Large-scale purification of membrane-containing bacteriophage PRD1 and its subviral particles and its subviral particles

VIROLOGY

181, 348-352

(1991)

Large-Scale

Purification

of Membrane-Containing and Its Subviral Particles

Bacteriophage

PRDl

JAANA K. H. BAMFORD AND DENNIS H. BAMFORD’ Department

of Genetics,

University

Received October

of Helsinki, Arkadiankatu 19, 1990; accepted

7, Helsinki SF-00 100, Finland

November

8. 1990

PRDl is a dsDNA virus that infects Escherichia coliand Salmonela typhimurium. The genome is a linear molecule with 5’ covalently linked terminal protein. The virus has a lipid membrane inside the protein coat. We describe the large-scale purification of the virus using a zonal rotor and the yields and quality of the virus and its subviral assemblies for 8 1991 Academic press. inc. subsequent biophysical measurements.

INTRODUCTION

lication uses a protein-primed mechanism as in the cases of adenovirus (Tamanoi, 1986) and phage 429 (Salas, 1988). A large number of nonsense mutants of PRDl and PR4 are available which affect vital functions such as replication, particle assembly, DNA packaging, and cell lysis (Mindich et al., 1982a,b; Davis and Cronan, 1983; Vanden Boom and Cronan, 1990). Using information obtained from the nonsense mutants, an assembly pathway was proposed in which the major coat protein multimer translocates the virusspecific membrane from the plasma membrane in a process analogous to clathrin action (Mindich and Bamford, 1988). Following the DNA packaging process the virus particle matures. The virus is composed approximately of 70% protein, 15% DNA, and 15% lipid (Wong and Bryan, 1978; Davis et al., 1982). The major coat protein constitutes about 80% of the viral protein and the membrane is approximately half protein and half lipid (Davis et a/., 1982). For our work toward an understanding of the structure and assembly of these viruses, we have initiated a series of studies using whole PRDl and its subassemblies. The techniques in use are analytical ultracentrifugation, Raman and circular dichroic (CD) spectroscopy, X-ray crystallography, and conventional and cryoelectron microscopy coupled with image-processing techniques. The first results with Raman spectroscopy demonstrate the presence of characteristic structure markers in the spectra for the macromolecular constituents of the virus: proteins, DNA, and lipids (Bamford et al., 1990). Very recently the conformations of these structures were also studied by CD spectroscopy (osterlund et al., 1991). The bases of successful structural studies are the quantity and the purity of the particles in use. In this paper we describe the largescale purification of PRDl -specific particles for physical studies.

Bacteriophages that contain lipid provide an opportunity to study membrane structure and biogenesis in systems where genetic, biochemical, and biophysical approaches can be combined (Mindich and Bamford, 1988). Recent studies have demonstrated that the basic principles of membrane biology are universal and can be studied in both eukaryotic and prokaryotic organisms (Saier et a/., 1989). Bacteriophage PRDl is a member of a group of very similar viruses infecting a variety of gram-negative hosts harboring an appropriate plasmid (Olsen et al., 1974; Bamford eta/., 1981). Among the hosts are Escherichia coli and Salmonella typhimurium. These viruses are so similar that results obtained with one member are applicable to the others. The best studied members are PRDl and PR4. In this virus group, the viral membrane resides inside an icosahedral protein coat (Lundstrom er al., 1979; Bamford and Mindich, 1982). The outer layer is composed of the major and minor coat proteins, proteins P3 (43.1 kDa) and P5 (34.3 kDa), respectively (Bamford and Bamford, 1990b). The membrane contains at least 15 phagespecific gene products (Bamford and Mindich, 1982) and phospholipids derived from the host phospholipid pool (Davis et al., 1982). The 15-kb linear dsDNA is located inside the membrane vesicle. The entire nucleotide sequence is known (manuscript in preparation). Both genome ends contain a 1 10-bp inverted repeat sequence and a covalently linked protein at the 5’ ends (Savilahti and Bamford, 1986; Bamford and Mindich. 1984). DNA rep-

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348

LARGE-SCALE

PURIFICATION

TABLE 1 BACTERIALSTRAINSAND BACTERIOPHAGESUSED IN THIS STUDY Relevant property

Reference

Bacteria S. ryphimurium

DS88

standard host

PSA (pLM2)

suppressor host for sus I suppressor host for su.s186

DB7154 (pLM2) Phages PRDl PRO1 sus I (P9-) PRDl sus186(Pl l-)

