A pilot protein participates in the initiation of P22 procapsid assembly

VIROLOGY 182, 673-681 (1991)

A Pilot Protein Participates in the Initiation of P22 Procapsid Assembly

DENNIS THOMAS’ AND PETER PREVELIGE, JR.’

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received October 30, 1990; accepted February 25, 199 1

The gene 16 protein of the bacteriophage P22 is required as a pilot protein aiding the transfer of DNA from the phage into the Salmonella typhimurium host cell. During assembly 1 O-20 copies of the 63,000-Da gp16 protein are incorporated into the procapsid shell prior to DNA packaging. The protein has been purified from isolated procapsids and behaved as a monomer in solution. Upon incubation with purified coat and scaffolding subunits in vitro, it assembled into procapsids with the correct stoichiometry. The addition of physiological quantities of gp16 resulted in an increased rate of procapsid assembly. Sedimentation of mixtures of coat and gp16 protein subunits revealed association/disso- ciation behavior. It is likely that the added gp16 is acting to stabilize a transient oligomeric coat protein species that functions as the in vitro initiation complex for procapsid assembly. 0 1991 Academic PEW, IN.

INTRODUCTION

The detailed pathway by which viral coat proteins assemble into virions remains obscure. In the best studied viral assembly system, the helical tobacco mosaic virus, the polymerization process has been differentiated into distinct initiation, propagation, and termination phases (Butler and Klug, 1971; Potschka et al,, 1988). In the case of icosahedral shell assembly these phases are less well defined, but there is evidence for distinct initiation and growth phases (Black and Showe, 1983; Prevelige e? al., 1988). In the assembly of dsDNA viruses the shell that is the direct product of the subunit polymerization reaction-the procapsid- is distinct in structure from and a direct precursor to the mature virion. The procapsid is a double shell con- sisting of an inner shell of hundreds of molecules of a scaffolding protein within the outer coat subunit shell (Casjens and King, 1975; Black and Showe, 1983; Casjens, 1985). The replicated genomic DNA is pack- aged into the procapsid, resulting in the release of scaffolding protein. The P22 procapsid is a shell of approximately 420 molecules of the 47-kDa coat protein (gp5) arranged in a T = 7 lattice around a core of approximately 300 molecules of the 33-kDa scaffolding protein (gp8) (Earnshaw et a/., 1976).

Although often described as having icosahedral symmetry, all of the dsDNA phages have one vertex

’ Current address: Program in Biophysics, Brandeis University, Waltham, MA, 02254. Supported by NIH Grant GM17980.

’ To whom correspondence should be addressed.

which is differentiated from the other 11. This portal vertex is the site of DNA packaging and later DNA injection (Bazinet and King, 1985). Although not always morphologically differentiated in the procapsid, the do- decameric ring of portal subunits, the channel for nucleic acid packaging, is already incorporated. Other proteins of the procapsid involved with DNA packaging and later with DNA injection are probably located at this vertex (Goldberg, 1983). In Bacillus phage 429 a small RNA molecule is also located at this vertex (Guo eta/., 1987). It is likely that definition of the portal vertex occurs during initiation of assembly.

In order to be biologically active P22 virions must also contain lo-20 copies each of the protein prod- ucts of genes 16, 20, and 7 (gp16, gp20, and gp7) (Botstein et al., 1973; Casjens and King, 1974). The use of conditional lethal mutants demonstrated that the protein product of gene 16 is not required for formation of the P22 virion but is required for infectivity (Botstein et al., 1973; Hoffman and Levine, 1975a,b). Gp16 functions as a pilot protein, assisting in delivery of infectious DNA to the host cell. The full complement of gp16 is already present in the procapsid (King et a/., 1973).

In an attempt to understand in detail the assembly of icosahedral virions, we have utilized our in vitro assembly system to examine the mechanisms controlling the incorporation of the minor protein gp16. We are espe- cially interested in the linked questions of how assembly initiates, and how minor proteins are incorporated. The experiments described in this paper demonstrate that purified gp16 can only be incorporated by growing procapsids, that it need not be present in excess for

673 0042-6822191 $3.00 CopyrIght 0 1991 by Academic Press, Inc All rights of reproducuon I” any form reserved.

