Poliovirus proteins associated with ribosomal structures in infected cells

VIROLOGY

69,

1-20

Poliovirus

(1974)

Proteins

Associated

with

Infected PETER

J. WRIGHT’s2

Ribosomal

Structures

in

Cells PETER

AND

D. COOPER

Department of Microbiology, John Curtin School of Medical Research, Australian National University, Canberra, Auslralia Accepted

November

26, 1973

Some poliovirus-specific protein in infected cell cytoplasm was found to have the same sedimentation coefficient and buoyant density in CsCl as the native 45 S subunits (1.48 g/cc), as viral ribonucleoprotein (1.40 g/cc) and as 60-80 S mono- or oligoribosome/vRNA complexes (1.50 to 1.54 g/cc). Cross-fixation artifacts resulting from glutaraldehyde treatment in the CsCl procedure could be controlled in these cases. Other structures carrying viral protein (1.44 and 1.47 g/cc) may be earlier polysome precursors qr may be cross-fixation artifacts. The viral proteins found in each case were those of the 65 S empty capsids (VPO, VPl, and VP3, in equimolar ratio), but were not due to empty capsid contamination. The label attached to the 45 8 subunit was removed by EDTA and could be recovered as a 6 S particle by RNase, EDTA, LiCl, and deoxycholate treatment; similar treatments of the other structures yielded only large ill-defined [VPO, VPl, VP31 aggregates. The presence of guanidine suppressed the addition of [VPO, VPl, VP31 to the 45 S and 7W?O S complexes, but induced the formation of an unidentified labile [VPO, VPl, VP31 multimer cosedimentimz with empty capsids. The findings are discussed in terms of the equestron model for poliovik regulation. INTRODUCTION

These polypeptides were also found in the replication complex of viral RNA (Wright and Cooper, in preparation), and are accordingly implicated as the candidate for the equestron.

We have pointed out elsewhere (Cooper et al., 1973) that the detailed growth kinetics of poliovirus are difficult to explain without a hypothetical regulator particle that we have termed the “equestron.” The equestron is supposed to have specific affinities for the smaller ribosomal subunit and for the replication complex of viral RNA; its functions are enlarged upon in the Discussion. This paper reports some of the results of a search for viral proteins attached to various nucleic acid components of infected cells. Certain structures with the properties of polyribosome precursors were found to carry a particular complex comprising equimolar amounts of the viral structural precursor polypeptides VPO, VP& and VP3.

MATERIALS

Cells and virus. All virus was poliovirus type 1, strain Mahoney unless otherwise indicated, but strain ts+ (Cooper et al., 1970a; Cooper, 1968) was used for some experiments. Virus was grown and assayed in U cells as before, and either purified and stored in CsCl (see below) or stored at -60” as spinner culture fluids at 4 to 8 X lo* PFU/ml. Cells were grown in Eagle’s medium or in Auto-Pow MEM (Flow Laboratories, Irvine, Scotland), autoclavable powdered Eagle’s medium (Yamane et al., 1968), supplemented with 10% calf serum, 1.25 mg/ml NaHCOa, 146 pg/ml L-glutamine and 100 pg/ml each of penicillin, neomycin, and streptomycin.

1 Present address: Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois. 2 Supported by a Studentship of the C.S.I.R.O. 1 Copyright All rights

Q 1974 by Academic Press, of reproduction in any form

Inc. reserved.

AND METHODS

2

WRIGHT

AND

Infection of cells and preparation of cytoplasmic extracts. Cells (1 to 5 X 10’ cells/ml) from an overnight spinner culture were stirred in Eagle’s medium for l-2 hr at 0” with virus (30-50 PFU/cell). In most experiments, a control (uninfected) culture received identical treatment except for omission of the virus. The cells were then diluted into growth medium in a 37” bath and stirred for a stated time, chilled, and resuspended in Eagle’s medium lacking all amino acids but containing labeled amino acids. At a given time the culture was chilled, and all subsequent operations were done at O-5”. Cells were washed twice with PBS, once with RSB (0.01 M NaCl, 1.5 rnlu MgCl,, 0.01 M Tris, pH 7.4), resuspended in RSB, and disrupted by Dounce homogenization; the nuclei were removed by centrifugation. Sodium deoxycholate (DOC) and then Brij 58 (Huang and Baltimore, 1970) were added to 5 mg/ml each, and samples were layered directly on the first sucrose gradients as indicated in the figures. Cultures were generally 5 X lo7 cells (106/ ml, or 4 X 106/ml during labeling periods) ; each sucrose gradient received the cytoplasm of not more than 5 X lo7 cells. Sucrose and CsCl gradient analysis. In all figures, the top of a gradient is at the right. All gradients depict acid-insoluble counts; unless otherwise stated 5 % of each fraction of sucrose gradients and 60% of each fraction of CsCl gradients were counted. Radioactivity was determined by drying SO- or 100-g fractions on Whatman 3 MM 2 cm disks, which were washed (5 min at O-5’) three times with 5 % trichloroacetic acid (TCA), once with ether:ethanol (1: l), and twice with ether. They were counted in toluene-based scintillation fluid in a Packard 3320 Liquid Scintillation Counter, appropriate cross-channel corrections being made where necessary. Sucrose concentrations are percentages (w/w) in F@B unless otherwise stated. Centrifuging conditions are given in the figure legends. These gradients were linear and were fractionated through a tube inserted through the gradients from the top. The procedure for the CsCl gradients followed that of Baltimore and Huang (1968). Port.ions of pooled sucrose gradient frac-