WI defect in DNA packaging soluble membrane

Bamford and Bamford, 1990 Mindich et al. 1982a Mindich et al., 1982a Olsen et a/., 1974 Mindich et al., 1982b Bamford and Mindich, 1982

MATERIALS AND METHODS Bacterial strains, bacteriophages, and media The bacterial strains, phages and their relevant properties are listed in Table 1. Plasmid pLM2 (Mindich et a/., 1976) is a derivative of the conjugative plasmid RPl making the bacteria susceptible to PRDl . Strain DS88 was kept on Luria-Bertani (LB) agar plates (Sambrook et a/., 1989) containing 25 pg/ml kanamycin. The suppressor strains PSA (pLM2) and DB7 154 (pLM2) were kept on LB ampicillin (150 pg/ml), tetracycline (20 pgl ml) plates to select for the suppression. The mutant virus stocks were grown on an appropriate suppressor host and the stocks had the following titers on suppressor and nonsuppressor hosts, respectively: sus 1, 2 x lo”/8 X lo4 pfu/mI, and sus186, 3 X lo”/1 X 1O5 pfu/mI.

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349

be obtained with SW rotors (Bamford et a/., 1981). The light-scattering viral bands from swinging bucket rotors were collected and both empty and full virus particles pelleted by differential centrifugation in the fixed angle T865 (SotvaIl) rotor (32,000 rpm, 3 hr, +5”). The resuspended pellets were designated 1X purified. When better separation between the empty and filled particles was needed, the swinging bucket rotor 1X purified material was recentrifuged in the rate zonal gradient and then pelleted as above. This material was designated 2X purified. In the case of susl particles the 1X purified material was further purified in a 30-70% sucrose equilibrium gradient in buffer A or C (Beckman SW41 rotor, 35,000 rpm, 18 hr, at +15“) from which the viral material was collected, diluted 1: 1 with buffer, and pelleted as above. Large-scale purification was carried out in a zonal TZ-28 (Sot-vall) rotor using a 5-30% (w/v) sucrose gradient (1300-ml gradient volume) and 45- to 50-ml sample volume. The centrifugation conditions were 24,000 rpm, 1 hr 10 min, +15’, and reograde mode 5 in Sorvall Combi ultracentrifuge. After centrifugation the gradient was pumped through an Uvicord S uv monitor (2.5-mm optical path length) operating at 280 nm. The gradient was fractionated and protein concentration, pfu, and percentage sucrose were determined. The peak fractions (about 400 ml) were pooled and the virus collected by differential centrifugation in a T647.5 (Sorvall) fixed-angle rotor (32,000 rpm, 3 hr, +5”). The pellet was resuspended in bufferA or C overnight on ice. This material was designated as 1X zonal purified virus. The final virus preparation was analyzed for protein concentration and pfu and for the protein pattern in SDSPAGE (see below).

Growth and purification of viral particles

Isolation of subviral components

Both wild-type virus and mutant particles were produced on DS88 using LB broth. The bacterial cultures were infected at cell densities of l-2 X 10’ cfu/ml using a multiplicity of infection of about 5. The virus particles from lysed and cleared cultures were concentrated with polyethylene glycol 6000 as previously described (Bamford et a/., 1981) and resuspended in buffer A or C (buffer A, 10 mfl/l K phosphate, pH 7.2, 1 pH 7.4, 1 mM mM MgCI,; buffer C, 10 mMTris-HCI, MgCI,) in 1:20 volume of the original lysate. The virus infectivity was the same in both buffers. Depending on the volume and purpose, the concentrated viruses were purified on 5-20% sucrose gradients in buffer A or C in different rotors as follows: 1.5-ml samples/36ml tubes were centrifuged for 24,000 rpm, 1 hr, at +15”, using the AH627 (Sorvall) swinging bucket (SW) rotor. The separation of empty and filled particles can

DNA. The DNA was isolated from 1X zonal purified wild-type virus. The virus preparation was diluted to contain approximately 0.5 mg protein/ml and then lysed by adding 0.2 vol of 10% SDS and 0.1 vol of predigested pronase (5 mg/mI) and incubating at 37” for 40-50 min. The mixture was extracted four times with neutralized phenol saturated with 0.1 MTris-HCI, pH 8.0, and then six times with ether. The DNA was precipitated from the aqueous phase by the addition of 0.1 vol of 3 M NaCl and 2 vol of ethanol. The pellet was resuspended in 10 mMTris-HCI, pH 7.4, 1 mlLl EDTA, and the uv spectrum from 200 to 400 nm was recorded. The DNA was reprecipitated and stored at -20” as a dry pellet. Capsid protein multirners. The major (P3) and minor (P5) capsid protein multimer fractions were isolated as previously described (Bamford and Mindich, 1982;

BAMFORD

350

sucrose %

AND BAMFORD

lated from A,,, using the approximation corresponds to 50 pg of dsDNA/ml.

that OD = 1

30

RESULTS AND DISCUSSION

25

I

0

5bo

1000 ml

FIG. 1. Bacteriophage PRDl purification using a zonal (TZ-28, Sorvail) rotor. The sedimentation in the figure is from the right to the left. A,,, (-). protein concentration (XX), pfu (-. -), and sucrose concentratron (w/v) (-&).

