Independent assembly of Qβ and MS2 phages in doubly infected Escherichia coli

VIROLOGY

40, 920-929 (1970)

Independent

Assembly

of Qfi and

Infected C. M. LING, Department

of Virology,

MS2

Escherichia

P. P. HUNG, Abbott Laboratories,

Phages

in Doubly

co/i

AND

L. R. OVERBY

North

Chicago, Illinois

60064

Accepted December 10, 1969 Cultures of Escherichia coli Q-13 infected with both Qp and MS2 phages led to infection of the cells by both phages. Upon simultaneous addition, the two phages interfered with each other and resulted in reduced infection by each phage; however, preinfection of the cell by one phage did not affect subsequent infection by the other. The phage progenies produced during double infection were authentic Qp andMS2; phenotypic mixing (mixed-coat particles) and genomic masking (hybrid particles) were not detected. In vitro phage reassembly showed that high species specificity in forming reassembled particles could be achieved when the proteins and RNAs of the two phages freely interacted with one another. INTRODUCTION

The bacteriophages, Q/3 and MS2, are serologically distinct RNA phages (Overby et al., 1966a), which induce unique, specific RNA replicases for their own replication (Haruna and Spiegelman, 1965). The RNAs and proteins isolated from these two phages readily reassemble into phagelike homologous particles (Hung and Overby, 1969; Sugiyama et al., 1967), as well as heterologous particles (Ling et al., 1969), indicating that there are no barriers for RNA and protein of different origins to form hybrid particles. It has been found that proteins from the plant virus CCMV (Hiebert et al., 1968), or coliphage fr (Hohn, 1969) can be assembled around RNA from a variety of sources, or induced to form empty capsids. Particles with mixed-protein coats have also been assembled in vitro from components of different plant viruses (Wagner and Bancroft, 1968). These studies suggest the possibility of phenotypic mixing in viva. To study this, we infected individual cells of Escherichia coli Q-13 with both Qp and MS2 phages. If phage particles are assembled from pools of RNAs and proteins within the dually infected cell, infection of the same cell by these two phages could lead to the production of genomically

masked (hybrid) or phenotypically mixed (mixed-coat) particles. However, as will be shown in this paper, only authentic Q/3 and MS2 particles were found. This suggests a cellular encapsulation mechanism providing a great deal of specificity of interaction between the phage RNAs and their own coat proteins. Additional studies of in vitro reassembly showed that high specificity between homologous RNA and protein in forming phage-like particles could be achieved. Thus, the chemical nature of the viral RNA and proteins can provide a specificity to ensure efficient encapsulation of infectious particles. MATERIALS

AND

METHODS

Bacteria and phages. Bacteriophages Qp and MS2 were prepared in Escherichia coli Q-13 cultures and purified as described previously (Overby et al., 1966a). Assay for infectivity. Plaque-forming units (PFU) for products of infection were determined with the standard two-layer agar technique (Adams, 1959; Overby et al., 1966a). Specific antiviral rabbit sera were used to determine the serotypes. Infective centers (infected host cells) were assayed in the same manner. Injection procedures. A culture of E. coli 920