COOPER

tions at 0’ were diluted to 0.8 ml with the same buffers as the sucrose. Their content of ribosomal material was estimated from the A260 using a value of 144 for E:z* (Petermann and Pavlovec, 1966). Glutaraldehyde (50% w/w) was diluted to 40% (w/v) or 8 % (w/v) at room temperature, and adjusted to pH 7.0 with 1 M NaHC03 . Within 10 min, 0.2 ml of diluted glutaraldehyde was added to the material to be fixed, to give final concentrations of 8% or 1.6%j which are the values quoted. The fixed material was layered within 15 min on preformed linear CsCl gradients at O-4” (usually 27-53 % w/w) in RSB + 0.6% w/w Brij 58. The gradients were centrifuged at 5” for 5 hr at 180,000 g in a Spinco SW 50.1 rotor and fractionated through a hole in the bottom of the tube. Densities were determined by refractive index. About 70% of the radioactivity layered was recovered in the fractions. Purified radioactive virus and empty capsids. Cells were infected and labeled as for the one-step growth experiments; label was generally present from 2 to 4.5 hr, when the cultures were frozen and thawed and centrifuged for 10 min at 10,000 g; virus plus empty capsids were sedimented from the supernatant (70 min at 200,000 g). This pellet was resuspended in 5 ml 0.02 M phosphate pH 7 + 2.5 g CsCl, then banded in the Spinco SW 65 rotor (20 hr at 170,000 g). The peaks of label at 1.29 g/cc (largely empty capsids) and 1.34 g/cc (virions) were rebanded in CsCl. The peak of label at 1.34 g/cc was taken as purified virus, and was dialyzed before use against 0.01 M phosphate pH 7 at 4”. The 1.29 g/cc fract’ions were dialyzed against 0.2 M NaCl, 0.07 M EDTA and 0.01 M Tns pH 7.4 (NEB2) and centrifuged through a 15-30% sucrose gradient in NEBB. The single peak of label (60-70 S) was taken as purified empty capsids. Polyacrylamide gel electrophoresisused the reduced-gel procedure described by Burrell et al. (1970). Gels were 15-20 cm and 7.5% acrylamide unlessotherwise stated, and contained 0.1% sodium dodecyl sulphate (SDS) and 0.5 M urea. In all figures the origin is to the left. Some sampleswere concent,rated by precipitation with cold 10 % TCA and

POLIOVIRUS

PROTEINS

ON

washed with 5 % TCA and acetone. All samples were heated in 0.01 M sodium phosphate pH 7.3,2 % SDS, 1% 2-mercaptoethanol, and 0.5 1M urea (90 see at 100”) for electrophoresis. Some samples were then dialyzed before electrophoresis, but this never affected the gel pattern and so was rarely done. RESULTS

In most experiments, cells were infected as described in Materials and Methods, then were given labelled amino acids or uridine at such a time that only virusspecified protein or RNA was being made (e.g., Summers et al., 1965; Fenwick, 1963). Unless otherwise stated, actinomycin D and preincubation in guanidine media were not employed. After incorporation of label, cells were disrupted, nuclei removed, and subcellular fractions were separated by a first zonal centrifugation through a sucrose gradient. Fractions were then examined for their content of viral protein or RNA by further centrifugation through sucrose or CsCl gradients and by gel electrophoresis. Viral

Proteins Attached to the Native Small Ribosomal Subunit of Poliovirus-Infected Cells

Figure 1 shows the typical amino acid labelling patterns obtained in first sucrose gradients of uninfected cells (A), compared with infected cells (B), and with infected cells to which guanidine was added just before the label (C). The greater content of free (74 S) ribosomes in infected compared with uninfected cells was a reproducible finding, as was the virtual elimination of new ribosomal protein synthesis (Fig. 1C). In a series of experiments (not shown here) it was demonstrated that the repression of synthesis of the ribosomal proteins, as a subclass of all the proteins of the cell, closely paralleled repression of overall protein synthesis. The kinetics of repression were very simiiar to those described by Penman and Summers (1965). Gel electrophoresis of preparations of ribosomes and subunits obtained at intervals during the first 2 hours of infection revealed that, with the partial exception of certain ribosomal proteins indicated

RIBOSOMAL

STRUCTURES

3

below, repression of synthesis occurred progressively and equally for all ribosomal proteins. The most noticeable peak of protein label in the infected-cell sucrose gradients (Figs. 1B and 1C) was at about 65 S. This material proved to comprise mainly empty capsids, as much of it was stable to RNase and EDTA: the stable portion cosedimented in sucrose gradients with empty capsids artificially producedfrom purifiedlabelledvirions (Van Elsen and Boeye, 1966), and contained exclusively VPO, VPl, and VP3 (Maize1 et al., 1967). These polypeptides were identified by coelectrophoresis with purified virions, as their molecular weight determined by coelectrophoresis with 1251-labelled standard proteins (Cooper and Wright, in preparation) reproducibly differed from previously published values. The sedimentation coefficient of .65 S, relative t.o ribosomes (74 S) and the larger subunits (60 S, or 50 S in EDTA) in sucrose at 4”, was reproducible but also differed from some previous values (73-80 S, Maize1 et al., 1967; Jacobson and Baltimore, 1968) ; it confirms an earlier report of 65 S (Penman et al., 1964). The method of McEwen (1967), by which an estimate of sedimentation coefficient in a solvent of the density and viscosity of water at 20’ can be derived from sucrose gradients, gave a mean and standard deviation (4 experiments; assumed density = 1.30 g/cc) of sZo,w = 77 =t 2 s. Despite the repression of ribosomal protein synthesis evident by 2 hr, later addition of label revealed in gradients like that of Fig. 1B significant amounts of radioactivity coincident with the native 45 S subunits (fractions 17-19) and at 70-80 S (fractions 5-10, considered below). Label in these fractions was shown to be in viral proteins by its nonproduction in presence of guanidine (Fig. 1C). On recentrifugation of the 45 S peaks from gradients like that of Fig. 1B through second sucrose gradients, a large peak of label coincided with the 45 S subunits (Cooper et al., 1973). Although quite separate from the position of empty capsids, its gel pattern was similar (mainly VP0 + VP1 + VP3). The first sucrose gradient of a duplicate

30 10

30.

-1 5 n

20-

0 ‘;

- 10

10L

5 v

i

5

25

8

(

0

00 50

10

0 c

Infected

and

gum&w

treated

30. 20. 35 25

15-

15 10

1.0 -

OS-

10

20 Fraction

30

No.

Fra. 1. First sucrose gradients of cytoplasmic cell extracts labelled with Suspension cultures of uninfected (A) or poliovirus-infected (B, C) cells were then labelled for 2 hr with 1 &X/ml W- or 5 &i/ml 3H-amino acid mixtures ods). One culture (C) received 2 mM guanidine hydrochloride 5 min before the cytoplasmic extracts were centrifuged through 15-300/c (w/w) sucrose gradients and 5”, Spinco 25.1 rotor).

4

radioactive amino acids. incubated for 3 hr at 37’, (see Materials and Methlabel. DOC/Brij-treated in RSB (16 hr at 50,000 g

POLIOVIRUS

Fraction

PROTEINS

ON RIBOSOMAL

No.

