for T4. Van Driel & Couture (19786) showed that the scaffolding protein of T4 could self-assemble under certain conditions into structures similar to the internal shell of the procapsid. They proposed’that such prior polymerization of the inner shell might be the normal pathway in T4. However, more detailed analysis of the conditions of aggregation leaves some ambiguity as to the physiological pathway (Van Driel, 1980). We did not detect any large intermediate in shell assembly, such as partially completed shells, or naked assembly cores, described by Van Driel & Couture (19783) for T4 procapsid proteins. However, we examined reaction mixtures only after overnight incubation, which would have allowed all initiated structures to proceed to completion. At high concentrations, the coat protein alone did polymerize into shell-like structures, although less efficiently than in the presence of scaffolding protein. Similar behavior was observed for the coat protein in wivo, in cells infected with mutants defective in scaffolding protein synthesis (King et al., 1973 ; Earnshaw & King, 1978). The formation of double shell structures when both proteins were incubated together and dialyzed to physiological conditions, suggests to us that the assembly process involves a copolymerization of the two proteins, with both shells growing simultaneously from a common initiation region, as indicated in Figure 13(a). (c) Initiation From the genetic analysis of P22 morphogenesis, we have been unable to identify a protein that is required for the initiation of the shell assembly process (Botstein et a2.. 1973; King et al., 1973; Poteete & King, 1977). Although four minor proteins must be incorporated into the procapsid for the formation of viable phage, removal of any of these by mutation does not interfere with procapsid assembly in vivo. Van Driel & Couture (1978a) have shown clearly that the gene 20 protein of T4, which is located at the proximal vertex (Muller-Salamin et al., 1977 ; Hsiao & Black, 1977), is required for the initiation of T4 procapsid assembly in vitro. The gene I protein of P22 was the most likely candidate for a gp20 equivalent. However, coat protein prepared from I - procapsids assembled in vitro just as well as coat protein prepared from I+ procapsids. Thus, gpl does not function as an essential initiator in vitro.

Nonetheless, within infected cells some mechanism is required to ensure the incorporation of the gene 1 protein and the gene 7, 16 and 20 proteins. Since the former is required for DNA packaging, and the latter three are required for DNA injection (Hoffman &. Levine, 1975: Bryant, 1978; Griffin-Shea & King, unpublished results), it seems most probable that they are located at the initiating vertex (Murialdo & Becker, 1978aJ). We suspect therefore, that in vivo these proteins form part of the initiating vertex, though in their absence, initiation can proceed from just the coat and scaffolding protein. The assembly of the purified coat and scaffolding subunits into procapsid-like

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particles in vitro appeared to proceed in the absence of minor structural prot,eins. host proteins or host structures. However, the sensitivity of our result is limited. A special initiator protein could be required in as few as five copies per particle. Such a host or early phage protein might not have been detected in our stained gels. (d) Role of guanidine

hydrochloride

When guanidine hydrochloride was dialyzed away from coat protein solutions tirst. and the coat and scaffolding proteins mixed together in its absence. the yield of procapsid-like particles was reduced. This suggests a possible role for Gu. H(‘1 itself in the assembly process. The coupling of the synthesis of coat and scaffolding proteins (King et al.. 1978) had suggested that the scaffolding protein might interact in viva with the nascent coat protein. If this is the case, Gu . HCl could br stimulating assembly by slightly unfolding the coat subunits so that they resemble the nascent state. Alternatively, the slightly unfolded coat subunit might substitute more efficiently for whatever protein species is responsible for procapsid initiation in ~vh:o. (e) The role of the scaffolding