674 THOMAS AND PREVELIGE

effective incorporation, and that it increases the rate of assembly.

MATERIALS AND METHODS

Purification of gp16

Procapsids were prepared as previously described (Prevelige et al., 1988) except that the phage employed was 2-amH200/20-amN20/13-amH101/cl7. The cl 7 mutation ensures entry into the lytic pathway. The mutation in gene 2 blocks DNA packaging and thereby results in procapsid accumulation. The mutation in gene 13 delays lysis. The gene 20 amber mutant codes for a truncated protein not incorporated into the procapsid.

Purified procapsids were made 0.5 M in GuHCl by adding an equal volume of 1 M GuHCl in 50 mM Tris, 25 mM NaCI, 2 mM EDTA, pH 7.6 (buffer B), at 4“ for 20 min. This procedure extracts the scaffolding protein (gp8) and all the minor proteins from procapsids leav- ing shells of coat protein. The extracts were pelleted to remove intact shells by centrifugation in a Beckman Ti-45 rotor at 35,000 rpm for 50 min (Fig. 1 lane 2). The supernatant still contained significant quantities of coat protein (lane 3). It was decanted and dialyzed against 2 X 2 of 100 mM NaCI, 50 mM Tris, pH 7.6, 2 mM EDTA at 4”. Following dialysis, the sample was centrifuged at 35,000 rpm for 40 min in a Ti-45 rotor to remove large coat protein structures (Fig. 1 lane 5) but residual coat protein was still present.

Approximately 65 ml of dialyzed sample at an A,,, = 0.5 was applied to a lo-ml CM Sepharose CL-6B column (Pharmacia LKB, Pleasant Hill, CA) equilibrated in the same buffer. Approximately 6-ml fractions were collected. Following sample application the column was washed with 2 column vol of 100 mM NaCl buffer, and then a 100 to 500 mM NaCl gradient totaling 100 ml in volume was applied. At this point the fraction volume was reduced to 1 ml. The column fractions were analyzed by SDS-PAGE. The gp16 eluted at a NaCl concentration of approximately 290 mM and was well separated from contaminating proteins. The fractions containing gp16 were pooled.

In order to concentrate the gp16 it was diluted two- fold with buffer B and applied to a 2-ml CM Sepharose column. The column was washed with buffer B, and then the gp16 was removed by eluting the column with 400 mM NaCI, 50 mMTris, pH 7.6, 2 mM EDTA. Frac- tions containing gp16 were pooled (Fig. 1 lane 7) dialyzed against buffer B, and stored at 4”. The yield from 3 liters of infected cells was approximately 2 mg of wl6.

Estimation of the extinction coefficient of gp16

An estimate of the extinction coefficient of gp16 was obtained by correlating its absorption spectrum with Coomassie blue staining intensity on SDS-PAGE. Known quantities of scaffolding protein (gp8) and coat protein (gp5) were applied to SDS-PAGE gels as standards. The extinction coefficient obtained in this man- ner for gp16 was E’&‘,,% = 0.4.

Purification of coat and scaffolding protein

Purification of coat and scaffolding protein was done as previously described (Prevelige et al., 1988) except that concentration of the scaffolding protein was done by ammonium sulfate precipitation at >35% saturation rather than by dialysis against PEG.

ln vitro assembly experiments

Purified coat and scaffolding protein subunits were maintained at 4”. Prior to assembly the proteins were separately equilibrated to room temperature. The room temperature proteins were rapidly mixed by vortexing and added to cuvettes containing gp16 or buffer as described in the text. For the kinetic determinations the two reactions were prepared simultaneously by two experimenters in order to eliminate staggering the mixing points. The cuvettes were installed in the spectrophotometer prior to mixing. The dead time between initiating the reaction and the beginning of data collec- tion was typically 15 sec.

Turbidity measurements

Turbidity measurements were made in a Gilford Re- sponse spectrophotometer as previously described (Prevelige et a/., 1988) except that the temperature was held constant at 20”, and data points were ac- quired at 20-set intervals.