2. Effect of EDTA on viral protein associated with the 45 S subunit. A suspension of poliovirus-infected cells was incubated for 2.5 hr at 37” in presence of 0.2 fig/ml actinomycin D, then labelled for 1.5 hr with 15 &i/ml %-methionine (see Materials and Methods). The cytoplasmic extract was made in 0.01 M NaCl, 5 mM MgCL, 0.01 M Tris pH 7.4 (HMB), then treated with DOC/Brij and centrifuged through a 15 to 30% sucrose gradient in HMB for 15 hr at 59,OCOg and 4” in a Spinco SW 27 rotor (A). The pooled fractions 1517 (45 S) of gradient A were diluted 2-fold and equal portions centrifuged in parallel through 15 to 30% sucrose gradients in HMB (B), or 0.01 X NaCl, 10 mM EDTA, 0.01 M Tris pH 7.4 (C), for 15 hr at 64,090 g and 4’ in a Spinco SW 27 rotor. FIG.

experiment prepared in 5 mn// MgClz , in which the subunits are more stable, is shown in Fig. 2A. The 45 S peak was recentrifuged through two parallel second gradients, in the absence and in the presence.of 10 mM EDTA (Figs. 2B and 2C, respectively). The protein label in the 45 S position of the secondgradients (fractions 13-15) was shifted in the presence of EDTA to the top of the gradient. The gel pattern of pooled fractions 13-15 (45 S) of Fig. 2B is given in Fig. 3

STRUCTURES

5

and shows that the major labelled polypeptides in the EDTA-labile 45 S region were again VPO, VP1, and VP3. Contamination from empty capsids in this pool, as judged by the EDTA-stable material in analogous portions of Fig. 2C, was negligible. The experiments on repression of ribosomal protein synthesis mentioned above indicate that the polypeptides around fraction 10 of Fig. 3 are probably host-coded. The gel pattern of a protein-labelled preparation of native 45 S subunits from uninfected cells in comparison with 1251-labelled standard proteins is shown in Fig. 4. None of the peaks corresponded in molecular weight with VPO, VPl, or VP3 (34,000, 30,000, and 22,000 daltons, respectively, in this system; Cooper and Wright, in preparation) in either this gel, or another (not shown) in which uninfected 45 S subunits were coelectrophoresed with 35S-labellcd empty capsids. Figure 5 compares the gel pattern of infected-cell material sedimenting just behind the 45 S peak (fractions 21-24 of the gradient of Fig. 1B) with those of empty capsids and virions. In this material there was more NCVP4 and VP2 than in the 45 S preparations (Fig. 3, and Fig. 1 of Cooper et al., 1973), indicating that the 45 S gel patterns could not be explained simply by contamination with slower sedimenting material. The 45 S peak of the first infected-cell gradient of Fig, 1B (pooled fractions 17-19) was further purified by centrifugat’ion through a secondsucrosegradient (30 to 40 % sucrose in RSB, 17 hr at 90,000 q in the Spinco SW 27 rotor at 4”). The second 45 S peak was large and well separated from empty capsids, yet its gel pattern closely resembled that of Fig. 3. This gel pattern and the second gradient have been presented elsewhere (Cooper et al., 1973). A portion of this purified 45 S material was fixed with glutaraldehyde and the density in CsCl was determined (Fig. 6) : 70 % of the recovered label banded at 1.45 g/cc and 30 % at the density of protein (1.29 g/cc). The density in CsCl of the 45 S subunits of uninfected cells was independently determined to be 1.48 g/cc (Table 1). The presence of label at 1.29 g/cc indicated either incom-

WRIGHT

AND COOPER

/

VP1

VP3

1 c

I

0 ‘;z

E Et v) v) 0

VP2

t

50

30

10

3

Fraction

NO

FIG. 3. Polypeptide composition of viral proteins associated with the 45 S subunit. The 45 S region (pooled fractions 13-15) of the second sucrose gradient of 45 S subunits from 3%-methionine-labelled infected cells (shown in Fig. 2B) were concentrated by acid precipitation and coelectrophoresed in polyacrylamide gels with purified virions labelled with aH-amino acid mixture.

‘H cpm 12c

Hamocyonin

Pepsin

Fraction

TMV

Cytochroma

c

No.

4. Polypeptide composition of 45 S subunits from uninfected cells. Procedure resembled that of Fig. IA, but labelling with SH-leucine. The 45 S region was concentrated by acid precipitation and coelectrophoresed in polyacrylamide gels with 1*61-labelled standard proteins. The calculated molecular weights of the peaks are indicated. FIG.

POLIOVIRUS

PROTEINS

ON RIBOSOMAL

150

100 E a v 50 I c-7 0

25C

0 Fraction

NO

FIG. 5. Polypeptide composition of viral proteins sedimenting near the 45 S subunits. Samples were electrophoresed concurrently in polyacrylamide gels under identical conditions. (A) purified poliovirus empty capsids and (B) virions; (C), pooled fractions 21-24 of Fig. 1B. Each sample was concentrated by acid precipitation before electrophoresis.

STRUCTURES

7

plete fixation or contamination of the 45 S preparation by unattached protein. If the latter was the case, then the level of contamination was insufhcient to account for the proportion of radioactivity in the VPO, VPl, and VP3 region (45 %: Fig. 1 of Cooper et al., 1973) but could explain the small amounts of other polypeptides throughout the gel. Although cross-fixation artifacts produced by glutaraldehyde have been found (seebelow), the small concentrations of material present in the 45 S regions and the densities of the components involved are not compatible with any cross-fixation in Fig. 6. Thus the polypeptides VPO, VPl, and VP3 were found at the same density and s value as the small ribosomal subunit, and are therefore presumed to be attached to it. The possibility that the labelled proteins had fortuitously attached to the 45 S subunits after homogenization of the cells was tested by mixing uninfected cell cytoplasm with the labelled cytoplasm of an equal number of infected cells (prepared as for Fig. lB, but without detergents) from which the ribosomal material had been removed by centrifugation (5 hr at 150,000 g). The mixture was treated with detergent and sedimented through a sucrosegradient as in Fig. 1, but no label was found associated with the ribosomes or subunits. This suggests that the presence of VPO, VPl, and VP3 on the small subunit had some functional significance in intact infected cells. The subunits in other portions of the 45 S material shown in Fig. 6 were disrupted by treatment with RNase (10 pg/ml, 10 min at 200), followed by 20 mM EDTA, 2 M LiCl and 0.2 mg/ml DOC. Lithium chloride was added as 10 M LiCl neutralized with NaOH. The sampleswere diluted 5-fold with water and centrifuged through a sucrose gradient. In Fig. 7, a typical sedimentation of the label from the disrupted 45 S subunits is compared with sheep IgG (ca. 7 S) and human serum albumin (4 to 5 S). Most of the label after disruption sedimented at about 6 S (80,000-100,000 daltons), a value corresponding to 1 molecule each of VPO, VPl, and VP3; the label recovered in the gradient was about 50 % of that in the sample treated.