protein,

The absence of efficient polymerization by coat protein alone indicates that, the scaffolding protein is involved in the initiation of polymerization as well as in shell growth. Our experiments do not separate the two phases. We presume that the scaffolding serves to increase the accuracy of determining the shell radius in the elongation phase. Whether this is due to more accurately locating the S-fold vertices, or more accurately specifying the radius of curvature in the faces of the icosahedron, is not quite clear. The scaffolding protein in T4 is involved bot,h in the specification of the width of the prolate phage capsid, and its length (Paulson & Laemmli, 1977: Van Driel & Couture, 1978a). Since the volume of the head determines the size of the genome for both T4 and P22. it is not, surprising t,hat accessory proteins are utilized to ensure accurate head dimensions. Earnshaw & Casjens (1980) have suggested that, among other functions, the scaffolding protein might serve to exclude cytoplasmic prot,eins from the int,erior shell volume, before the incorporation of the DNA. In addition, the exit of the scaffolding protein might be coupled to the initiation of DNA4 packaging. particularly in the laying down of the first coil of DNA against the inner capsid lattice (Earnshaw et al., 1978; Earnshaw & Casjens, 1980). However, lambda procapsids that have lost their scaffolding can efficiently package DNA, ruling this out as a general function (Muriafdo & Becker, 197&J). The observation that all of the double-stranded DNA phages utilize a scaffolding protein in the assembly of the initial shell (Wood &, King, 1979) indicates that the requirement for accessory proteins to accurately specify the dimensions of’ structures that are large with respect t,o their individual subunits may he a relatively general phenomenon. We t,hank P. B. Berget, A. R. Poteete and D. Botstein This manuscript was prepared with the skilled assistance supported by National Institutes of Health grant number (M.T.F.) was supported by National Institutes of Health

for helpful discussions and advice. of Diane DePaulo. The work was GM 17,980-10 (to J.K.). One of us grant number (:M 07287.

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REFERENCES Andrews, P. (1964). B&hem. J. 91, 222-233. Berget, P. B. & Poteete, A. R. (1980). J. Viral. 34, 234-243. Botstein, D., Waddell, C. H. & King, J. (1973). J. Mol. Biol. 89, 669-695. Bryant, J. L. (1978). Ph.D. thesis, Massachusetts Institute of Technology. Casjens, S. (1979). J. Mol. Biol. 131, 1-13. Casjens. S. & King, J. (1975). Annu. Rev. Biochem. 44, 555-611. Caspar, D. L. D. (1976). III Structure-function Relationships of Proteins, Proc. Third John Innes Symposium, L\‘oru:ich, England (Markham, R. & Horne, R. W., eds), pp. 85-99, North-Holland, Amsterdam, New York and London. C&par, D. L. D. 8: Klug, A. (1962). Cold Spring Harbor Symp. Quunt. Biol. 27, l-24. Crowther, A. R. & Klug, A. (1975). Annu. Rev. Biochem. 44, 161-182. Earnshaw, W. & Casjens, S. (1980). Cell, 21, 319-331. Earnshaw, W. & King, J. (1978). J. Mol. Biol. 126, 721-747. Earnshaw, W., Casjens, S. & Harrison, S. C. (1976). J. Mol. Biol. 104, 387-410. Earnshaw, W., King, J.. Harrison, S. & Eiserling, F. (1978). Cell, 14, 559-568. Earnshaw, W., Hendrix. R. & King, J. (1979). J. Mol. Biol. 134, 575-594. Fish, S. It., Hartman. K. A., Fuller, M. T., King. ,J. & Thomas, G. J. (1980). Biophys. J. 32, 234-237. Fuller, M. T. & King. J. (1980). Biophys. J. 32, 381-401. Fuller, M. T. & King, J. (1981). Virology, 112, 529.-547. Griffin-Shea, R. (1977). Ph.D. Thesis, Massachusetts Institute of Technology. Hoffman, B. & Levine, M. (1975). J. ViroZ. 16, 1547-1559. Hsiao, C. L. & Black, L. W. (1977). Proc. Nut. Acad. Sci., U.S.A. 74, 3652-3656. Kellenberger, E. (1972). In PoZymerizution in Biological Systems (Wolstenholme, G. E., ed.), pp. 189-206, Elsevier, Amsterdam. Kellenberger, E. (1980). Biosystems, 12, 201-223. Kikuchi, Y. &, King, J. (1975). J. Mol. Biol. 99, 645-672. King, J. (1980). In Biologic& Regulation and Development (Boldberger, R., ed.), vol. 2, Plenum Press Inc., New York. King. *J. & Casjens, S. (1974). LVature (London), 251, 112-119. King, J. & Laemmli, U. K. (1971). J. MoZ. Biol. 62, 465-477. King, J., Lenk, E. 1’. & Botstein, D. (1973). J. Mol. Biol. 80, 697-731. King, J., Hall, C. & Casjens, S. (1978). Cell, 15, 551-560. Klug. A. (1972). In Polymerization in Biological Systems (Wolstenholme, (:. E., ed.), Elsevier, Amsterdam. Laemmli, U. K. (1970). Nuture (London), 227, 680-685. Laemmli. U. K., Amos, L. A. & Klug, A. (1976). Cell, ‘7, 191-203. Lenk, E., Casjens, S., Weeks, J. & King, J. (1975). Virology, 68, 182-199. Martin, R. & Ames, B. (1961). J. Biol. Chem. 236, 1372-1379. Muller-Salamin, L., Onorato, L. & Showe, M. K. (1977). J. Viral. 24, 121-134. Murialdo, H. & Becker, A. (1977). Proc. Xat. ilcad. Sci., UJS.~~. 74, m-910. Murialdo, H. & Becker, A. (1978a). Microbial. Rev. 42, 529-576. Murialdo, H. & Becker, A. (1978b). J. Mol. Biol. 12.5, 57-74. Murialdo, H. & Becker, A. (1979). Virology, 96, 341-367. Oosawa, F. & Asakura, S. (1975). Thermodynamics of the Polymerization of Protein, Academic Press, London. Paulson, J. R. & Laemmli, U. K. (1977). J. Mol. Biol. 111, 459-485. Poteetr. A. R. & Botstein, D. (1979). Virology, 95, 565-593. Poteetr, A. R. & King, J. (1977). Virology, 76, 725-739. Poteete, .4. It., Jarvik. V. & Botstein, D. (1979). Virology, 95, 550-564. Showe, M. K. & Black, L. W. (1973). Nature Neu: Biol. 242, 7&75. Showe, M. K. & Kellenberger, E. (1976). In Control Processes in Virus Multiplication (Burke, D. C. & Russell, W. C., eds), pp. 407-438, Cambridge University Press, Cambridge. Siegel, L. & Monty, K. (1966). B&him. Biophys. Acfa, 112, 346-362.