ASSEMBLY

OF Qfi AND

Q-13 was allowed to grow exponentially in a nutrient broth [L-medium (Overby et al., 1966a)] at 37” to approximately 2 X IO8 cells per milliliter. The culture was infected with phage lysates at a multiplicity of 10. At intervals after infection the number of infected cells were determined. To neutralize free phage, an aliquot of 0.1 ml was treated at room temperature with 2.4 ml of specific antiviral serum diluted 1 to 1000 in 0.01 M Tris-HCI, pH 7.1, plus 0.5 % bovine serum albumin. After 5 min the sample was appropriately diluted and plated on agar for infected centers. In case of infection of a culture with both Qp and MS2 phages, both anti-&p and anti-MS2 sera were added in the first dilution tube to neutralize unreacted phages. In addition, 0.1 ml of 1: 10 dilution of antiserum was added to the agar plate to inhibit the progenies of one or the other phage. Four sets of plates were used as follows: (A) without serum, (B) with anti-MS2 serum, (C) with anti-Q@ serum, (D) with both sera. The plates were examined for plaques after overnight incubation at 37”. Normally, (D) gave no plaques or some very small plaques easily distinguishable from normal Q/3 and MS2 plaques; (A) gave plaques indicating the total infected centers; (C) gave the infected centers containing MS2 phage; and (B) gave the infected centers containing Q/3 phage. An infected center containing both Qfl and MS2 phages produced one plaque respectively in (A), (B), and (C); therefore, the number of doubly infected centers was the difference (B) + (C) - (A). When the yields of progenies or single bursts were to be measured, the infected cultures were diluted in a nutrient broth and incubated at 37” with constant shaking for 3 hours before assaying for plaque-forming units. Spheroplast assay of phage RNA. Infectivity of phage RNA was assayed in spheroplasts prepared from cells of E. coli Q-13 (Pace and Spiegelman, 1966). The phages produced by double infection were separated electrophoretically on agarose columns into Qp and MS2 phenotypes. The RNA of the phenotypes was extracted from the recovered particles. Approximately 1 pg of each RNA (0.2 ml) was used to infect 0.2 ml of spheroplasts. After infection of the spheroplast with

MS2 PHAGES

921

the RNA preparations, the mixtures were plated for infected centers or incubated at 37” for 2 hours to allow phage replication. The latter were then shaken with l/10 volume of chloroform to lyse thespheroplasts, and then assayed for plaque-forming progenies. Isolation of RNA and protein. Purified phage particles were dissociated into RNA and protein by sodium dodecyl sulfate (SDS) and phenol extraction (Gierer and Schramm, 1956; Overby et al., 1966b). The phenol phase containing the interface was reserved for protein isolation (see below). Phage RNA was recovered from the aqueous phase by precipitation with ethanol in the presence of 0.2 1Mpotassium acetate. The RNA preparations thus obtained showed an ~~0,~of approximately 30 S and were infectious in spheroplasts (Pace and Spiegelman, 1966). In reconstitution with phage proteins the RNA also gave infectious particles (Hung and Overby, 1969). Radioactive phage RNA, labeled with 3H or 32P, was prepared from radioactive phages grown in cultures containing uridine-3H or orthophosphateJ2P (Overby et al., 196613). An appropriate amount of carrier RNA was usually added to the radioactive RNA before purification. The yield of RNA was estimated by absorbance at 260 rnp using E:T; = 250. The phenol phase from the phenol extraction mentioned above was further extracted twice with one-half volume of 0.01 M Tris buffer, pH 7.1, to remove trace amounts of RNA and the aqueous layers were discarded. Phage proteins were recovered from the phenol layer by precipitation at -20” with 5 volumes of methanol containing 3% sodium acetate. The collected precipitate was washed twice with cold methanol and dissolved in 5 1M guanidine-HCl, plus 0.5 M mercaptoethanol in 0.1 M Tris-HCI buffer, pH 7.1. The protein solution was stored at -20’ and used within 2 weeks, Radioactive proteins labeled with l4C or 3H were obtained from radioactive phages grown in cultures containing 14C-amino acid mixtures or 3Htyrosine. The yield of phage protein was estimated by lyophilizing and weighing an aliquot of the recovered material. Reassembly of phage particles. The conditions for phage reassembly were essentially