Sucrose gradient analysis and SDS-PAGE

Sucrose gradient analysis and SDS-PAGE were per- formed as previously described (Prevelige et a/., 1988). Approximately 150 ~1 of sample was loaded on a 5-ml 5-20% (w/w) sucrose gradient and centrifuged in an SW50.1 rotor at 20”. After centrifugation the gradients were fractionated into 250~~1 fractions through a pinhole in the bottom of the tube. When necessary fractions were concentrated by precipitation with 10% tri- chloroacetic acid (TCA). Samples were analyzed for protein content on SDS gels as described by Laemmli (1970) and King and Laemmli (1971) except using a slab gel apparatus (Studier, 1973). Quantitation was done by scanning the Coomassie blue-stained gel on

INITIATION OF PROCAPSID ASSEMBLY 675

an LKB laser densitometer. When present, 5 ~1 of marker proteins at 10 mg/ml were mixed directly with the sample prior to layering on the sucrose gradient.

Electron microscopy

Protein samples were applied to carbon-coated grids stained with 2% uranyl acetate and air dried. The grids were examined in a JEOL 100B microscope at 80 kv.

RNase digestion

Purified gp16 at 0.35 mg/ml was treated with bovine pancreatic RNase A (Sigma Chemical Co.) at a final concentration of 0.01 mg/ml at room temperature for 15 min. In order to directly test the activity of the added RNase, 10 ~1 of the gpl G/RNase digestion reaction was removed and added to 0.5 ml of tRNAA,,, = 0.75 (Sigma Chemical Co.). This represents a 50-fold lower concentration of RNase than was used in the pretreat- ment of gp16. After a 30-min digestion at room temperature, the sample was assayed for (TCA) soluble nu- cleotides. In the sample to which the RNase/gpl6 mixture was added approximately 85% of the RNA was rendered TCA soluble.

Trypsin digestion

Purified gp16 at 0.35 mg/ml was treated with TPCK- treated bovine pancreatic trypsin obtained from Sigma, made up in 1 mg/ml aliquots in distilled water, and stored at -20”. Treatment of purified gp16 was per- formed at a final trypsin concentration of 0.01 mg/ml at room temperature for 15 min. The digestion was termi- nated by the addition of soybean trypsin inhibitor to a final concentration of 0.04 mg/ml. SDS-PAGE analysis demonstrated that the trypsin digestion was complete.

RESULTS

Purification of gp16

Procapsids contain 1 O-20 molecules of the 60,000 molecular weight gene 16 protein (King et a/., 1973) most likely contained internally, perhaps as a complex with the product of gene 20 (Botstein et al,, 1973). We found it difficult to recover pure gp16 from procapsids that contained both gp16 and gp20. The two proteins cofractionated presumably in a complex. While this complex proved to be active in assembly, it displayed limited solubility and was difficult to dissociate, thwart- ing our efforts to study it directly. Since in vivo gp16 is incorporated into procapsids that lack gp20, we prepared procapsids from cells infected with a nonsense mutation in gene 20 (King et al., 1973). These procap-

1 23 4 5 6

FIG. 1. SDS-PAGE of samples obtained during purification of gp16. Lane 1: procapsids from 2-/20-/l 3--infected cells (10 pg loaded); gpl , portal protein; gpl6, pilot protein; gp5, coat protein; gp8, scaffolding protein. (The unidentified band below gp8 is most likely a proteolytic fragment of gp8.) Lane 2: shells of coat protein after extraction with 0.5 M GuHCl (5 rg loaded). Lane 3: 0.5 M GuHCl extract of procapsids (50 pg loaded) still containing significant quantities of coat protein. Lane 4: 0.5 M GuHCl extract of procapsids after dialysis into 100 mM NaCl buffer (50 pg loaded). Lane 5: dialyzed extract after recentrifugation to pellet coat protein shells (40 pg loaded). Lane 6: purified gp16 from carboxymethyl cellulose column (2.3 pg loaded) showing trace amounts of gp8 and the proteolytic fragment. The estimates of protein loaded were obtained from uv spectra using an extinction coefficient E$&% of 0.8 (lanes 1 and 2) 0.6 (lanes 3-6). and 0.4 (lane 7). reflecting estimates of the relative contributions from the constituent proteins. The gel was in- tentionally overloaded in order to visualize minor compontents.

sids were morphologically indistinguishable from those that contained gp20 (data not shown).