WRIGHT

8

BUOYANT DENSITIES (g/cc)

AND COOPER

TABLE 1 IN CsCl OF RIBOSOMES AND SUBUNITS FROM UNINFECTED CELLS'

Buffer RSB (0.01 M NaCl, 1.5 mM MgC12, 0.01 M Tris pH 7.4) HMB (0.01 M NaCl, 5 mM MgC12, 0.01 M Tris pH 7.4) HMB + 50 mM EDTA RSB*

74 s

60s

45 s

50 s

30 s

1.54

1.56

1.48

-

-

1.47

-

-

1.49

1.53 -

1.44 -

1.54 1.57

1.54

0 The particles are identified by their sedimentation coefficient. The appropriate peaks of AZ60 from sucrose gradients of detergent-treated cytoplasmic extracts from uninfected cells labeled with SH-leutine were fixed in 8% glutaraldehyde and banded in CsCl. Each value in the first three rows is the mean of two determinations; the density difference between fractions was 0.014 g/cc, hence the values are only accurate to *O.a07 g/cc. * Data from Huang and Baltimore (1970).

Viral Proteins Attached to Ribonucleoprotein Complexesof 60-80 S in Injected Cells Material of density 1.47 g/cc. The 70-80 S regions of sucrose gradients from infected cells, e.g., fractions 5-8 of Fig. lB, apparently contained labelled protein that could not be explained simply by a spreading of the adjacent peak of empty eapsids. To determine the nature of the species present, appropriate fractions were fixed with glutaraldehyde and centrifuged t,o equilibrium through CsCl density gradients. The densities of the ribosomesand subunits of uninfected cells as determined by Huang and Baltimore (1970), and in this work using both amino acid and uridine label, are given in Table 1. However, some difficulties with the fixation procedure must be illustrated at this point. In one experiment, 14C-aminoacid-labelled 74 S ribosomes or 60s subunits of uninfected cells were mixed with analogous portions of a first sucrose gradient from 3H-amino acidlabelled infected cells, and the mixture was fixed in a final concentration of 8 % glutaraldehyde. The amount of ribosomal material fixed in these mixtures was calculated from the AZW, as described in Materials and Methods. In both mixtures (Fig. 8) most of the aH-viral protein banded at 1.47 to 1.48 g/cc. There was no doubt that material of this density contained VPO, VPl, and VP3: for example, a portion of the preparation from infected cells fixed in Fig. 8A was coelectrophorescd wit.h 35S-labeIledempty capsids (Fig. 9), when the large proportion of

‘H cpm 300

200

0 Fraction

No

6. Buoyant density in CsCl of viral protein associated with the 45 S subunit. After hation in 8% glutaraldehyde (see Materials and Methods) a portion of pooled fractions 23-25 from the gradient of Fig. 1 (Cooper et al., 1973) was centrifuged to equilibrium through a CsCl gradient. FIG.

3H-protein in VPO, VPl, and VP3 demonstrated the presence of these proteins in the 1.47 to 1.48 g/cc complex. Similar gradients were obtained with the 3H-labelled viral material on its own, except that most of the label was in a sharp peak at 1.50 g/cc. However, it is clear in Fig. 8 that some of the added uninfected 14C-labelled ribosomes or subunits also banded at 1.47 to

POLIOVIRUS

PROTEINS

ON RIBOSOMAL

STRUCTURES

9

2

d 8

1

0

1

2

3

A

0

ml. of gradient

7. Sucrose gradient centrifugation of 3H-labelled viral protein from disrupted 45 S subunits from infected cells (see text), in comparison with human serum albumin labelled with I261 and sheep IgG. Samples were centrifuged through separateidentical 10-45% sucrosegradientsin RSB for 16 hr at 195,ooO q and 10” in the Spinco SW 50. 1 rotor. Fraction 1 comprised a pad of 66% sucrose; counts per minute represent the radioactivity of the whole fraction. FIG.

1.48 g/cc, whereas in the absence of infected material a single peak at 1.54 or 1.56 g/cc, respectively, was obtained (results not shown). These observations suggested the possibility that the material at 1.47 to 1.48 g/cc contained a nonspecific aggregate of ribosomal material and viral protein, representing an artifact caused by fixation. The viral protein was possibly free (density 1.30 g/cc) in the unfixed material, or complexed with ribosomal structures having a density other than 1.47 to 1.48 g/cc in the absence of aggregation. A similar result was obtained with uridine label. Cells were given 3H-uridine from 3 to 5 hr after infection, a period when virtually all RNA synthesis was viral. The cytoplasmic extract of one experiment analyzed on a sucrose gradient as in Fig. 1B is shown in Fig. 10. The large peak in fractions 23-25 is double-stranded RNA. The slight peak at fraction 17 (45 S) reflected residual labelling of ribosomal RNA, as this material when hxed with glutaraldehyde cobanded in CsCl with W-labelled subunits from uninfected cells at 1.48 g/cc (results not shown). When 42 pg of a mixture of ribosomal material from the 70-80 S regions of the gradient of

Fig. 10 (fractions 7 and 8) and uninfected 14C-aminoacid-labelled ribosomes (74 S peak from the gradient of Fig. 1A) were fixed and analyzed in a CsCl gradient, the viral uridine label banded at 1.53 and 1.47 g/cc (Fig. 11A). However, some ‘*C-ribosomes also banded at 1.47 g/cc. When only 25 ccg of ribosomal material (3H label from a subsequent experiment) was used, the 1.47 g/cc band was not found (Fig. 11B). As shown by Huang and Baltimore (1970), treatment with EDTA gave a single peak at 1.41 g/cc corresponding to vRNP (Fig. 11C). Cross-$x&g with glutaraldehyde. The following experiment demonstrated that naturally occurring empty capsids could be hxed by glutaraldehyde into material of density higher than protein, at the concentrations of ribosomal material normally found in gradient fractions. A preparation of purified 3%-methionine-labelled empty capsids in CsCl (see Materials and Methods) was dialyzed for 24 hr against RSB. The preparation was mixed with a cytoplasmic extract of uninfected cells labelled with 3H-uridine, and the mixture was centrifuged through a sucrose gradient (Fig. 12A). The peaks of tritium coinciding with labelled ribosomal

WRIGHT

AND

COOPER

VP0

8-

VPI

15 c!

