Production of defective MS2 virions in resting Escherichia coli

VIROLOGY

68, 164-175 (1974)

Production

of Defective

MS2

Virions

C. PROPST RICCIUTI’ Microbiology

Department,

Yale

University Accepted

AND

School

in Resting

coli

A. >I. HAYWOOD

of Medicine,

November

Escherichia

LVew Haven,

Connecticut

06510

16, 1973

Starvation of Escherichia coli for nitrogen, glucose, or sulfur was initiated by cellular consumption of the nutrient in a medium designed to contain only a limiting amount of nitrogen, glucose, or sulfur. MS2 infection of nitrogen-starved cells resulted in a normal yield of plaque-forming units. MS2 infection of sulfur-starved or glucose-starved E. coli, however, resulted in a lOO-fold decrease in the yield of plaque-forming units. Further investigation of MS2 infection in sulfur-starved E. coli showed the major cause of the decrease in yield of plaque-forming units was the synthesis of noninfective virions. INTRODUCTION

RNA phage infection in resting cells was investigated. Since the RNA phage, MS2, can code for only a polymerase, a maturation protein and a coat protein, it, is very dependent upon host cell syntheses. Therefore changes in host cell syntheses can be expected to be reflected in changes in phage syntheses. Both RNA and protein turn over in resting cells (Mandelstam, 1960; Pine, 1972). During starvation the proteins synthesized are more unstable than those synthesized during growth (Pine, 1965), and ribosomal RNA breaks down (Ben-Hamida and Schlessinger, 1966; Nath and Koch, 1971). While new ribosomes are made in at least some starved cells, the synthesis of ribosomal proteins is more depressed than the synthesis of soluble proteins and the rate of assembly of ribosomes is greatly slowed in starved cells (Schlessinger and Ben-Hamida, 1966). The details of macromolecular turnover in starved cells vary according to the nutrient factor limited (Ecker and Schaechter, 1963; Pine, 1965). MS2 infection of resting cells in some of 1 Present address: Miles Laboratories, Inc., 400 Morgan Lane, West Haven, Connecticut 06516. 2 Present address: Biophysics Unit, Agricultural Research Council, Babraham, Cambridge, CB2 4AT, England

the standard media and of resting cells st’arved for sulfur, glucose, or nitrogen was studied as a method of probing the host’s regulatory mechanisms and probing how the virus utilizes host’s syntheses. MATERIALS

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

METHODS

Bacteria and bacteriophage. MS2 was originally obtained from Alvin Clark. For purification the virus was suspended in CsCl (p = 1.385) and centrifuged for 24 hr in a type 65 angle-head rotor at 40,000 rpm. The virus band was put on a Sephadex G-75 column and eluted with 0.1 IM NaCl and 0.01 M Tris, pH 7.6. E. coli 3000 (Hfr thi- rel-) was obtained from R. L. Sinsheimer. Chemicals. Uracil-5-3H, 25.4 Ci/mmole, and uracil-2-14C, 52 mCi/mmole, were obtained from Schwarz BioResearch, Inc. Leucine-4, 5,- 3H, 6 Ci/mmole, was obtained from New England Nuclear Corporation. Deoxyribonuclease I, electrophoretically purified, and lysozyme, 18,600 units/mg, were obtained from Mann Research Laboratories. Media. GSO medium is a modification of the medium of Garwes, Sillero, and Ochoa (1969) and contains per liter: 4.97 g NaCl, 7.46 g KCl, 0.047 g KH2P04, 12.1 g Tris base, 1.07 g NH&I, 0.023 g NazSO,, 0.368 g CaC12, 0.51 g MgC12, 10 mg thiamine, and 5 g glucose. The pH was adjusted to 7.5