The gp16 protein together with other internal proteins were extracted from the procapsid shell by treatment with 0.5 M GuHCI. The 0.5 MGuHCl extract was dialyzed to remove the GuHCl and centrifuged to remove shells of coat protein. This procedure resulted in the recovery of gp16, gpl (the portal protein), and gp8 (the scaffolding protein) in the supernatant (Fig. 1 lanes 2 and 3). Although these proteins are all associated with the procapsid, they are probably not integral parts of the procapsid lattice. The dialyzed extract was applied to a Sepharose CM column and equilibrated with 100 mM NaCI, 50 mMTris, pH 7.6, 2 mM EDTA. The scaffolding protein and gpl passed through this column, while the gp16 was bound. The gp16 eluted upon application of a gradient of NaCl at 290 mM. The purified gp16 was soluble and stable when stored at 4”.

Incorporation of gp16 in procapsids formed in vitro

In order to determine whether the purified gp16 retained biological activity, we tested its ability to be incorporated into procapsids assembled in vitro. Soluble coat and scaffolding subunits do not self-assemble,


No gp16 mately 3.5-fold stoichiometric excess. The other two cuvettes had equivalent volumes of buffer. The three reaction mixtures were incubated at 20” for 1 hr, dur- ,,

w coat ing which time we monitored the turbidity at 350 nm. rp

scaffolding

c”.., The increase in turbidity proceeded at rates similar to

Q::;, s’*:; 1 ,.‘,“y, that described previously (Prevelige et al., 1988) al- # s**.: :. i . . though the highest level was obtained in the 16+

Gp 16 Added Early t =0 sample.

When the increase of turbidity had leveled off, (approximately 1 hr), purified gp16 was added to one control reaction to 3.5-fold stoichiometric excess, and equivalent volumes of buffer were added to the other two (Fig. 3). The buffer addition resulted in the expected drop in turbidity due to dilution. The addition of gp16 resulted in an increase in turbidity. After an addi-

Added Late t = 1 hour tional hour had passed, the shell structures were fractionated from subunits by sucrose gradient centrifugation and analyzed by SDS-PAGE, (Fig. 2).

Unassembled coat and scaffolding subunits were re- covered from the tops of all the gradients, and unassembled gp16 from those samples to which it was added. The procapsids sedimented about 3/4 the length of the gradients. Gp16 was only successfully incorporated into procapsid-like particles when present during the initial stages of the reaction. If it was added after procapsids had assembled (>l hr) no de-

FIG. 2. Sucrose gradient analysis of procapsid particles produced in the presence and absence of gp16. Coat and scaffolding protein were mixed and then either buffer (top and bottom) or gp16 (middle) was added immediately. Assembly was allowed to proceed for 1 hr. tectable gp16 was incorporated into the procapsids. and then gp16 was added to the sample marked “added late” (bottom), and buffer was added to the other samples (top and middle).

These results indicate that in order for gp16 to be-

After an additional hour had passed 150 PI of the samples was cen- come incorporated into the procapsid, it must be pres-

trifuged through a 5-20% sucrose gradient. The gradient was frac- ent during the initial stages of assembly. If gp16 could

tionated, and the protein composition of the fractions analyzed by SDS-PAGE. The gp16 was present in 3.5.fold stoichiometric excess.

0.3 I

C

but when mixed together activate each other for polymerization into procapsid-like shells (Prevelige et al., 1988). The purified gp16 subunits were added to freshly prepared coat and scaffolding subunits and incubated at room temperature. The shell structures formed were fractionated by sucrose gradient centrifugation and analyzed by SDS gel electrophoresis. A substantial fraction of the input gene 16 protein was found associated with the procapsid-like particles sedimenting in the 200 s region of the sucrose gradient. This can be seen in the second panel of Fig. 2.