10

0 7

5 0

s b

k U I C-J I

f

Fraction

IO 5 0

10

20 Fraction

30

No

FIG. 8. Buoyant densities in CsCl of mixtures (made before fixation in 8% glutaraldehyde) of uninfected ribosomes or subunits (“C-amino acid label, gradient of Fig. 1A) with analogous regions from an infected-cell gradient (SH-amino acid label, gradient identical to that of Fig. 1B). (A) 7 rg of uninfected ribosomes (74 S peak) plus 22 rg of infected-cell ribosomes (74 S peak) ; (B) 5 rg of uninfected subunits (60 S peak) plus 22 rg of infected-cell subunits (60 S peak).

RNA were at 74 S, 60 S, and 45 S, i.e., fractions 7, 13, and 20, respectively. As reproducibly found, the 35S-labelledempty capsids sedimented between the 74 S and 60 S peaks. A portion of fraction 8 containing 33 pg of ribosome material, was fixed in 8 % glutaraldehyde and banded in CsCl (Fig. 12B). With complete resolution of the speciesoriginally added, peaks of tritium at 1.56 and 1.54 g/cc and of 3sSat 1.30 g/cc would be expected. However, the labelled protein was fixed into an aggregate of higher density (1.44 g/cc), and the density of the ribosomal material was decreased. Reducing the glutaraldehyde concentration to 1.6 % and the amount of material fixed to 8 pg (Fig. 12C) greatly decreased the amount of

No.

9. Polypeptide composition of aH-viral proteins in the 74 S material analyzed in Fig. 8A, in polyacrylamide gel coelectrophoresis with purified empty capsids labelled with 36S-methionine. FIG.

protein at higher densities, although there was still some distributed at high densities throughout the gradient. Evidence for ribosomal complexescarrying viral protein. Following the preceding and similar tests with a variety of fixation conditions, subsequent glutaraldehyde fixation was done in 1.6 % glutaraldehyde and < 15 pg of ribosomal material, in presence of a mixture of purified 35S-methionine-labelled virions and empty capsids in about equal portions, to test for cross-fixation. In addition, cultures were labelled for a shorter time (3-3.7 hr) to reduce the proportion of label in empty capsids. The first sucrosegradients of such experiments were virtually identical to that of Fig. 1B; 15 erg of ribosomal material from the 60-70 S region were fixed in 1.6 % glutaraldehyde in the presence and in the absenceof the 35S-labelledmixture, then banded in CsCl (Fig. 13). The addition of the 36S-labelled mixture did not alter the fixation pattern. In Fig. 13A, 44% of the 3H-label was recovered in a broad peak between fractions 3 and 9 (1.49 to 1.56 g/cc). The 3H content of the identical region of

POLIOVIRUS

PROTEINS

ON

RIBOSOMAL

STRUCTURES

11

Fraction No. 10. First sucrose gradient of cytoplasmic extracts from infected cells labelled with SH-uridine (see Materials and Methods). A suspension of infected cells was incubated for 3 hr at 37”, then labelled for 2 hr with 8 #i/ml 3H-uridine. The DOC/Brij-treated cytoplasmic extract was centrifuged through a 15 to 30% sucrose gradient in RSB for 13.5 hr at 60,000 9 and 5” in a Spinco SW 25.1 rotor. A sample of each fraction (0.1 ml) was tested for resistance to 1 p&/ml ribonuclease (10 min at 37”) (O---O) before spotting onto discs for the acid wash.

FIG.

Fig. 13B was 43 %, and it contained no peak of g5S. Thus there was no cross-fixation of empty capsids or virions in this experiment, and viral protein is shown to occur in native structures that, from their density, must contain ribonucleoprotein. The composition of the 35S-labeled virion and empty capsid mixture is monitored in a 2238% CsCl gradient (Fig. 13C). The polypeptide composition of the 3Hlabelled material illustrated in Figs. 13A and 13B is compared with that of s5S-labelled empty capsids (Fig. 14; in contrast to most other amino acids, methionine label gives a predominant peak of VP3). As 71% of the recovered 3H-label was in VPO, VPl, and VP3 (fractions 35 to 55) in Fig. 14, it is apparent that these polypeptides were present in the ribonucleoprotein complexes of 1.50 to 1.56 g/cc shown in Figs. 13A and B. To ascertain the nature of these dense complexes carrying viral polypeptides, some of the material of Fig. 13A was treated with 20 mM EDTA before fixation and CsCl banding (Fig. 15A). The proportion of protein label at density 1.50 to 1.54 g/cc decreased from 44 % to 21%, most appearing as free protein (1.30 g/cc) but some as viral

ribonucleoprotein (vRNP, 1.40 g/cc, Baltimore and Huang, 1970; Huang and Baltimore, 1970). A similar preparation was treated with 50 mM EDTA, a concentration that destroys viral polysomes completely (Miller, 1972). A peak at the density of vRNP (1.39 g/cc) with a shoulder at the density of the small ribosomal subunit (in EDTA, 1.43 g/cc-Table 1) was obtained (Fig. 15B), although most of the label was now free protein (1.31 g/cc). Interpretations. These results demonstrated that, when cross-fixation artifacts were avoided, viral protein was found attached to native particles of 60-70 S with a variety of densities between 1.50 and 1.54 g/cc. These structures were labile to EDTA [most viral protein being liberated (1.30 g/cc), but some remaining attached to vRNP (1.40 g/cc)], and were therefore likely to comprise strands of vRNP complexed to a small number of ribosomes, i.e., incipient polyribosomes. Their density, S, value, and behaviour in EDTA differed from that of the replication complex (Wright and Cooper, in preparation). The attached viral protein was again VPO, VPl, and VP3 in approximately equimolar ratios. The possibility that the

WRIGHT

12

AND

COOPER

Huang and Baltimore (1970) noticed uridine-labelled components of 1.44 and 1.47 g/cc during growth of poliovirus, and suggested that they may represent, a vRNPsmall subunit complex and a vRNP-ribosome complex, respectively. The quite sharp 1.44 and 1.47 g/cc peaks that we obtained wiith amino acid and uridine label and using these authors’

fixation

procedure

nevertheless

con-

tained some cross-fixed material, a finding raising some doubt as to whether such complexes do exist & viva. On the other hand, not all of the 1.44 and 1.47 g/cc complexes found in our experiments need be the result of cross-fixation. The question of t,he existence of naturally occurring 1.44 and 1.47 g/cc complexes thus remains open; if they do exist, however, they appear to carry VPO, WI, and VP3. The E$ect of Guanidine on Ribonueleoprotein ComplexesCarrying Viral Protein

5

15 Fraction

25 No.

FIG. 11. Buoyant densities in CsCl of mixtures (made before fixation in 8% glutaraldehyde) of uninfected ribosomes (W-amino acid label, 74 S peak from the gradient of Fig. 1A) and anaIogous regions from infected cell gradients (3H-uridine label, 74 S peaks). (A) uninfected ribosomes + pooled fractions 7-8 of the gradient of Fig. 10 (42 pg total ribosomal material) ; (B) similar mixture, but infected material from a replicate experiment (‘25 pg total ribosomal material); (C) 5 ag of the 74 S infected-cell peak treated with 50 mM EDTA before fixation. structure comprised a complex of vRNP (density 1.40 g/cc) with only a small ribosomal subunit (density 1,.48 g/cc) is unlikely, as such a complex is not expected to have a

density higher than either of its constituents. Some of the protein-labelled material of density 1.54 g/cc may have represented free 74 S ribosomes that had VPO, VPl, and VP3 attached to the small subunit.