164 Copyright All rights

AND

DEFECTIVE

MS2

with HCl. G:rowth of E. coli 3000 ceases at OD45o 1.5 in this medium. To determine the composition of media which would be limiting for nitrogen, sulfur, and carbon, each substance was reduced until growth ceased near ODdj,, 0.5. The limiting concentrations are NH&l, 120 pg/ml; NazSO1, 3.2 pg/ml; glucose, 330 pg/ml. Starvation for a substance was initiated by cellular consumption of the substance present in limited amounts. In experiments in which the limiting substance was returned to starved cells, the substance was returned to the concentration in the complete GSO medium. Robert’s C medium (Roberts et al. 1955) contains per liter: 3.0 g NaCl, 6.0 g Naz. HPOJ, 3.0 g KH2PO+ 2.0 g NH&I, 0.26 g Na2S04, and 12.1 g Tris base. The pH was adjusted to 7.3 with HCl. This was supplemented with 10 mg thiamine, 1.0 g Casamino acids, 0.119 g MgClz and 5 g glucose. MS agar plates (Davis and Sinsheimer, 1963) were used for plating. Growth conditions. E. coli was grown from a slant in GSO medium until the culture reached stationary phase. This culture was then diluted 1:200 or more into fresh GSO medium. Growth was at 37°C in a shaking water bath, and was followed by determining the optical density (450 nm) of the culture in a Bausch and Lomb Spectronic 20. Cell numbers were determined using a Petroff -Hausser counting chamber. A minimum of 250 cells per point was counted. Virus assays and infective centers. To determine infective virus (intracellular plus extracellular phage), infected bacteria were artificially lysed using a 1ysozymeEDTA procedure (Haywood and Harris, 1966). Dilutions of the lysed sample were then plated on MS agar plates with a lawn of E. coli 3000. To determine infective centers, i.e., virusinfected bacteria, an aliquot of the culture was diluted I : 10 into antiserum .5 min after infection. After 5 min at 37°C in antiserum, the cells were quickly diluted and plated onto a lawn of E. coli 3000. Under these conditions each virus-infected bacterium appears as a plaque. The antiserum, a gift of David Scott, had a K value of 1617 for MS2. Assay for virus-infected bacteria which

IN

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Coli

produce colonies. Virus-infected bacteria were sequentially diluted through cold antiserum and plated on SIS agar plates at 37°C. The resultant colonies were replica plated onto plates with a soft agar overlay seeded with E. coli 3000. Colonies producing bacteriophagc appeared as colonies with a plaque halo surrounding them. RESULTS

MS2 Production by Logarithmic and Stationary Phase E. coli To determine whether stationary phase E. coli have the capacity to produce infective MS2, aliquots of a culture of E. coli 3000 grown in Robert’s C medium plus 0.1% casamino acids were removed at different t#imes in growth and infected with MS2. Infective centers and MS2 progeny synthesized 60 min after infection were determined. Figure 1 shows that, while the number of infective centers stays nearly constant, when the culture reaches stationary phase, the number of infective hIS2 progeny produced drops almost’ lOO-fold. Similar experiments using other standard media, TPG plus 0.1% casamino acids (Haywood et al. 1969) and MS broth (Davis and Sinsheimer, 1963) show greater than a RIO-fold drop in the yield of infective progeny phage when the cultures arc infected in stationary phase. MS2

Production by Nitrogen-, Glucose-Limited E. coli

Sulfur-

or

The limiting factor in the media used in the previous experiments is not known. It seemed possible, however, that production of MS2 by stationary phase cells might vary according to the factors responsible for the cessation of cell growth. For this reason cultures of E. coli 3000 were grown in GSO medium which was deliberately made limiting for nitrogen, sulfur, or glucose. Experiments similar to those described above were performed with each of the limiting media. At different times in the growth of the culture, aliquots were removed and infected with MS2. Infective centers were determined at 5 min after infection. In addition, shortly after infection the aliquot was halved, and an excess of the limiting substance was returned to one-half of the aliquot. Infective

RICCIUTI

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IIAYWOOD

10' 1

2 HOURS

3

4

FIG. 1. MS2 infection of logarithmic and stationary phase E. coli. At different times in the growth of a culture of E. coli 3000 in Robert’s C Medium plus 0.1% casamino acids, aliquots were removed and infected with MS2 at a multiplicity of 5. Seven minutes after infection samples were taken to determine infective centers. At 60 min after infection aliquots were titered for extracellular and intracellular MS2. C-0, ODdsa; X----X, infective centers; O-Cl, MS2 titer.