We were particularly interested in whether the gp16

could associate with newly formed completed procapsids or whether its incorporation was coupled to the shell assembly process. Soluble coat and scaffolding subunits were freshly prepared and equilibrated to room temperature. The proteins were mixed together and immediately distributed into three cuvettes. One cuvette contained purified gp16 subunits in approxi-

0.01 m ’ 0 50 100

Time (min)

FIG. 3. Assembly of coat and scaffolding proteins in the presence and absence of gp16. Coat and scaffolding proteins were mixed together, and either gp16 (C) or buffer (A and B) was added immediately. After approximately 55 min gp16 was added to B and an equivalent volume of buffer was added to A and C. The final concentration of coat and scaffolding protein in cuvette A, lacking gp16, was 6% lower than the two gp16+ samples. The increase in turbidity with time was monitored at 350 nm. The gp16 was present in 3.5-fold stoichiometric excess.


be added to assembled procapsids, or were binding nonspecifically, it would be expected to cosediment with the procapsids even when added after 1 hr of assembly had elapsed.

Although the gp16 subunits were not incorporated when added late in the reaction, there was an increase in turbidity upon addition of gp16 after 1 hr. This increase corresponded to about 20% of the turbidity produced in the initial assembly phase. We have previously determined that for purified procapsids turbidity is directly proportional to procapsid concentration. However, the yield of procapsids (determined from the SDS gels) was similar (36, 34, and 33%) for all three samples. As an additional 20% procapsid yield would be easily detectable by the sucrose gradient technique employed, the increase in scattering may be due to stimulation of off pathway complexes that are not spherical procapsids. The presence of a small quantity of highly scattering material would be hard to detect by the sucrose gradient techniques employed.

The turbidity curve suggests that the rate of assembly in the presence of gp16 is greater than in its absence. In this experiment we did not attempt to obtain data for the earliest time points. Kinetic experiments designed to examine the initial rate of assembly are reported below.

Gp16 is incorporated in stoichiometric amounts during in vitro assembly

To examine the control of gp16 incorporation, purified gp16 was added to assembly reactions to final concentrations in the reactions corresponding to the in viva stoichiometric ratio and IO-fold excess. The assembly reactions proceeded for 75 min, and then the reactions were analyzed by sucrose gradient centrifugation (Fig. 4).

At stoichiometric gp16 concentration all the gp16 supplied was incorporated, indicating that the purified gp16 was essentially all biologically active. Under con- ditions of excess gp16 (lo-fold), most of the gp16 remained at the top of the gradient. The gpl6/gp5 stain intensity ratio in the region of the sucrose gradient corresponding to procapsids averaged 0.04, which is the same as that obtained for in viva procapsids sedimented in parallel. This result suggests that there is a mechanism to control the stoichiometry of gp16 incorporated into procapsids and this mechanism is built into the coat and scaffolding proteins alone, requiring no other cofactor.

Kinetics of assembly of particles in the presence and absence of gp16

The kinetics of polymerization of coat protein into large assemblies from subunits can be followed by the

No gp16 2;procapsids

limiting gp16

gp16

coat

scaffold

Bottom TOP

FIG. 4. Sucrose gradient analysis of procapsid particles produced in the presence of excess and limiting gp16. Coat and scaffolding protein were mixed, and buffer (top) or gp16 in limiting quantities (middle) or lo-fold excess (bottom) was added. After reaction had proceeded for 75 min, 150 ~1 of sample was centrifuged through a 520% sucrose gradient and fractionated, and the fractrons were analyzed by SDS-PAGE.

increase in turbidity at 350 nm. The turbidity of purified gp16, coat protein, scaffolding protein, and mixtures of both gp16 with coat protein and scaffolding protein remained unchanged during the course of the experiments (data not shown). This result demonstrates that the gp16 is neither self-assembling nor triggering an assembly reaction between itself and either coat or scaffolding proteins.

To determine the effect of gp16 on the rate of shell assembly, coat and scaffolding proteins were mixed at a weight ratio of 1: 1, (0.6 mg/ml each) and then rapidly added to two cuvettes, one of which contained gp16 the other containing an equal volume of buffer. The final concentration of both coat and scaffolding proteins was 0.5 mg/ml. The final concentration of gp16 was 0.02 mg/ml, which corresponds to the stoichiometric ratio found in procapsids produced in viva. The increase in turbidity was monitored as a function of time (Fig. 5).