The cytoplasm of cells labelled with amino acids from 3 to 5 hours after infection in t’he presence of guanidine typically gave a sucrose gradient pattern (Fig. 1C) different from that of cytoplasm labelled without guanidine (Fig. 1B). Figure 16 shows a duplicate experiment using 35S-methionine. The peak of label at 60-65 S was considerably increased, and there was no peak of acidinsoluble radioactivity at 45 S, nor an obvious shoulder at 7c-80 S. Analysis in CsCl after

fixation

with

8%

glutaraldehyde

of

the 60-65 S material showed a single large peak at 1.47 g/cc (results not shown), but opportunity was not available in this instance to examine

the effects of cross-fixation.

Although this 60-65 S material corresponded in sedimentation coefficient and polypeptide composition to empty capsids, consisting exclusively of VPO, VPl, and VP3 (results not shown), it also contained another more labile structure (seebelow and Fig. 18B). The material (70-80 S) sedimenting just ahead of this peak in the gradient of Fig. 16 was fixed and banded in C&l (Fig. 17A). The major component (47% of the label) banded at 1.40 g/cc, the density of vRNP. The gel pattern of this material (Fig. 17B) showed that 64 % of the label was recovered in the polypeptides VPO, VPl, and VP3

POLIOVIRUS

a b T E,

PROTEINS

ON

RIBOSOMAL

13

STRUCTURES

15

15

10

10

5

5

0

10

20

30

0

70 -2 c z ” v) a

8

- 1I

-0 .5

-f 3 5

15

25

5 Fraction

15

25

NO.

FIG. 12. Sucrose and CsCl gradients of a mixture of a cytoplasmic extract 3%methionine-labelled labelled for 16 hr with 2 &X/ml 3H-uridine, and purified mixture was centrifuged through a 15 to 30y0 sucrose gradient in RSB for 15 hr at Spinco SW 25.1 rotor (gradient A). Portions of fraction 8 from gradient A were CsCl gradients: (B) 33 rg of ribosomal material, fixed in 8% glutaraldehyde; (C) terial, fixed in 1.6% glutaraldehyde.

(fractions 2644), so that the vRNP in the sample analyzed contained at least some of these three polypeptides. Thus guanidine appeared to induce the formation of a novel, labile 65 S structure, and to inhibit the formation of the two species of ribosome-viral protein complexes : the native 45 S subunits no longer carried VPO, VPl, and VP3, and in place of an obvious shoulder of ribosome-vRNP complex at 70-80 S, there was only a small amount of material, most of which had a density of 1.40 g/cc corresponding to vRNP. However, there was sufficient VPO, VPl,

from uninfected cells empty capsids. The 65,006 g and 5’ in the fixed and analyzed in 8 pg of ribosomal ma-

and VP3 in the gel pattern to show that the vRNP did carry these proteins. Attempts

to Dislodge

VPO, VPl,

and VP.3 Cmn-

from the 60-80 S Ribonucleoprotein

plexes Attempts to obtain from the 60-80 S region of gradients like those of Figs. 1B and 1C a 6 S particle, similar to that obtained from the 45 S region (Fig. 7), were not successful. Figure 18 A shows a sucrose gradient analysis after treatment with RNase and 2 M LiCl of viral protein structures sedimenting ahead of the empty capsids.

WRIGHT

AND

B 1 r

COOPER

1.6

Fraction

1.5

g/cc

1.31

-7 0 T-

I.4

\

1.54 1 1.50

1.3

R

Ea 5"

m :: p 0

0

10

5

1

0 5

15

Fraction

25

No.

FIG. 13. Buoyant densities in CsCl of viral protein attached to ribosomal structures, showing lack of cross-fixation. (A) 15 pg of the 6(t70 S region (pooled fractions 10-12) from the first sucrose gradient of an experiment similar to that of Fig. 1B (14 pCi/ml 3H-leucine + 6 &i/ml 3H-lysine, given 3-3.7 hr after infection) fixed in 1.6% glutaraldehyde; (B) the same, but a mixture of purified 36S-methionine-labelled virions + empty capsids added before fixation; (C) the mixture of virions + empty capsids fixed and monitored in a lower density of CsCl.

Most of the labelled protein sedimented in a broad band of W-160 S (calculated as described by McEwen, 1967). Apparently viral protein from the disrupted ribosome-

No.

FIG. 14. Polypeptide composition of viral protein attached to ribosomal structures. The 60-70 S pool analyzed in Fig. 13 (3H-amino acid label, concentrated by acid precipitation) was coelectrophoresed in polyacrylamide gels with purified a%-methionine-labelled empty capsids.

vRNP complexes had reaggregated into structures with a range of sedimentation constants. Empty capsids do not behave in this way. In a second experiment, the 65 S peak made in presenceof guanidine (Fig. 16) was treated with RNase and adjusted to 0.2 M NaCl, then centrifuged through a second sucrose gradient containing DOC to minimize aggregation (Fig. 18B). Two rather broad peaks were obtained at 85 S and 65 S, with a minor peak at 20 S. The gel patterns of the two main peaks of Fig. 18B are shown in Fig. 19. Both consisted almost entirely of VPO, VPl, and VP3. We expect that the peak at 65 S comprised empty capsids, which were resistant to the treatments, and that the other peak was comprised of aggregated material from some other, more labile, structure present in at least equal amounts. Jacobson and Baltimore (1968) show that the intracellular accumulation of the 65 S peak in the presence of guanidine is reversed when guanidine is removed. Both experiments constitute additional evidence that a major part of the protein label in the 60-80 S region, although containing the same polypeptides (VP0 + VP1 + VP3) as empty capsids, is nevertheless in physically quite distinct structure(s). DISCUSSION

Figure 20 summarizes certain characteristics of the ribosomal complexes de-

POLIOVIRUS

PROTEINS

ON RIBOSOMAL

STRUCTURES

15

6 t

Fraction

1.5

3 1.4:

1.3 0

6

No.