progeny virus were determined at 40 min after infection for both halves of the aliquot. Figure 2 shows that the MS2 yield decreases in sulfur-limited cells as the cells consume the sulfur and become sulfur-starved. Results with limiting glucose are similar to those with limiting sulfur. On the other hand, nitrogen-starved E. coli continue to produce infective MS2. The number of infective centers formed in the different media is similar. Thus, cessation of cell growth and division per se is not responsible for failure to produce infective virus. Rather the lack of plaque-forming units appears related to the cells’ response to the depletion of some required substances. Virus Growth Curves in Resting E. coli In the previous experiments virus production was measured at only one time after infection. To be sure these times were repre-

sentative of virus production under conditions of limited nutrition, one-step growth curves were performed. Figure 3 shows the results of MS2 infection of nitrogen-starved and sulfur-starved cells. In each case the medium’s limiting compound was returned to half of the infected culture. Consistent with the previous experiments the growth curve in nitrogen-starved cells was not significantly different from that in logarithmic phase, and did not change when nitrogen was returned. Under conditions of sulfur starvation, the number of plaque-forming units was greatly reduced. The MS2 growth curve in glucose-starved cells was similar to that in sulfur-starved cells. The return of sulfur or glucose to these cells resulted in normal yields of phage. Even under conditions of sulfur or glucose deprivation, however, cells are capable of producing a few virus.

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FIG. 2. MS2 infection of sulfur-limited and nitrogen-limited E. co&. At different times in growth, aliquots of a culture of E. coli 3066 grown in sulfur-limiting or nitrogen-limiting GSO medium were infected with MS2 at a multiplicity of 5. Five minutes after infection, samples were taken to determine infective centers, and the limiting substance returned to half of each aliquot. Virus titers were taken at 46 min after infection. (A) Infection in sulfur-limiting medium. (B) Infection in nitrogen-limiting medium. O--O, ODkSO; X----X, infective centers; O-0, virus production in starved cultures; in cultures with limiting substance returned. A . . . . . A, virus production 167

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3. Virus growth curves. Sulfur-starved and nitrogen-starved E. cali ~OCIO, about 1.5 hr after they stopped growing, were infected with MS2 at a multiplicity of 5. The cultures were halved, and the limiting substance returned to half the culture at 5 min after infection. At different times after infection the MS2 yield was determined. (A) Sulfur-starved cells. (B) Nitrogen-starved cells. O--D, MS2 titer in starved cells; A.. . . . A, MS2 titer in cells with limiting substance returned. FIG.

Virus Adsorption to Logarithmic Resting E. coli

Phase and

It has been reported that resting E. coli show decreased functions associated with pili (Hayes, 1965). Rappaport (1965) and Valentine et al. (1969) have shown, however, that there is only a minor decrease in adsorption of RNA phage to resting cells. This was also tested directly using the method of Newbold and Sinsheimer (1970). It was found, when MS2 was added at a multiplicity of 0.07 to 0.2, logarithmic phase cells, sulfur-starved, carbon-starved, and nitrogen-starved cells adsorbed 93 %, 87 %, 83 %, and 94% of the input MSP, respect.ively . As can be seen from Fig. 2, the number of infective centers does drop some as the cells go into stationary phase, but the drop is always less than 50%. This drop could result from a drop in some step secondary to adsorption, such as penetration, or could be only an apparent drop if the infected cells did not ultimately release virus. Colony Formation by ilJS2 Infected E. coli The mechanism of cell death caused by RNA bacteriophage infection is not known. Previous workers (Davern, 1964; Hoffman-

Berling and MazC, 1964; Knolle, 1964) have described conditions where E. coli can replicate and still retain the viral genome so that the cell or its progeny can produce phage. This is sometimes referred to as a carrier state or persistent infection. Since the yield of infective virus is greatly decreased in sulfur-starved and glucose-starved cells, it seemed possible these infections may not result in cell death. In addition, cell death could be a result of interruption of a series of syntheses which may not occur in resting cells. Therefore, to determine in infected logarithmic phase cells and in infected sulfurstarved, glucose-starved, and nitrogenstarved cells how many cells did not die but could yield both colonies and progeny phage, samples of the cultures were diluted through MS2 antiserum and plated t’o produce colonies. The colonies were then replicaplated onto a bacterial lawn of MS2 sensitive E. coli to detect viral production. Table 1 shows that in logarithmic phase and nitrogen-starved bacteria the number of colonies producing MS2 ranges from 6 to 8% of the total number of infective centers. In sulfurstarved and glucose-starved cells, however, this number increases 2-fold to 5-fold. It is hoped this type of RNA bacterio-

DEFECTIVE TABLE COLONY

FORMATION Medium

BY

1 MS2

Growth

MS2 IN RESTING

E.