922

EING, HUNG,

the same as reported previously (Hung and Overby, 1969). A typical 0.5-ml reaction mixture consisted of 500 pg of phage proteins, 125 pg of phage RNA, 2.5 mmoles of guanidine-HCl and 25 ~1of mercaptoethanol, in 0.1 M Tris-HCl buffer, pH 7.1. The mixture was dialyzed against two changes of Tris-acetate (TA) buffer (0.1 M Tris, pH 7.2; 0.05 M KCl; 0.02 M magnesium acetate) at 4” over a period of about 20 hours. The dialyzed mixtures were centrifuged at 10,000 9 to remove precipitates before further analyses. Density-gradient centrifugation. The reassembly reaction mixtures were purified by gradient centrifugation in 3-18% sucrose in Tris-EDTA buffer (0.01 izI Tris, pH 7.1; 0.005 M EDTA) with a Spinco SW 41 rotor at 40,000 rpm for 110 min at 4”. Fractions were collected from the bottom of the tube and analyzed for radioactivity, infectivity, or absorbancy at 260 mp. The peak fractions were pooled, and the particles were precipitated by ammonium sulfate (2 M). The precipitates were dissolved in minimum volumes of 0.01 M Tris-HCl buffer, pH 7.1, and dialyzed against the same buffer. Electrophoresis. Electrophoreses on agarose plates were carried out in a LKB immunoelectrophoresis apparatus. Agarose plates were prepared by pouring 25 ml of a 0.8% solution of melted agarose in 0.0125 M sodium phosphate buffer, pH 7.4, plus 0.005% sodium azide onto 2 X 10.5 inch glass plates, The same buffer was used in the electrode chambers. Ten micrograms of sample was placed in each well, and electrophoresis was performed with lo-15 mA per plate for 45-60 min at room temperature. The plates were then subjected to overnight double diffusion at 37” against diluted antisera (25 ~1 of 1: 10 dilution). The antigenantibody precipitation bands were stained for 15 min with 0.1% brilliant blue in 5 % acetic acid after the agarose plates had been soaked in buffer for at least 5 hours and dried under warm air. When direct staining of the phage particles was desired, the plates were dried after the electrophoresis, and stained with the same dye. The stained agarose plates were washed with 5% acetic acid and air dried. Electrophoresis on agarose-gel columns was carried out on 75 mm gels in 9

AND

OVERBY

(id) X 125 mm glass tubes in a disc electrophoresis apparatus at 7.5 mA per column for 45-60 min. Phosphate buffer and 0.8% melted agarose were the same as in agarosegel plate electrophoresis. Samples to be electrophoresed were dialyzed against 0.01 ilf Tris-HCl buffer, pH 7.1, and adjusted to 10% sucrose by adding sucrose crystals. Aliquots of 0.2-0.4 ml of the samples were layered on each agarose-gel column and 0.2 ml of a 5 % sucrose solution was layered on top of the sample to provide a disking effect. After electrophoresis, the gel was stained for 1 hour with 0.1% Amido Black in 7 % acetic acid and destained in 7% acetic acid in the same electrophoresis apparatus at 10 mA per column. For preparative purposes, the area of the gel which contained the phage was excised and phage particles were extracted by shaking with 0.01 M Tris-HCl buffer for 5 hours. Radioactivity counting. Solutions containing radioactive phage particles were precipitated with trichloroacetic acid, filtered onto nitrocellulose filters, and counted in a scintillation spectrometer. The distribution of radioactivity on agarose plates used for immunoelectrophoresis was estimated as follows. The agarose slabs were sliced at 1.5 mm intervals, and each slice was heated in 1 ml of 0.01 ill Tris-HCl buffer, pH 7.1, at 80” for 20 min to melt the agarose. An aliquot of 5 ml of ice-cold 5% trichloroacetic acid was then added to each tube with vigorous mixing. The tubes were then counted as usual after filtration onto nitrocellulose filters. The heating and vigorous mixing were essential for 3H-label because of high quenching by agarose. RESULTS

Double Infection When a culture of E. coli Q-13 was treated with both Qfl and MS2 at multiplicity of 10 for each phage, most of the cells were infected by both phages. The sums of the infective centers (IC) produced by Q@ (assayed with anti-XS2 antibody) and by MS2 (assayed with anti-Q@ antibody) were much greater than the total IC (assayed without antibody) (Table 1). Double infection took place when the two phages were added to the cell culture simultaneously (experiment I) or in sequence

ASSEMBLY

OF Qp AND TABLE

DOUBLE

Expt.

INFECTION

Phages

NO.