FIQ. 16. First sucrose gradient of a cytoplasmic cell extract from infected cells labelled in presence of 2 m&f guanidine. Procedure as for Fig. lC, except that cells were labelled in the presence of *%-methionine (10 pCi/ml), and the gradient was centrifuged for 14.5 hr at 59,000 g in the Spinco SW 27 rotor.

likely to be attached to these structures in the infected cell. In addition (Maize1 et al., 1967; Phillips et al., 1968), they exist in apparently monomeric combination (5-6 S, equivalent to 1 molecule of each), in oli10 20 30 gomers thereof (up to 14 S) and in a large Fraction no. FIG. 15. Buoyant density in CsCl of 6G36 S specific 65 S multimer related to the virion (native empty capsids). material (EH-amino acid label) from infected cell Viral polypeptides attached to the 45 S extracts after treatment with EDTA. (A) 15 pg of the 6%70 S pool illustrated in Figs. 13 and 14, subunits were recovered as a 6 S particle by treated with 20 mM EDTA then fixed in 1.6% disruptive treatments (Fig. 20B), but similar glutaraldehyde; (B) 22 pg of the 74 S peak illustreatment of the other complexes (which contrated in Figs. 8 and 9, treated with 59 mM EDTA, tained much more protein) has only yielded then fixedfin 8% glutaraldehyde. novel large structures resembling aggregates of [VPO, VPl, VP31 monomers. The “stickscribed above, and illustrates some interpreiness” and tendency to aggregate of this tations. Our results show that the polypep- monomer appears to be a pronounced feature tides VPO, VPl, and VP3, products of the (unpublished results). poliovirus structural protein gene, occur in Amino acid label associated with the equal molar ratio and quite ubiquitously ribosomal material did not represent nascent throughout the infected cell. They are found protein, as it consisted of 3 discrete and with the same sedimentation coefliicient and small proteins (<35,000 daltons) of reprodensity as the native 4FjS subunit (Fig. 20A, ducible size, rather than material of a specF), as incipient polyribosomes or oligosomes trum of sizes extending to that of NCVPl (Fig. 20 C, D, E, J, L), as viral ribonucleo- (ca. 105,000daltons). The absenceof NCVPB protein (Fig. 20 G, H), and as the replica- and other viral polypeptides indicated that tion complex (Wright and Cooper, in prepa- the attachment of [VPO, VPl, VP31 was ration). The complex [VPO, VPl, VP31 is specific. It is not known whether these par-

16

WRIGHT

AND

COOPER

A

1.50 Density g/cc

1.40

1.40

1

b T E a” v) 2

50-

1.52 I 1.48

4

0

10

20

30

VP3

p! 0 T;;

E 3 v) 2

2.

I

Fraction

No.

FIG. 17. Buoyant densities in CsCl and polypeptide composition of 70-80 S viral protein structures made in presence of guanidine. Pooled fractions 5 and 6 from the gradient of Fig. 16 were: (A) fixed in 8% glutaraldehyde (11 ag ribosomal material) and banded in CsCI, and ($3) concentrated by acid precipitation and coelectrophoresed in polyacrylamide gels with purified *H-amino acid-labelled virions.

titular VPO, VPl, and VP3 molecules had existed free in the cytoplasm and rejoined as an initiation factor, or whether a small proportion of the NCVPl molecules (from

which they are derived by peptide bond cleavage, Jacobson et al., 1970) had remained, and were subsequently cleaved, on the ribosome that produced t,hem.

A 80 S I

160s I

85 S I

65 s

10

20 Fraction

30 No.

FIG. 18.USucrose gradient analysis of viral proteins from disrupted 60-80 S structures made in the absence (A) and in the presence (B) of 2 mM guanidine. (A) A portion of the 70-80 S region (fractions 5-10) from a gradient similar to that of Fig. 1B was treated with 10 pg/ml RNsse (10 min, 37”) then 2 it4 LiCl, diluted a-fold and centrifuged through a 15 to 30% sucrose gradient in RSB for 5.5 hr at 60,000 9 and 5’ in the Spinco SW 25.1 rotor. (B) A portion of the 60-70 S peak from a gradient similar to that of Fig. 16 was treated with 20 pg/ml RNase (10 min, 37’), adjusted to 0.2 M NaCl then centrifuged through a 25 to 40% sucrose gradient in 10 mil4 Tris.HCl (pH 7.3), 0.2 M NaCl, and 0.1% DOC (3 hr at 309,000 9 and 20” in the Spinco SW 65 rotor) ; fraction 1 was a pad of 6070 sucrose.

Fraction

No

19. Polypeptide composition of %-viral proteins from a disrupted 6&70 S structure made in presence of guanidine, in polyacrylamide gel coelectrophoresis with aH-amino acid-labelled empty capsids. (A) The 85 S peak, and (B) the 65 S peak, of the gradient of Fig. 18B. 17 FIG.

WRIGHT

18 0

VPO,

.

Lobelled

Labelled

VPl,

VP3 VPO.VPl.VP3

Species

Found

-

vRNP

-

Labelled

AND

COOPER

vRNP

Labelled EDTA

*

0

455

Ribosomal

Subunit

0

605

Rlbosomal

Subunit

Species

after

Treatment

ic)

Density

g/cc 1.48 1.30 1.50

1.54 1.44

1.40

1.53

FIG.

20.

Diagrammatic

summary

of the labelled

ribosomal

structures

found in poliovirus-infected

cells.

The question arises of the functional meaning, if any, of the many apparently specific locations found for [VPO, VP1 , VP3]. It happens that these locations closely follow those predicted for the equestron (Cooper et al., 1973), a hypothetical subviral particle proposed as a regulator for poliovirus. This regulator is suggested to repress synthesis of host protein by combining with the 45 S subunit and thereby blocking its link with host mRNA. By a second aflinity for the 5’ end of vRNA, it is also suggested to encourage the vRNA45 S combination and spur on translation of vRNA, a vital acceleration if viral protein synthesis is to

keep pace with that of vRNA. This 5’ ailinity is also suggestedto effect the equally vital limitation of excess synthesis of complementary RNA, and of reining back vRNA synthesis to keep within the required 1: 60 ratio to structural protein (Cooper and Bennett, 1973). Finally, the equestron should be labile, to free vRNA ultimately for maturation. The 6 S [VPO, VPl, VP31 monomer is thus implicated as the candidate for the equestron. In strong support of this, genetic evidence shows that the configuration of a product of the poliovirus structural protein gene controls both the rate of vRNA sgn-