INFECTED Min;t~ infection

COW

Infected colonies as percent of infective centers

Complete GSO medium Limiting nitrogen

Logarithmic

25

8

Stationary

Limiting

sulfur

Stationary

Limiting

glucose

Stationary

11 25 11 25 11

6 7 17 33 27

25

42

a Cultures of E. coli 3000 in logarithmic phase (ODtao 0.1) in complete GSO medium, and cultures l-lx hr after entering stationary phase in nitrogen-limiting, sulfur-limiting, and glucoselimiting media were infected with MS2 at a multiplicity of 5. Six minutes after infection, aliquots were removed for infective centers. At 11 and 25 min after infection, aliquots were removed to determine, as described in Materials and Methods, the number of MS2-infected bacteria that could make bacterial colonies. phage infection may serve as a useful model for mammalian RNA virus persistent infection (Walker, 1964; Rustigian, 1966; Rawls,

1968; Horta-Barbosa et al., 1969). In many instances of mammalian virus persistent infection, defective is shown below,

viruses are made which, as is also true for RNA bac-

teriophage in resting cells. Incorporation of Labeled Precursors RNA and Protein in Resting Cells

into

To determine whether the difference in the of infective MS2 in nitrogen-starved and sulfur-starved cells was due to the capacity of the cells to synthesize RNA and protein, the -uptake of 14C-uracil into RNA and 3H-leucine into protein in uninfected cells was measured at different times in cell growth in nitrogen-limiting and sulfurlimiting media (Fig. 4). The net uptake of course depends upon both the syntheses and the pool sizes. Nevertheless, since the uptake in the dierent cultures is nearly similar, it seems reasonable to conclude that the differences in infective phage yields in nitrogen-starved and sulfur-starved cells are not yield

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due to differences in the cells’ general synthetic capacities. Petroff-Hausser cell counts in Fig. 4 outline the single, synchronized division which routinely occurs as cells enter the nongrowing state. A comparison of the growth curves of sulfur-limited cells in Fig. 2 indicates that the phage decrease in these cells occurs close to the time of last cell division. Synthesis of Single-Stranded Progeny MS2 RNA in Sulfur-Starved Cells To determine whether the failure of sulfurstarved E. coli to produce MS2 virus was due to a failure to synthesize viral RNA, a sulfur-starved culture and a culture to which sulfur had been returned (parts of the same culture used for Fig. 3A) were labeled with 14C-uracil immediately after infection. At 15, 30, and 45 min after infection, samples were removed and the RNA extracted and subjected to electrophoreses on acrylamide gels to separate the progeny single-stranded MS2 RNA from the replicative RNA and host RNA. The results of this experiment show that the incorporation of label into single-stranded MS2 RNA is decreased about 2-fold up to 30 min after infection and then is decreased 6-fold at 45 min after infection in sulfur-starved cells as compared with cells to which the sulfur has been returned (Table 2). If the decrease in label results from an equivalent decrease in RNA synthesis and not just from changes in pool size, the decrease still does not account for the lOO-fold decrease in viral titer. Electrophoresis of 3H-labeled MS2 RNA from a sulfur-starved culture mixed with 14C-labeled MS2 RNA from a culture to which the sulfur had been returned showed that the RNA’s from these two cultures had the same electrophoretic mobility and therefore would appear, within the limits of this method, to be identical. Production of Defective MS2 Sulfur-Starved E. coli To determine what made under conditions sulfur-starved E. coli MS2 and labeled with 32 min after infection.