Qb' + MS2

QP MS2 Qp then MS2

MS2 then Qp

QS MS2

1

OF Escherichia

Antibodies used in assay

0

(4

anti-MS2 anti-&b 0 0

(B) (C)

0 anti-MS2 anti-Q@ 0 anti-MS2 an&&b 0 0

(A) (B) (C) (A) (B) (C)

923

MS2 PHAGES

coli Q-13 BY QP AND MS2 Infective IC with center9 both phages (K/ml X 10e8) (B + C - A)

Degree of double infection (%)

1.6 1.1 1.4 1.6 1.7

0.9

56

1.3 1.3 1.3 1.4 1.4 1.2 1.2 1.3

1.3

100

1.2

86

n Total cell concentration was 1.6 X lOa/ml. * Both phages added simultaneously. c Cultures were first infected with one phage for 5 min. Unreacted phages were inactivated by specific antiserum, and then the second phage was added. Infective centers were assayed 10 min after the second infection.

(experiment II). However, the percentage of dually infected cells was lower when both phages were added simultaneously (56% vs. 100% and 86%). Infection by either phage, upon simultaneous addition of the two phages, was much lower than that observed in singly infected cultures when the infected centers were determined after infection for 10 min or less. However, when the two phages were added sequentially the extent of infection was indistinguishable from that of single infections (Fig. 1). Sequential additions allowed the host cells to contact only one phage species at a time because the unreacted particles of the preinfecting phage were neutralized by the specific antibody before the addition of the second phage. Thus, preloading the host cells with phages of one species did not affect the entry of phages of the other species. With simultaneous additions, competition by two phages for sites of entry might be the cause of delay in infection by either phage. Single Cell Burst Experiments E. coli Q-13 cultures were infected with both Q/3 and MS2 at a multiplicity of 10 for

each phage. Ten minutes after infection they were serially diluted in broth to 1 cell per 10 ml of medium. One milliliter of the culture was then dispensed into each of 200 tubes and incubated with constant shaking at 37” for 3 hours. All the tubes were assayed for PFU and only about one-tenth of the tubes were found to contain phages. With the aid of specific antibodies, the serotypes of the phages derived from each phage-containing tube (representing 1 host cell) were determined. The result of a typical single-burst experiment on a doubly infected E. coli Q-13 culture is shown in Table 2. Of 200 tubes assayed, 18 tubes contained phages, of which 4 tubes had Qp only, 4 tubes had MS2 only, and 10 tubes had both Q/3 and RIS2. Therefore, in this particular experiment, 10 out of 1s host cells were infected by both phages. Nearly 100 % of double infection was found when the time allowed for infection was prolonged to 20 min. Longer duration for infection was avoided because some lysis of the cells could be observed as early as 30 min after infection. In these single bursts of doubly infected cells, both phages replicated themselves in a common host. The products of mixed as-

924

LING,

HUNG,

AND

OVERBY TABLE

2.0IMS2 1.5

MS2

lmin 1Omin lmin 1Omin TIME AFTER INFECTION FIG. 1. Mutual effects of bacteriophages Qp and MS2 on infection of Escherichia coli Q-13. (A) and (B) are two separate experiments in which the number of infected cells were determined at 1 min and 10 min after addition of the phages at multiplicities of 10 each. (A) Simultaneous infection series: open bars, Qp or MS2 used alone for infection; hatched bars, Qp and MS2 used together. (B) Sequential infection series: open bars, &p or MS2 used alone; hatched bars, cultures were first infected with one phage for 5 min; unreacted viruses were inactivated by specific antiserum and then the second qhage added; the number of cells infected by the second infecting species was determined.

sembly, in which phage particles were composed of coat protein subunits of both phages, was considered. If such penotypitally mixed particles were produced and were infectious, they should be inhibited by both Q@ and MS2 antibodies. As shown in Table 2, the sums of the PFU of Q/3 and MS2 phenotypes, determined after treating the lysates, respectively, with MS2 antibody and Q/3 antibody (column D, Table 2), were reasonably close to the PFU determined without antibody treatment (column C). Thus, if infectious phenotypically mixed particles were formed, they were not numerous enough to detect.