POLIOVIRUS

PROTEINS

ON

thesis (Cooper et al., 1970b) and the repression of host protein synthesis (Steiner-Pryor and Cooper, 1973). Lability is given to this candidate by its content of VPO, which is cleaved ultimately to VP2 + VP4 in the structural unit (Jacobson et al., 1970). Apart from this, there is no direct evidence yet that the [VPO, VPl, VP31 monomer is the equestron. The present results do not indicate whether the monomer was at or near the 5’ end of its complex with vRNA (assumed for the purposes of Fig. ZO), or attached to the 45 S subunit in the polysome, although the latter idea is favoured by the release by EDTA of some viral protein in a particle corresponding to the smaller subunit (Fig. ZOF). The monomer may be a rather basic protein (unpublished results), and its attachment to vRNA may not be restricted to the 5’ terminus; conceivably, it could also stick to other cellular structures without having any function. Also, with only the minor change of cleavage of VP0 to VP2 + VP4, it must ultimately complex with vRNA and with other cleaved monomers as the structural unit. Accordingly, its basicity, general affinity for RNA, and tendency to aggregate are all to be expected. A pronounced feature of the effect of guanidine was the lack of viral protein on the 45 S subunit and on the ribosomal-vRNP complexes, revealing a small amount of vRNP that probably carried [VPO, VPl, VPSJ. An explanation of these findings is possible if, as has been proposed (Cooper et al., 1973), guanidine acts to increase the affinity of the equestron ([VPO, VPl, VP3]?) for the 5’ end of vRNA and also the tendency of [VPO, VPl, VP31 to aggregate, thus removing these polypeptides from complexes with ribosomal material. Alternatively, conformational changes in these polypeptides induced by guanidine may have prevented their combination with ribosomes. The presence of a labile unidentified 60-65 S component carrying [VPO, VPl, VP31 that accumulates in presence of guanidine suggests that the role of empty capsids as such, as direct precursors to the virion (“procapsids,” Jacobson and Baltimore, 196S), could be reconsidered.

RIBOSOMAL

19

STRUCTURES REFERENCES

BALTIMOPE,

D.,

and HUANG,

A.

S. (1968).

ISO-

pycnic separation of subcellular components from poliovirus-infectedand nTrrna1HeLa cells. h’cience162, 572-574. BALTIMORE, D., and HUANG, A. S. (1970).Interaction of HeLa cell proteinswith RNA. J. Mol. Biol. 47, 263-273. C. J., MARTIN, E. M., and COOPER, P. D. (1970). Posttranslationai cleavage of virus

BURRELL,

polypeptidesin arbovirus-infectedcells.J. Gen. Viral. 6, 319-323. P. D. (1968). A genetic map of poliovirus temperature-sensitive mutants. Virology 35,

COOPER,

584-596.

COOPER, P. D., and BENNETT, D. d. (1973). Genetic and structural implications of tryptic peptide analysis of poliovirus structural protein. J. Gen. Viirol. 20, 151-160. COOPER, P. D., SUMMERS, D. F., and MAIZEL, J. V. (1970a). Evidence for ambiguity in the posttranslational cleavage of poliovirus protein. vi~ozogy 31, 4os-418. COOPER, P. D., WENTWORTH, B. B., and McCAHON, D. (1970b). Guanidine inhibition of poliovirus: a dependence of viral RNA synthesis on the configuration of structural protein. ViTology 40, 486493. COOPER, P. D., STEINER-PRYOR, A., and WRIGHT, P. J. (1973). A proposed regulator for poliovirus: the equestron. Inten&-ology 1, l-10. FENWICK, M. L. (1963). The influence of poliovirus infection on RNA synthesis in mammalian cells. Virology 19, 241-249. HUANG, A. S., and BALTIMORE, D. (1970). Initiation of polyribosome formation in poliovirusinfected HeLa cells. J. MOE. Biol. 47, 275-291. JACOBSON, M. F., and BALTIMORE, D. (1968). Morphogenesis of poliovirus. I. Association of the viral RNA with coat protein. J. Mol. Biol. 33, 369-378. JACOBSON, M. F., Asso, J., and BALTIMORE, D. (1970). Further evidence on the formation of poliovirus proteins. J. Mol. Biol. 49.657-669. MCEWEN, C. R. (1967). Tables for estimating sedimentation through linear concentration gradients of sucrose solution. And. Biochem. 20, 114-149. MAIZEL, J. V., PHILLIPS, B. A., and SUMMERS, 33. F.

(1967).

Composition

of artificially

pro-

duced and naturally occurring empty capsids of poliovirus type 1. Virology 32, 692-699. MILLER, A. 0. A. (1972). Study of RNA extracted from HeLa cell polysomes. Isopycnic centrifugation of cytoplasmic particles extracted from poliovirus-infected HeLa cells and normal,

20

WRIGHT

AND COOPER

mouse A9 cells. Arch. Biochent. Biophys. 150, 282-295. PENMAN, S., and SUMMERS, D. F. (1965). Effects on host cell metabolism following synchronous infection with poliovirus. Virology 27, 614420. PENMAN, S., BECKER, Y., and DARNELL, J. E. (1964). A cytoplasmic structure involved in the synthesis and assembly of poliovirus components. J. Mol. Biol. 8, 541-555. PETERMANN, M. L., and PAVLOVEC, A. (1966). The subunits and structural ribonucleic acids of Jensen sarcoma ribosomes. Biochim. Biophys. Acta 114, 264-276. PHILLIPS, B. A., SUMMERS, D. F., and ,MAIZEL, J. V. (1968). In vitro assembly of poliovirusrelated particles. Virology 35, 216-226. STEINER-PRYOR, A., and COOPER, P. D. (1973).

Temperature-sensitive poliovirus mutants defective in repression of host protein synthesis are also defective in structural protein. J. Gen. Viral. 21, 215-225. SUMMERS, D. F., MAIZEL, J. V., and DARNELL, J. E. (1965). Evidence for virus-specific noncapsid proteins in poliovirus-infected HeLa cells. Proc. Nat. Acad. Sci., U.S. 54, 505-513. VAN ELSEN, A., and BOEYI~, A. (1966). Disruption of type 1 poliovirus under alkaline conditions: role of pH, temperature and sodium dodecyl sulphate (SDS). Virology 28, 481-483. YAMANE, I., MATSUYA, Y., and JIMBO, K. (1968). Autoclavable powdered culture medium for mammalian cells. Proc. Sot. Exp. Biol. Med. 127, 335336.