The final level of scattering reached was higher for the sample which had gp16 added. However, analysis of the product by sucrose gradient centrifugation fol-

678 THOMAS AND

0 t 2 3 4 5

0.00' . n ' ' J 0 20 40 60 80

Time (min)

FIG. 5. Kinetics of assembly in the presence and absence of gp16. Coat and scaffolding protein were mixed and then buffer or gp16 at a final weight ratio of 0.04/l was added. The kinetics of assembly were monitored by following the development of turbidity as a function of time. Inset, initial rate of assembly in the presence and absence of gpl6. The absorbance value was plotted as the percentage of final value attained, and tangent to the curve fitted by eye.

lowed by SDS-PAGE demonstrated that the yield of procapsid-like particles was the same in both cases. Two of the possible explanations for this results are: that the scattering signature of a procapsid containing gp16 is different from the scattering signature of one which does not, or that the presence of gp16 results in the production of a small number of highly polymerized structures which contribute to the scattering. We saw no systematic increase in the amount of highly polymerized material in assembly reactions containing gp16, but did observe a morphological difference between procapsids assembled in the presence and absence of gp16 (see below), which could result in a difference in scattering signature. We believe it likely that the differences in level of scattering observed are due to a difference in scattering signature.

The effect of added gp16 on the rate of assembly can be evaluated from the inset on Fig. 5, which plots the percentage of the reaction toward completion ver- sus time for the first 5 min. Plotting the progress curve as approach to completion, rather than absolute A,,, values, scales for the difference in signal levels. Tan- gents drawn to the curves by eye suggest that the initial rate of assembly is approximately three times greater in the presence of gp16 than in its absence.

Morphology of particles assembled in the presence of gp16

Electron micrographs of negatively stained procapsid particles assembled in vitro in the presence and

PREVELIGE

absence of gp16 were compared to procapsids isolated from host cells infected with 2-/20-/13- phage (Fig. 6). The 16- procapsids produced in vivo (Fig. 6 panel D) displayed a well defined uranyl acetate-ex- cluding outer shell of coat protein. Within this shell was a region of stain exclusion due to the presence of scaffolding protein. The inside edge of the coat protein shell was well defined and distinct from the scaffolding protein core. The particles assembled in vitro in the presence of purified gp16 displayed a similar morphology (Fig. 6 panel A). The inner core of scaffolding protein was not quite as well defined as in the in viva case, but the inner edge of the coat protein shell was well defined.

Particles assembled in vitro in the absence of gp16 (Fig. 6, panel B) had a thicker shell with a diffuse inner edge, and did not display a well defined scaffolding protein core.

In order to verify that these differences were real and not the result of staining artifacts or viewer bias, we mixed both types of particles in a 1:5 and 5:l ratio in solution and prepared grids. In a blind experiment the particles were then classified and counted. Approxi- mately 1000 particles were classified for each mixture. Fifteen to thirty-five percent of the particles could not be clearly categorized. However, in the 1:5 mixture of gpl 6+:gpl6- particles, 19% were categorized as morphology A (having well defined inner walls), while in the 5:l mixture, 539/o were categorized as having morphology A. As can be seen in the higher magnification inset, the differences between the two morphologies are more clearly apparent when they lie adjacent on the grid. Thus we believe that the observed morphology differences are significant.

Is there a special carrier molecule?

It has been suggested that initiation of P22 procapsid assembly may involve the presence of RNA (Bazinet et a/., 1990) and a small RNA fragment is essential for DNA packaging by phage 429 (Guo et al., 1987). In order to eliminate the possibility that there were trace amounts of RNA contaminating the gp16, we treated the purified gp16 with RNase prior to adding it to the reaction mixture. This had no effect on the kinetics of assembly. In order to directly demonstrate that the kinetic effect was due to a protein, we digested the purified gp16 with trypsin and then added trypsin inhibitor. SDS gel analysis of the trypsin-treated gp16 demonstrated that the digestion was complete. Coat and scaffolding protein mixtures to which trypsin-digested gp16 was added assembled at the control rate, demon- strating that the rate enhancement was due to the presence of protein, presumably gp16.


FIG. 6. Electron micrograph displaying morphology differences between procapsid particles. ln vitro particles assembled in the presence (A) and absence of (B) of gp16. (C) Mixture of particles independently assembled in the presence and absence of gp16. Particles marked with arrows were assigned as 16-. The inset is a two-fold enlargement of one particle assumed to be 16+ (left) and one particle assumed to be 16- (right) from panel C. (D) Procapsids assembled in viva and purified from Salmonella infected with 2-/20-/13- P22 phage. Particles were negatively stained with uranyl acetate and examined on a Jeol 1 OOB microscope at 80 kV.