Bamford and Bamford, 1990b). In short, 1X zonal virus was incubated with guanidine hydrochloride (GuHCI, 2.5 M) for 5 min at room temperature. An equal volume of buffer A or C was added and the sample was loaded on top of a 1O-40% sucrose gradient in an appropriate buffer and centrifuged in the AH627 rotor for 35,000 rpm, 45 hr, at +l 0”. Gradients were fractionated and protein content analyzed by SDS-PAGE. Pooled fractions were concentrated with spectrapor membrane (MW 30,000 cutoff) using 4 atm nitrogen pressure. The concentrated material was subjected to protein assay and SDS-PAGE. Membrane. The wild-type membrane readily aggregates when removed from GuHCl (Bamford and Mindich, 1982). Nonaggregating membranes were obtained from sus 186 particles lacking the membrane protein Pl 1. The mutant particles were treated with 2.5 M GuHCl and the membranes separated through a 1O-40% (w/v) sucrose-buffer A gradient (SW41 rotor, 30,000 rpm, 2 hr, +20”). The membrane pellet obtained was resuspended in buffer A. The protein composition in SDS-PAGE and the protein concentration were determined. Analytical techniques. Protein was determined with Coomassie brilliant blue (Bradford, 1976) using BSA as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out as previously described (Olkkonen and Bamford, 1989). To record the uv spectra the specimens were dialyzed against buffer A at +4’, and diluted to give equal concentrations of the major coat protein in the virus, the empty particle, and the major capsid protein multimer preparations. DNA was diluted to correspond to the DNA concentration in the virus preparation. The uv spectra were recorded on a Gary 2 19 spectrophotometer using a 1 .O-nm slit. DNA concentration was calcu-

The zonal rotor used for the large-scale purification is designed to be loaded and unloaded statically. As a consequence, the gradient reorients during acceleration and deceleration. Although we were not able to recover the virus band with the 5-200/o gradient used in the SW rotor, a virus peak was observed in a steeper 5-30% sucrose gradient (Fig. 1). Possibly, reorientation caused loss of gradient capacity at the heavy end. With the 5-30% gradient, the virus zone stays in the steep portion of the gradient. With the zonal rotor, uvabsorbing impurities migrated further than in the SW rotor, and we were not able to recover the empty particle zone. Virus from a l-liter culture could be processed in a single run leading to the recovery of 7.5-l 0 mg 1 X purified virus (Table 3A). To purify the same amount of virus in the 36 ml SW tubes, 33 tubes are needed. The wild-type infection using DS88 produces about 20% empty DNA-less particles (Table 3A) which can be separated in the SW rotor. After one rate zonal separation, typically some 49/o infective particles are found in the empty particle fraction, as calculated from the specific infectivities (Table 2). When 1X purified empty particles were centrifuged again on a similar gradient, typically only about 0.1 o/owere infective particles. Due to the smaller amount of empty particles produced, contamination of the infective peak by empty particles is markedly smaller. Based on specific infectivities (Table 2), the quality of zonal 1x purified virus preparation equals that obtained from the virus purified in the SW rotor. The yields of viral particles from a 1-liter culture are given in Table 3A. Empty particles with better yield can be obtained from the packaging deficient sus 1 mu-

TABLE 2 SPECIFICINFECTIVITIESOF FILLEDAND EMPTY PHAGE PARTICLES FROMWILD-TYPE INFECTION’

Purificatior? 1 X zonal purified 1 X purified

2X purified

Virus

Empty particles

100 x (empty particles/virus)

1.2 x 1O’O 1.1 x 10’0 9.0 x lo9

4x lo* 1 x 10’

3.6% 0.1%

a pfu/pg protein. Protein determined using Bradford (1976) assay using BSA as standard. ’ Particles purified using rate zonal sucrose gradients as described under Materials and Methods. 2X purified material has gone twrce through the rate zonal purification procedure.