2

YIELD AND SEROTYPES OF PHAGE PROGENIES DERIVED FROM SINGLE CELLS IN CULTURES INFECTED WITH Qp AND MS2 PHAOES~ Tube No.”

(4 + anti-Q@

3 7 19 25 26 31 48 62 87 92 108 115 128 130 151 168 186 195

0 960 0 860 2240 980 0 540 0 880 1160 1250 1690 480 1820 1180 520 1840

(B) ‘I$:-

NdZtiSerum

1040 2770 1580 1970 0 1810 910 1570 1410 2220 3770 0 1480 1870 0 0 1890 4790

1040 4110 1530 2700 2310 2670 1060 2100 1380 2900 4970 1150 3440 2400 2260 1280 2800 5720

CD) (A) + 09

3730 2830 2790 2110 3100 4930 3170 2350

2410 6630

a Values are expressed as plaque-forming units. b Of 200 tubes assayed in duplicate, 18 tubes contained phages.

Electrophoresis and Immunoelectrophoresis Purified phage particles from doubly infected cultures were electrophoresed on agarose-gel slides followed by direct staining, or staining after double diffusion with specific antibodies. The electrophoretic patterns of the phage particles (Fig. 2B) showed only 2 stainable spots, equivalent to authentic Qp and MS2. The immunoelectrophoretic pattern also produced only 2 precipitation bands equivalent to the authentic parent phages. There was no evidence of particles with mixed coat proteins, which logically would have electrophoretic mobilities between the two authentic phages, and would form precipitation bands with either antiserum. Search for Infectious Particles In (Ling that RNA

Genomically

Masked

in vitro self-assembly experiments et al., 1969) we have previously shown Qp RNA and MS2 protein or MS2 and Qp protein readily form phagelike

ASSEMBLY

OF Qp AND MS2 PHAGES

r

925

origin

-

(MS2+Qb

) Control

-

(MSP/QB)

Superinf.

-

(MS2/QB)

Superinf.

-

(MS2+QB

-

(MS2)

-

(0~3 ) Control

-

(MS2/Q@

(B)

) Control

(0 Control

.”Z’. (“” .“,b

) Superinf.

”

(+I

(-1

FIG. 2. Electrophoresis and immunoelectrophoresis of phage particles produced during double infection of Escherichia coli Q-13. (A) Immunoelectrophoretic patterns; (B) direct staining of (A) without superinf., purified double diffusion with antibodies; (C) agarose column electrophoresis. (ML%/&@ particles produced during double infection by MS2 and &a phages; a&, anti-&b rabbit serum; uM, anti-MS2 rabbit serum.

particles. It is thus theoretically possible for genomically masked hybrids to be assembled during double infection by Qp and MS2 phages. Figure 3 shows 6 types of hypothetical particles that could form by free interaction of the RNAs and proteins of these 2 phages. The two complex (phenotypically mixed) types consisting of Qp or MS2 RNA and both Q/3 and MS2 proteins do not form in vitro (Ling et al., 1969) and were not detected as products of double infection, as mentioned above. The possible existence of the other four types of particles (2 normal types and 2 hybrid types) was tested in the following procedure (Fig. 3): lysates of cultures originally infected with Q/3 and MS2 were treated with Qp antibody (A) or with MS2 antibody (B); the survivors of (A) would be particles having MS2 protein and that of (B), Qfl protein, both enclosing either Q/3 or MS2 RNA; when used to infect a

culture, the progenies of both (A) and (B) would be both Qp and MS2 if infectious hybrids were present in the parent population. PFU assays on the progenies indicated that (A) produced only authentic MS2, and (B) produced only authentic Q/3. Therefore, it is concluded that double infection of E. coli Q-13 by Q/I and MS2 produced only authentic Qp and MS2 as infectious progenies. Infectivity of RNAs Extracted from Phage Particle Phenotypes Produced in Double Infection The lack of infectious genomically masked hybrids in the phage progenies from double infection did not exclude the possibility that noninfectious hybrid particles were produced. In vitro self-assembled hybrid particles were found to possess no, or exceedingly low infectivity, as compared to the in vitro self-assembled homolgous particles (Ling et