Virions

in

MS2 particles are of sulfur starvation, were infected with 14C-uracil from 16 to A lysate of the in-

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DEFECTIVE TABLE SYNTHESIS

Minutes after infection

15 30 45

1,406 44,300 65,200

IN

2

OF SINGLE-STRANDED MS2 SULIWR-LIMITED E. co@ MS2 RNA labeled in sulfur-starved cells @pm)

MS2

MS2

RNA

IN

RNA labeled when sulfur returned (dpm) 2,400 107,800 388,200

Q E. coli 3000 grown in sulfur-limiting medium were labeled with 3H-uracil, 68 nCi/ml and 0.38 rig/ml in logarithmic phase (OD4m 0.1). This amount of uracil is totally incorporated into ribosomal RNA within a generation time. One and a quarter hours after the culture entered stationary phase, it was infected with MS2 at a multiplicity of 5, and %-uracil, 0.25 &i/ml and 0.5 Kg/ml, was added. The culture was divided, and sulfur returned to one-half. At 15, 30, and 45 min after the addition of 14C-uracil, samples were removed from both halves of the culture, chilled and lysed. The RNA was extracted by a modification of the method of Kirby (1965). The 3H labeling was used to determine that the yield of RNA from all samples was equal. The RNA was subjected to electrophoresis at 6 mA/gel and 5 V/cm for 3 hr at room temperature on 2.4% acrylamidea).5a/o agarose gels with a buffer containing 0.04 M Tris base, 0.02 M sodium acetate, 0.002 M EDTA, 0.2% SDS, and enough glacial acetic acid to bring the pH to 7.5. As described by Adesnik and Levinthal (1969), the gels were sliced, exposed to Kodak single-coated blue sensitive medical X-ray film, and the bands were read in a Joyce-Loebl recording microdensit#ometer. Standard gels containing known amounts of radioactivity were used to calculate the disintegrations per minute (dpm) from band density.

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fected cells was centrifuged on a 5-30% sucrose gradient. Figure 5 shows that label is incorporated into the 81 S phage particles in sulfur-starved E. coli. The amount of label in 81 S phage particles in sulfur-starved cells (Fig. 5A) is about one-third that in cells to which the sulfur has been returned (Fig. 5B). That 81 S particles are made implies that cells have a normal or nearly normal capacity to make MS2 coat protein. Fractions 15-25 contain ribosomal subunits and ribonucleoprotein particles containing MS2 RNA (Haywood et al., 1969; Cramer and Sinsheimer, 1971). The amount of label in these fractions is also decreased 2- to 3-fold in sulfur-starved cells. This 2- to S-fold decrease in labeling is consistent with the decrease in labeling of viral R9A described in Table 2. As previously stated this small decrease in RNA labeling obviously does not account for the lOO-fold decrease in MS2 titer in sulfur-starved E. coli. When, however, the 81 S fraction was titered, the titer of the 81 S fraction from the sulfur-starved culture was less than >
In this work it was shown that MS2 infection results in a normal yield of plaque-

FIG. 4. Incorporation of labeled precursors into RNA and protein in logarithmic phase and in nitrogen-limited and sulfur-limited E. coli. E. coli 3000 were grown into stationary phase in nitrogen-limited and sulfur-limited media. At intervals aliquots were removed and “C-uracil 0.1 &i/ml and 0.2 l.rg/ml, or 3H-leucine, 2 pCi/ml and 0.06 pg/ml, added. After 5 min 0.1 M Na azide and 100 Kg/ml of unlabeled uracil or 200 fig/ml of unlabeled leucine were added, and the aliquots were chilled in a dry ice-ethanol bath. Samples were put on Whatman 3 MM filters. Those samples labeled with uracil were treated with trichloroacetic acid (TCA) in the cold as previously described (Haywood et al., 1969). Those samples labeled with leucine were first left for 20 min in 5% trichloroacetic acid at 80-9O”C, and then treated similarly. Filters were counted in a Liquifluor-toluene scintillation fluid. The amount of radioactivity incorporated into each aliquot during the 5 min labeling period is expressed as cpm/108 cells. (A) Sulfurlimited cells. (B) Nitrogen-limited cells. O-0, OD 450; X-----X, Petroff-Hausser cell counts; m-m, JH-leucine; A.. . *A, 14C-uracil.