Evidence for an association of gp16 with coat protein in solution

The observation that gp16 is incorporated into growing procapsids suggests that the gp16 must interact with the coat protein, the scaffolding protein, or a complex of both. In order to detect the presence of such an interaction we cosedimented gp16 and either coat or scaffolding protein through sucrose gradients. All gradients contained phosphorylase b and carbonic anhydrase as internal standards. The sucrose gradients were fractionated and the fractions analyzed by SDS- PAGE and densitometry (Fig. 7). A strong and stable interaction would be expected to produce a faster sedimenting species containing the proteins involved. This was not observed. We did observe a significant increase in the s value of gp16 when sedimented in the presence of coat protein. The s value determined for gp16 sedimenting alone was 3.1 1 + 0.03 (average of two determinations). The s value determined for the coat protein was 3.31, which agrees well with the previous determinations (Prevelige et al., 1988; Fuller and King, 1981). The s value for gp16 sedimenting in

the presence of coat protein was determined at five different gpl G/coat protein ratios. The values obtained ranged from 3.4 to 3.8 s. These results suggested that gp16 interacts with coat protein in a weak and reversible fashion. Sedimentation velocity experiments and boundary analysis in the analytical ultracentrifuge provided further evidence for a gpl6/gp5 association-dis- sociation reaction that is reversible on the time scale of sedimentation experiments (Thomas Laue, personal communication). A sedimentation equilibrium study is currently underway (in collaboration with Thomas Laue) in order to obtain the stoichiometry and dissocia- tion constants for the reaction.

There was no increase in the sedimentation constant of gp16 when cosedimented with scaffolding protein.

DISCUSSION

The protein product of gene 16 in P22 is not required for procapsid assembly or subsequent maturation of procapsids into phage (Botstein eta/., 1973). However, it is required for the resulting phage to be infectious


i

phos b carbonic v

P\

. onhydrose

gp16 O-O

(3.7 s)

phos b

v

carbonic anhydrose

gp16 o-o

i

0

(3.1s) 0

I’

\ 0

\,

bottom top

FIG. 7. Sucrose gradient analysis of gp16 in the presence and absence of coat protein. Purified gp16 was centrifuged through a 5-209/o sucrose gradient in the presence of coat protein (top) or in its absence (bottom). The concentration of gp16 in both samples was 0.09 mg/ml. In the case where coat protein was present (top) the concentration of coat protein was 0.77 mg/ml. The total sample volume applied was 200 ~1. Sedimentation was at 45,000 rpm in an SW50.1 rotor at 20” for 12 hr. The gradients were fractionated into 250-pl fractions through a pinhole. The fractions were run on SDS- PAGE, and the area in each band was obtained by densitometry of the Coomassie blue-stained gel. The sedimentation positions of the markers proteins phosphorylase b and carbonic anhydrase (which were mixed with the samples prior to centrifugation) are indicated by the arrowheads.

(Hoffman and Levine 1975a,b). Temperature-sensitive mutants of gp16 produce virions that are capable of binding to host cells, but are noninfective. Infectivity of 16- phage can be restored by complementation with 16+ noninfectious phage during infection (Hoffman and Levine 1975b; Bryant and King, 1984). It has been demonstrated that greater than 90% of the gp16 is ejected from the phage after adsorption to the host cell (Israel, 1977). These results suggest that the gp16 function leaves the 16+ infecting phage and ensures transport of 16- phage DNA into the host cell, in an infectious form.

Since the presence of gp16 is essential to phage infectivity there must be a mechanism to guarantee its incorporation during phage morphogenesis. The sim- plest mechanism is for gp16 to associate with the pre- formed virion or procapsid. Previous attempts to complement 16- phage particles with a lysate containing gp16 were unsuccessful, (Botstein et al., 1973) sug- gesting that the gp16 had to be added at some earlier point during the assembly process. Our experiments indicate that assembled procapsids are not capable of binding gp16. However, if purified gp16 is provided

during the assembly process it is efficiently incorporated. The implication is that gp16 must be built into the growing procapsid before it becomes topologically closed.