926

LING,

AND OVERBY

HYPOTHETICAL PARTICLES

SURVIVORS

QB

HUNG,

w

MS2

SURVIVORS

QB

MS2

Normal

Normal

Normal

MS2

@ Hybrids

Hybrids

Hybrids mti MS:

hti

06 I)

4 Complex +

+

infect

Progenies

Progenies

CIAO FIG.

Qp

proteins

3. Scheme for identification

Infect

: ‘. MS2

proteins

QflRNA

a

MS2

RNA

of possible infectious products from double-infected

al., 1969). If in viva hybrids were formed they might also be deficient in an infection mechanism. By means of agarose-gel column electrophoresis, 200 pg quantities of phage particles from double infection were separated into &@-phenotype and MS2-phenotype based on their electrophoretic properties (Fig. 2C). The RNAs isolated from these two electrophoretic phenotypes were used to infect spheroplasts prepared from E. coli Q-13. The infected spheroplasts were incubated at 37” for 2 hours and then assayed for the serotypes of the phage progenies derived from the RNAs. The results are shown in Table 3. RNA from the MS2-phenotype reproduced only MS2 phage as its progeny; and that of &P-phenotype, predominantly Qp phage as its progeny. The 1% MS2 plaques that appeared in the progeny of the &P-phenotype was probably due to contamination inherent in the electrophoretic separation. In the course of electrophoresis, MS2 phenotype, the faster moving species,

TABLE

cultures.

3

IDENTIFICATION OF GENOTYPE OF RNA ExTR.~CTED FROM 2 ELECTROPHORETIC PHAGE PHENOTYPES DERIVED FROM DOUBLE INFECTION coli Q-13 BY Qp AND MS2 PHAGES OF Escherichia

Pha6e phenotype

QP MS2

Infected spheroplasts/ml

7.1 x

104

7.3 x 105

Phage progenies from lysed spheroplasts Genoty e of PFU, phw R NA Antiserum plate

None anti-&B anti-MS2 None anti-&p anti-MS2

515

QP

5

490 66 63

MS2

0

passed through the slower moving species, Q/3 phenotype. The data from the spheroplast assay led to the conclusion that all phage particles produced in double infection by Qp and MS2 were phenotypically and genotypically either Qp or MS2.

ASSEMBLY

OF Qfl AND

927

MS2 PHAGES

Reconst.

(MS2+QB

$I (A)

) Control

Reconst. @ (B)

FIG. 4. Immunoelectrophoresis of phagelike particles reassembled from mixtures of RNAs and proteins of Qp and MS2 phages. Equal amounts of Qp and MS2 RNAs (0.125 mg each) were reconstituted with Q/3 and MS2 proteins (0.375 mg each). After reassembly, the mixtures were purified by sucrose density gradient centrifugat.ion and concentrated by ammonium sulfate precipitation before immunoelectrophoresis. Reconst. + (A), mixture contained ““P-&p RNA, 3H-QP protein, and ‘%-MS2 protein. Reconst. b (B).., mixture contained 32P-MS2 RNA, ‘H-Q@ protein, and ‘%-MS2 protein. See Fig. 5 for profiles of radioactivity.

Specificity of Phage RNAs and Pr*oteins in #elf-Assembly The failure to detect genomically masked and phenotypically mixed phage particles in the progenies of doubly infected cultures leads to the question: how is this high degree of species specificity achieved in vivo when hybrid particles of the two phages are easily produced in vitro (Ling et al., 1969)? If the absolute species specificity achieved in vivo did not involve phage- or cell-mediated processes, conditions should exist under which the high degree of species specificity could be demonstrated in vitro. As shown in Fig. 4, the particles reassembled in vitro from a four-component mixture of radioactive RNA and protein of Q/3 and MS2 gave an immunoelectrophoretic pattern similar to that of a mixture of authentic Q/3 or MS2 phages. The radioactivity profiles (Fig. 5) revealed the distribution of the individual components. The nearly perfect superimposition of the homologous RNAs and proteins indicates that a high degree of species specificity was achieved under free interaction of the 4 components. It must be pointed out, however, that such a high specificity could be demonstrated only at the protein to RNA