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DEFECTIVE

MS2

forming units in resting cells starved for nitrogen, but a greatly decreased yield of plaque-forming units in cells starved for sulfur. In the cells starved for sulfur defective virus particles are made. Argetsinger and Gussin (1966) showed that defective virus particles are made if maturation protein mutants of R17 are grown in a nonpermissive host and that these noninfective particles contain intact RNA, if they are purified in the presence of Mg2* to inhibit RNase I, or if they are grown in an RNase I- host. Kaerner (1970) showed that similar noninfective virus particles were made when fr-infected cells were deprived of histidine which is required for maturation protein synthesis but not for coat protein synthesis. He interpreted his results to mean that association of the viral RNA with maturation protein is an early step in the process of phage maturation, and, if the maturation protein is not available for assembly, this step is passed by and defective particles result. Formation of defective particles occurs in many mammalian RNA virus infections (von Magnus, 1954; Huang and Baltimore, 1970). Very often these defective particles contain only a specific fragment of viral genome. Once formed, the incomplete genome of the mammalian virus defective particle often can be replicated. It is not known whether the initial defective particle originates by an error in RNA synthesis or by an error in assembly which leaves the RNA vulnerable to RNases. The fact that the RNA phage defective particles when

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exposed to RNase I also results in defective particles containing discrete fragments of the viral genome (Lodish et al., 1965; Argetsinger and Gussin, 1966; Heisenberg, 1966) suggests the origin of the RNA phage and RNA mammalian virus-defective particles may be similar. The RNA and coat protein in the defective phage particles made in sulfur-starved cells appear normal, since gel electrophoresis shows the RNA extracted from the defective particles has the same mobility as the RNA from infective particles and since the coat protein is capable of assembling into particles about the size of infective particles. Therefore, it is likely that the defective particles found in sulfur-limited cells, like other defective RNA phage particles which have been described, lack maturation protein. This could happen with wild-type MS2, if in sulfur-starved cells maturation protein either could not be made or could not be assembled onto the RNA. Inability to make maturation protein would imply selective translation of the coat cistron of the phage genome. There is some evidence to suggest that selective translation can occur (Lodish, 1970; Kozak and Nathans, 1972; Yoshida and Rudland, 1972). In starved cells there is considerable breakdown of ribosomes, depression of ribosome protein synthesis and delay in ribosome assembly (Ben-Hamida and Sehlessinger, 1966). Maturation of ribosomal RNA, which has been postulated tfo be linked to ribosomal assembly (Haywood and McClellen, 1973; Chang and Irr, 1973) is delayed in

FIG. 5. Production of noninfective virious in sulfur-starved E. coli. One hour after the cells ceased growing, E. coli grown in sulfur-limiting medium were infected with MS2 at a multiplicity of 5. The culture was immediately divided, and sulfur returned to one half of the culture. At 16 min after infection i4C-uracil, 0.5 &i/ml and 1.0 pg/ml, was added to both halves of the culture. At 32 min after infection, 100 pg/ml uracil and 0.1 M Na azide were added, and the cultures were chilled in a dry ice-ethanol bath. The cells were pelleted and resuspended in 0.01 M Tris, pH 8.0, 12% sucrose, 3 mg/ml EDTA, and 1 mg/ ml lysozyme. After 5 min the cells were centrifuged, resuspended in 0.005 M Tris pH 8 and 2.57, Brij were added. The lysates 58. After lysis 0.01 M Mg acetate, lOrg/ml DNase and finally 0.5j0oI deoxycholate were layered on linear gradients of 5-30y0 sucrose in 0.04 M KCl, 0.01 M Tris, pH 7.6, and were centrifuged for 3.5 hr at 7” in an SW 41 rotor at 37,006 rpm. Fractions were collected from the bottom of the tubes alternately onto 3 MM filters (4 drops) for radioactivity and into 1 ml 0.1 M NaCl and 0.01 M Tris, pH 7.8 (7 drops) for MS2 titer. The samples on the filters were precipitated with cold trichloroacetic acid and counted in Liquifluor-toluene scintillation fluid. (A) Lysate from sulfur-starved cells. (B) Lysate from cells to which sulfur has been returned. O-----O, X-labeled RNA; U-m, MS2 plaque-forming units (note difference in scales in A and B).