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Steven, A. C.. Couture, E., Aebi, U. 8: Showe, M. K. (1976). J. Mol. Biol. 166. 187-221. Uratani. Y. S., Asakura, S. & Imahori, K. (1972). J. Mol. Biol. 67, 85-98. Van Driel, R. (1989). J. Mol. BioZ. 138, 27-42. Van Driel, R. & Couture, E. (1978a). J. Mol. Biol. 123. 115-128. Van Driel, R. & Couture, E. (19786). J. Mol. Biol. 123, 713-719. Van Holde. K. E. (1971). Physical Biochemistry, Prentice-Hall. Inc.. Engelwoods Cliffs, Sew Jersey. Wood. W:. B. (1979). Harvey Lect. 73. 203. Wood. U’. & King, J. (1979). Comprehcnsiae l’irology (Fraenkel-Conrat & Wagner. eds), vol. 13. pp. 581-633, Plenum Press, New York.

Edited

by A. Kluy

Xoiotaadded in proof: In a recent paper Driedonks et a/. (Driedonks, K. A., Engel, A.. ten Heggler, B. & van Driel, R., J. Mol. Biol., 1981, 152.64-662) described a complex of the gene 20 protein of T4, part of the initiating vertex, which had 12-fold rotational symmet,ry. Also Carrascosa et al. (Carrascosa, J. L., Vinuela, E., Garcia, N. & Santisteban, A., J. Mol. Biol. 1982, 154, 31 l-324) have reported that, the head-tail connector of bacteriophage +29 has an inner 6-fold and outer 12-fold rotational symmetry. These findings suggest that thr hypothesized in vivo initiator for P22 procapsid assembly has a more complex structure than that. shown in Figure 13.