ratio (w/w) of approximately 3. Higher protein to RNA ratios tended to produce Q/3 phenotypes with both species of RNA; while a lower protein to RNA ratio yielded particles of Qp phenotype too heterogeneous electrophoretically to be separated from particles of MS2 phenotype. DISCUSSION

Cells of E. coli Q-13 were doubly infected by Q/3 and MS2 phages as evidenced by the finding (Table 1) that the sum of the infective centers carrying Q/3 and MS2 genotypes was much greater than the total number of infective centers. Double infection by the two phages was further confirmed by single cell burst experiments (Table 2) in which most of the individual infected-cells gave rise to both Qp and MS2 as progenies. Infection of the host cell by one phage was not affected by the preinfection of the host by the other phage (Fig. 1B). However, when cultures were simultaneously infected with two phages, mutual interference on the infection by each phage was evident (Fig. 1A). Interference was also noticed for the entry of z2P-labeled RNA of the two phages into the host cells (unpublished data). Some form

928

LING,

HUNG,

200

100

z G * .= .; z

8z

200

100

0

t-1

2

1 CM

3 (+I

FIG. 5. Agarose-gel electrophoresis of phagelike particles reassembled from mixtures of proteins and RNAs of both Qp and MS2 phages. The reconstitution samples were the same as used in Fig. 4, with the proteins and RNAs labeled as indicated. (A) MS2 RNA not labeled; (B) Q/.? RNA not labeled. Original radioactivity inputs were approximately 100,000 cpm for each isotope in the reconstitution mixtures.

of competition for sites of entry is suggested by these findings. This implies that the two phages share the same or adjacent sites of entry which are present in only a very small number per cell. Previously we have shown that Q@ and MS2 protein, during self-assembly, did not cooperate to form phenotypically mixed particles (Ling et al., 1969). As shown in the present studies, infectious phage progenies produced during double infection by the two phages fell into two serological categories, one sensitive to Qfl antibody, the other to MS2 antibody, but none of then sensitive to both antibodies (Table 2). Electrophoresis and immunoelectrophoresis (Fig. 2) of the double infection products also revealed that all the physical particles were electrophoret-

AND OVERBY

ically and antigenically identical to Qp or MS2. Presumably, mixed-coat particles would have electrophoretic mobilities smaller than MS2 but greater than Q/3, and would react with both Q,Band MS2 antibodies. It is, therefore, concluded that no phenotypically mixed particles, infectious or noninfectious, were made during double infection. Electrophoretic separation of the double infection product into Qfl and MS2 phenotypes (Fig. 2) did not exclude the possibility of the existence of genomically masked particles, consisting of RNA from one phage and protein from the other. Hybrids made in vitro were immunophoretically similar to the phage from which the protein moieties were derived (Ling et al., 1969). By testing the parents and progenies of the products of double infection with specific antisera, it was clearly shown that no infectious hybrid particles were formed. The spheroplast assay of RNA isolated from the Qp and MS2 phenotypes produced during double infection revealed that RNA from the Qp phenotype gave rise only to Qp progeny and that of MS2 phenotype only MS2 progeny (Table 3). It is, therefore, certain that no genomitally masked particles containing intact RNA, were produced in double infection. These findings were unexpected in view of the lack of specificity found during in vitro reassembly of various virus systems (Hohn, 1969; Hung and Overby, 1969; Ling et al., 1969; Wagner and Bancroft, 1968). Further studies on reassembly of Q/3 and MS2 demonstrated (Fig. 5) that at low protein to RNA ratios (approximately 3), nearly complete specificity in mixed phage assembly could be achieved. Therefore, if in the course of phage replication during double infection, phage proteins were not synthesized in large excess, the specificity in vivo should be as high as that demonstrated in vitro (Fig. 5). This suggests that the chemical natures of the RNAs and proteins of Q/3 and MS2 may be sufficient to express the specificity in in vivo phage assembly. Alternatively, the specificity can be explained by assuming that in vivo phage assembly does not involve free interaction between pools of RNA and protein. Hung et al. (1969) have found that a strand of QP or MS2 RNA and a few molecules of phage