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AND

nitrogen-starved cells but is much more slowed in sulfur-starved cells (Ricciuti and Haywood, unpublished data). If factors needed for the synthesis of one or more ribosome proteins were also needed for the synthesis of maturation protein, then one would expect to see defecOive particles and depressed ribosomal assembly under the same conditions. Alternatively, the turnover of ribosomes in sulfur-starved cells might result in the absence of a selected class of ribosomes needed for synthesis of maturation protein. Inability to assemble maturation protein into a virus particle could result if the host plays a role in the assembly of phage. That a host function may be involved in RNA phage assembly is suggested by the fact that the yield of R17 is decreased in E. coli with a mutation which causes defective production of lambda phage heads (Sternberg, 1973). This E. coli locus has been postulated to code for a membrane protein which is involved in the assembly of several DNA phages (Takano and Kakefuda, 1972; Sternberg, 1973). This is consistent with the observation that the initial steps of MS2 assembly take place on membrane (Haywood et al., 1969) and that the initial step in assembly involves maturation protein (Kaerner, 1970; Cramer and Sinsheimer, 1971). The formation of defective virus particles after infection with wild-type RNA phage hopefully can serve as a general model to study the host’s role in the production of defective RNA viruses. ACKNOWLEDGMENTS This work was supported by U.S.P.H.S. Grant AI 07788. C.P.R. held U.S.P.H.S. Predoctoral Grant 5 FOl GM 36985. A preliminary report of this work has been presented [Ricciuti and Haywood, (1973) Amer. Sot. Microbial. Abstr., p. 2291. REFERENCES ADESNIK, M., and LEVINTHAL, C. (1969). Synthesis and maturation of ribosomal RNA in Escherichia coli. J. Mol. Biol. 46, 281303. ARGETSINGER, J. E., and GUSSIN, G. N. (1966). Intact ribonucleic acid from defective particles of bacteriophage R17. J. Mol. Biol. 21,421434. BEN-HAMIDA, F., and SCHLESSINQER, D. (1966). Synthesis and breakdown of ribonucleic acid in

HAYWOOD Escherichia coli starving for nitrogen. Biochim. Biophys. Acta 119, 183-191. CH.~NG, B., and IRR, J. (1973). Maturation of ribosomal RNA in stringent and relaxed bacteria. ,l’ature (London) New Biol. 243,35-37. CRAMER, J. H., and SINSHEIMER, R. L. (1971). Replication of bacteriophage MS2. X. Phagespecific ribonucleoprotein particles found in MS2-infected Escherichia coli. J. Mol. Biol. 62, 189-214. DAVERN, C. I. (1964). The isolation and characterization of an RNA bacteriophage. Aust. J. Biol. sci. 17, 719-725. DAVIS, J. E., and SINSHEIMER, R. L. (1963). The replication of bacteriophage MS2 I. Transfer of parental nucleic acid to progeny phage. J. Mol. Biol. 6, 203-207. ECKER, R. E., and SCNAECHTER, M. (1962). Bacterial growth under conditions of limited nutrition. Ann. N.Y. Acad. Sci. 102,549-563. GARWES, D., SILLERO, A., and OCHOA, S. (1969). Virus-specific proteins in Escherichia coli infected with phage Qs. Biochim. Biophys. Acta 186, 166-172. HAYWOOD, A. M., and HARRIS, J. M. (1966). Actinomycin inhibition of MS2 replication. J. Mol. Biol. 18, 448-463. HAYWOOD, A. M., and MCCLELLEN, R. E. (1973). Ribosomal ribonucleic acid maturation and syn thesis after ribonucleic acid bacteriophage infection. Biochemistry 12, 2905-2909. HAYWOOD, A. M., CRAMER, J. H., and SHOEMAKER, N. L. (1969). Host-virus interaction in ribonucleic acid bacteriophage infected Escherichia coli I. Location of ‘[late” MS2-specific ribonucleic acid synthesis. J. Viral. 4, 364-371. HAYES, W. (1965). “The Genetics of Bacteria and Their Viruses,” 2nd ed. Wiley, New York. HEISENBERG, M. (1966). Formation of defective bacteriophage particles by fr amber mutants. J. Mol. Biol. 17, 136-144. HOFFMANN-BERLING, H., and MAZY, R. (1964). Release of male-specific bacteriophages from surviving host bacteria. Virology 22,305-313. HORTA-BkRBos.4, L., FUCCILLO, D. A., SEVER, J. L., and ZEMAN, W. (1969). Subacute sclerosing panencephalitis: Isolation of measles virus from a brain biopsy. Nature (London) 221, 974. HUANG, A. S., and BALTIMORE, D. (1970). Defective viral particles and viral disease processes. Nature (London) 226,325-327. KAERNER, H. C. (1970). Sequential steps in the in viva assembly of the RNA bacteriophage fr. J. Mol. Biol. 53, 515-529. KIRBY, K. S. (1965). Isolation and oharacterization of ribosomal ribonucleic acid. Biochem. J. 96, 266-269. KNOLLE, P. (1964). Cellular responses of bacterial