LARGE-SCALE

PURIFICATION

TABLE 3 A. Yields of particles obtained from 1-liter culture 1X 1X 1X 2X 2X 1X 2X 1X 1X

zonal purified wild-type virus purified wild-type virus purified wild-type empty particles purified wild-type virus? purified wild-type empty particles’ purified sus 1 empty particles purified sus 1 empty particlesb purified sus 186 virus purified sus 186 empty particles B. Yields of subviral components

P3 multimer P5 multimer DNA membrane

7.5-l 0.0 mg 5.0-8.0 mg 1 .O-2.0 mg 3.0-3.5 mg 0.3-0.6 mg 4.7-6.5 mg 3.8-5.0 mg 1.8-3.3 mg 1.5-3.4 mg obtained from 1 mg of virus

obtained 230-250 /.tgC 10-15 pg” 110-140 rgc 240-300pgd

theoreticale 550 PS 20 f&i 15orc.l 280 PCs

’ 2X rate zonal purified. b Rate zonal purified followed by equilibrium purification. ’ From 1 X zonal purified wild-type virus, d From 1 X purified sus 186 empty particles. e The theoretical calculation is based on approximations of 15% DNA, 15% lipid, and 70% protein, out of which 81% is in the coat proteins and 19% in the membrane. The calculated amount of P5 is based on the ratio of 1100 P3/40 P5 in PR4 (Davis et a/., 1982) in which the corresponding proteins are designated P2 and P3.

tant missing the structural protein P9. A sus186 mutant, deficient in membrane aggregation, forms approximately equal amounts of filled and empty particles. The yields of subviral particles obtained as described under Materials and Methods, from one mg of 1X zonal purified virus, are given in Table 3B. The protein patterns of theviral particles and subviral components are shown in Fig. 2. The small membrane-associated proteins are not indicated in the figure, due to their large number and diffuse appearance in SDS-PAGE. The identity of these proteins are, however, addressed in conjunction with the DNA sequence of the virus genome (manuscript in preparation). Impurity protein bands are indicated in Fig. 2. The zonal rotor-purified virus preparation contains the same impurity bands as the slower sedimenting empty particles obtained using the SW rotor. The 2X purification of the empty particles only marginally reduced these impurities. Only minimal amounts of impurities can be seen in SW 1X or 2X purified virus particles in a Coomassie blue-stained gel. Also, the wild-type empty particles showed a greatly reduced amount of the packaging factor P9. The membrane fraction containing the phospholipids and the proteins associated with the membrane (Bamford and Mindich, 1982) had residual major coat protein contamination (Fig. 2, lane i) but

OF PRDl

351

no DNA due to the usage of empty sus 186 particles as starting material. This, however, increases the protein impurity bands in this preparation. Figure 3A displays the uv spectra of viruses, empty particles, and DNA. When the spectrum of the empty particles is subtracted from the virus spectrum, the calculated spectrum overlaps with the measured DNA spectrum, confirming the absence of DNA in the empty particle. The concentration of the major coat protein in Fig. 3B was set to correspond to approximately 80% of the total protein in the empty particles. The subtraction of the major coat protein spectrum from the empty particle spectrum yields the calculated spectrum for the virus membrane with a maximum around 250 nm.

CONCLUSIONS In scaled-up culture conditions, milligrams of purified virus and empty particles from wild-type and mutant virus infections can be obtained for physical studies. The Raman spectra of 1X and 2X SW rotor-purified viruses were identical (Bamford et al., 1990) indicating an adequate quality of the 1X purified viruses for spectroscopic studies. The zonal rotor purification allows us to obtain enough virus material so that the major

s_,

~ ,,

4

16.0

4

9.5

; ‘. i , 0,

FIG. 2. 17% SDS-PAGE of purified PRDl particles and subviral components. Lane a, 1 X zonal purified virus; lane b, 1 X purified virus; lane c, 1 x purified empty particles; lane d, 2X purified virus; lane e, 2x purified empty particles; lane f, 2X purified susl (P9-) empty particles: lane g, P3 capsid protein; lane h, P5 capsid protein; lane i, sus186(Pl l-) membrane. The molecularweight standards at the right are bacteriophage 66 structural proteins the sequences of which are available (Mindich and Bamford, 1988). The impurity bands are indicated with an asterisk.