ASSEMBLY

OF Qp AND

coat protein formed complexes that were able to assemble into phagelike particles only with the protein identical to the one used in the original complex. The existence of such specific nucleoprotein complexes, designated as assembly initiation center, could account for the complete seggregation of Qp and MS2 proteins in forming phagelike particles in the mixed assembly experiments. Likewise, the phage RNA in a transcriptiontranslation complex (Hotham-Iglewski et at., 1969) should have a good opportunity to inherit a few molecules of newly synthesized coat protein and be discharged as a nucleoprotein complex. This complex, an assembly initiation center, could be rapidly encapsulated by a cooperative interaction with additional molecules of the same coat protein. Mechanisms of nonmistaken assembly within the host cell are thus provided. REFERENCES AD.~MS, M. H. (1959). Bacteriophages. Wiley (Interscience), New York. GIERER, A., and SCHRAMM, G. (1956). Die Infektiositgt der NucleinsSiure aus Tabakmosaikvirus. Z., Naturforsch. llb, 13&142. HAIKJN~, I., and SPIEGELMAN, S. (1965). specific template requirements of RNA replicases. Proc. Nutl. Acud. Sci. U.S. 54, 579-587. HIEBERT, E., BANCROFT, J. B., and BRACKER, C. E. (1968). The assembly in vitro some small spherical viruses, hybrid viruses and other nucleoproteins. Virology 34, 492-508. HOTHAM-IGLEWSKI, B., PHILLIPS, L. A., and FRSNKLIN, R. M. (1969). Viral RNA transcrip-

929

MS2 PHAGES

tion-translation complex in Escherichia co& iItfected wit,h bacteriophage R17. Nature 219, 700-703. HOHN, T. (1969). Role

of RNA

in the assembly

process of bacteriophage fr. J. Mol. Biol. 43, 191-200. HUNG, P. P., and OVERBY, L. R. (1969). The reconstitution of infective bacteriophage Qp. Biochemistry 8, 82G828. HUNG, P. P., LING, C. M., and OVERBY, L. 12. (1969). Self-assembly of QP and MS2 phage par-

titles: possible function of initiation complexes. Science 166, 1638-1640. LING, C. M., HUNG, P.

P., and OVERBY, L. R. (1969). Specificity in self-assembly of bacteriaphages Qp and MS2. Biochemistry 8, 4464-446~.

OVERBY, L. R., BARLOW, G. H., DOI, R. H., JACOB, M., and SPIEGELMAN, S. (1966a). Com-

parison of two serologically distinct ribonucleic acid bacteriophages. I. Properties of the viral particles. J. Bacteriol. 91, 442-448. OVERBY, L. R., BARLOW, G. H., DOI, R. H., JACOB, M., and SPIEGELMAN, S. (1966b). Comparison of two serologically distinct ribonucleic

acid bacteriophates. II. Properties of the nucleic acids and coat proteins. J. Bacterial. 92,739-745. PACE, N. R., and SPIEGELMAN, S. (1966). The synthesis of infectious RNA with a replicase purified according to its size and density. Proe. Nutl. Acad. Sci. U.S. 55, 1608-1615. SUGIYAMA, T., HIEBERT, 11. R., and HARTMSN, K. A. (1967). Ribonucleoprotein complexes formed between bacteriophages MS2 RNA and MS2 protein in vitro. J. Mol. Biol. 25, 455-463. WAGNER, G. W., and BANCROFT, J. B. (1968). The self-assembly of spherical viruses with mixed coat proteins. Virology 34, 74&756.