DEFECTIVE

MS2 IN RESTING

hosts to contact with particles of the RNA phage fr. Virology 23, 271-273. KOZAK, M., and NATHANS, D. (1972). Differential inhibition of coliphage MS2 protein synthesis by ribosome-directed antibiotics. J. Mol. Biol. 70,41-55. LODISH, H. F. (1970). Specificity in bacterial protein synthesis: role of initiation factors and ribosomal subunits. Nature (London) 226, 705-707. LODISH, H. F., HORIUCHI, K., and ZINDER, N. D. (1965). Mutants of the bacteriophage f2 V. On the production of noninfectious phage particles. Virology 27, 139-155. MANDELSTAM, J. (1960). The intracellular turnover of protein and nucleic acids and its role in biochemical differentiation. Bacterial. Rev. 24, 289-308. NATH, K.,

and KOCH, A. L. (1971). Protein degradation in Escherichia coli. II. Strain differences in the degradation of protein and nucleic acid resulting from starvation. J. Biol. Chem. 246,

69564967. NEWBOLD,

J. E., and SINSHEIMER, R. L. (1970). Process of infection with bacteriophage +X174. XXXIV. Kinetics of the attachment and eclipse steps of the infection. J. Viral. 5.427-431. PINE, M. J. (1965). Heterogeneity of protein turnover in Escherichia coli. Biochim. Biophys. Acta 194,439456. PINE, M. J. (1!)72). Turnover of intracellular proteins. Annu. Rev. Microbial. 26,103-126. RAPPAPORT, I. (1965). Some studies of the infectious process with MS2 bacteriophage. Biochim. Biophys. Acta 103, 486494.

E.

175

CoZi

W. E. (1968). Congenital significance of virus persistence.

RAWLS,

rubella: Progr.

the Med.

Viral. 40, 238-286. ROBERTS, R. B., ABELSON, P. H., COWIE, D. B., BOLTON, E. T., and BRITTEN, R. J. (1955). Studies of biosynthesis in Escherichia coli. p. 5. Carnegie Inst. Wash. Publ. 607. RUSTIGIAN, R. (1966). Persistent infection of cells

in culture by measles virus. I. Development and characteristics of HeLa sublines persistently infected with complete virus. J. Bacterial. 92, 1792-1804. SCHLESSINGER, D., and BEN-HAMIDA, F. (1966). Turnover of protein in E. coli starving for nitrogen. Biochim. Biophys. Acta 119,171-182. STERNBERG, N. (1973). Properties of a mutant of Escherichia coli defective in bacteriophage X head formation (gro E). J. Mol. Biol. 76,1-23. TAKANO, T., and KA~EFUDA, T. (1972). Involvement of a bacterial factor in morphogenesis of bacteriophage capsid. Nature (London) New Biol. 239, VALENTINE,

34-37.

R. C., SILVERMAN, P. M., IPPEN, K. A., and MOBACH, H. (1969). The F-pilus of Escherichia coli. Advan. Microbial. Phys. 3,2-51. VON MAGNUS, P. (1954). Incomplete forms of influenza virus. Advan. Viru..s Res. 2,59-79. WALKER, D. L. (1964). The viral carrier state in animal cell cultures. Progr. Med. Viral. 6, lll148. YOSHIDA, M., and RUDLAND, P. S. (1972). Ribosomal binding of bacteriophage RNA with different components of initiation factor F3. J. Mol.

Biol.

68,465.481.