The procapsid is formed of approximately 420 coat protein subunits and 300 scaffolding subunits. The incorporation of 12-20 gp16 molecules is controlled solely through protein/protein interactions, and the ul- tracentrifugation results demonstrate an interaction between coat protein and gp16. It makes sense to con- sider the symmetry of the capsid in an attempt to understand the mechanism of gp16 incorporation. Disposition of the gp16 at hexameric, trimer, or dimeric clusters of coat protein in the lattice would result in over incorporation. If binding of gp16 was limited to the 12 pentameric vertices, then the number of molecules of gp16 incorporated would be a multiple of 12, with either 12 or 24 molecules likely possibilities.

An alternative model is for a large multimer of gp16 to be bound at a single vertex, analogous to the loca- tion of a dodecamer of portal protein at a single vertex. If binding of gp16 was limited to one vertex, without a symmetry mismatch, it could yield 5 X 2 or 5 X 3 subunits giving the required stoichiometry.

If polymerization of viral shells initiates at a vertex, as seems likely, the complex would display fivefold symmetry. The difficulty of forming such complexes by dif- fusion means that in the absence of other factors this initiation step is liable to be rate limiting. It also suggests that additional template orjig information may be necessary for efficient initiation. If gp16 binding stabi- lizes a transient pentameric cluster involved in the rate determining step of assembly, the result will be an increase in the overall rate of assembly and a kinetic bias toward particles containing gp16. It has been observed that gp16 is present in three- to fourfold excess early in the period of infection (Casjens and Adams, 1985). The presence of excess gp16, coupled with a kinetic bias, should result in efficient incorporation in ho.

The nature of the interaction of gp16 with the coat protein is consistent with this model. The sedimentation studies show that the interaction between gp16 and coat protein is not a strong one, as no oligomers of gp16 and coat protein that were stable enough to sedi- ment away from the bulk of the protein were formed. We believe that the binding sites for gp16 are actually generated upon formation of the coat protein oligomer, and that this binding is reasonably strong. Thus the weak interaction between gp16 and the coat protein measured in the ultracentrifuge is actually a measure of the instability of the oligomeric complex in the absence of scaffolding protein.

We have also observed a morphology difference between procapsids assembled in vitro in the presence


and absence of gp16. The procapsids assembled in the presence of gp16 more resemble those produced in viva, displaying a sharp inner edge of coat protein and a well defined scaffolding protein core. In particles produced in vitro in the absence of gp16 these fea- tures are replaced by a diffuse inner edge of the shell. Since the ratio of coat to scaffolding protein is the same in all of the particles, it seems likely that a diffuse inner edge arises from a difference in the distribution of scaffolding protein. Such a difference is not seen between 16+ and 16- procapsids produced in viva.

It appears that there is significant kinetic control of assembly in the case of P22 procapsids and it is likely that there are subtle differences in the architecture of the procapsids depending upon the kinetic pathway followed. It has been proposed that the length determi- nation of the T4 head is controlled by a kinetic mechanism (Showe and Onorato, 1978). Analysis of the observed P22 differences will have to wait for high resolu- tion structural data on the disposition of both the scaffolding protein and the minor proteins within the procapsid shell.

In the process of performing these studies on purified gp16 we observed some additional properties. The purified gp16 retained biological activity even after a cycle of boiling and cooling. This is a remarkable prop- erty for a protein with a molecular weight of 60,000. We also noted that gp16 self-polymerized into large structures in a concentration-dependent reversible reaction. This reaction was driven by heating and did not occur to any appreciable extent below 30”. Although attempts to determine the morphology of these poly- mers were unsuccessful, the degree of scattering suggests that they were long rods. It is possible that this behavior is related to the protein’s suggested ability to form membrane spanning pores. Gp16 has recently been sequenced (P. Berget, personal communication). It will be interesting to analyze the sequence in light of these observed properties.

ACKNOWLEDGMENTS

These investigations were supported by NIHGMS Grant GM 17980 to Jonathan King, whom we thank for many valuable dis- cusslons and general encouragement.

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A pilot protein participates in the initiation of P22 procapsid assembly

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