BAMFORD

352

A 0.800

0.800 0.6oc 0.4oc 0.2oc

235

255

275

295 nm

FIG. 3. UltravIolet spectra of PRDl and its subviral particles. A. Infective virus (-a -); empty DNA-less particles (---); DNA (. . e); infective particle spectrum - empty particle spectrum (A). B. Empty particle spectrum (---); capsid protein multimer (P3) spectrum (r); empty particle spectrum - capsid protein multimer spectrum (V).

coat protein P3, the DNA, and the membrane are available in milligram amounts. The minor coat protein P5 trimer with the collagen-like motif (Bamford and Bamford, 1990a,b) is available only in microgram amounts.

ACKNOWLEDGMENTS We thank Dr. Leonard Mindich for the mutant viruses used in this study. This investigation was supported by grants from the Academy of Finland to D.H.B. and J.K.H.B.

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AND BAMFORD BAMFORD, D. H., and MINDICH, L. (1984). Characterization of the DNA-protein complex at the terminl of the bacteriophage PRDl genome. /. Viral. 50, 309-315. BAMFORD, D. H., ROUHIAINEN, L., TAKKINEN, K., and S~DERLUND, H. (1981). Comparison of the Ilpid-containing bacteriophages PRDl , PR3, PR4, PR5 and L17. /. Gen. Viral. 57, 365-373. BRADFORD,M. M. (1976). A rapld and sensitive method for the quantltation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. DAVIS, T. N., and CRONAN, J. E., JR. (1983). Nonsense mutants of the Ilpld-containing bacteriophage PR4. Virology 126, 600-613. DAVIS, T. N., MULLER, E. D., and CRONAN, J. E., JR. (1982). The virion of the lipid-containing bacteriophage PR4. virology 120,287-306. LUNDSTROM, K. H., BAMFORD, D. H., PALVA, E. T., and LOUNATMAA, K. (1979). Lipid-contalnlng bacteriophage PR4: Structure and life cycle. /. Gen. Viroi. 43, 583-592. MINDICH, L., and BAMFORD, D. H. (1988). Lipid-containing bacteriophages. in “The Bacteriophages” (R. Calendar, Ed.), Vol. 2, pp. 475-520. Plenum, New York. MINDICH, L., BAMFORD, D., GOLDTHWAITE,C., LAVERTY,M.. and MACK~ ENZIE, G. (1982a). Isolation of nonsense mutants of lipid-containing bacteriophage PRDl /. Viral. 44. 1013-l 020. MINDICH, L., BAMFORD, D., MCGRAW, T., and MACKENZIE, G. (198213). Assembly of bacteriophage PRDl: Parttcle formation with wildtype and mutant viruses. /. Viral. 44, 1021-l 030. MINDICH, L., COHEN, J., and WEISBURD, M. (1976). Isolation of nonsense suppressor mutants in Pseudomonas. /. Bacferiol. 126, 1777182. OLKKONEN, V. M., and BAMFORD, D. H. (1989). Quantitation of the adsorption and penetration stages of bacteriophage 46 infection. Virology 171, 229-238. OLSEN, R. H., SIAK, J., and GRAY, R. H. (1974). Characteristics of PRDl, a plasmid-dependent broad host range DNA bactenophage.1 Vkoi. 14, 689-699. SAIER, M. H., JR., WERNER, P. K., and MULLER, M. (1989). InsertIon of proteins Into bacterial membranes: Mechanism, characteristics, and comparisons with the eucaryotlc process. Microbial. Rev. 53, 333-366. SALAS, M. (1988). Phages with protein attached to the DNA ends. In “The Bacteriophages” (R. Calendar, Ed.). Vol. 1, pp. 169--186. Plenum, New York. SAMBROOK, J., FRITSCH, E. F., and MANIATIS, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, New York. SAVILAHTI, H., and BAMFORD, D. H. (1986). Linear DNA replication. Inverted terminal repeats of five closely related Escherichia co/i bacteriophages. Gene 49, 199-205. TAMANOI, F. (1986). On the mechanism of adenovirus DNA replication. In “Adenovirus DNA” (W. Doerfler, Ed.), pp. 97-128. Nijhoff, Boston. VANDEN BOOM, T., and CRONAN, J. E., JR. (1990). Nonsense mutants defining seven new genes of the Ilpld-contatning bacteriophage PR4. firology 177, l l--22. WONG, F. H., and BRYAN, L. E. (1978). Characteristics of PR5, a lipidcontalnlng plasmid-dependent phage Canad. /. Microbial. 24, 8755882. OSTERLUND, E., &,ERLUND, K., BAMFORD, J. K. H., and BAMFORD, D. H. (1991). Circular dlchrolc spectroscopy of vlrion, empty virus particles, nucleic acid and major capsld protein of bacteriophage PRDl. Submitted for publication.