J. gen. ViroL (I976), 3 o, 317-328

317

Printed in Great Britain

Morphogenesis of Picornaviruses: Characterization and Assembly of Bovine Enterovirus Subviral Particles By R. T. SU* AND M. W. T A Y L O R t Department of Microbiology, Indiana University, Bloomington, Indiana 47401, U.S.A.

(Accepted 24 October 1975) SUMMARY

Bovine enterovirus-I (BEV-I) infection results in the production of a low amount of infective virus. A large number of non-infectious virus particles can be detected in BEV-1 lysates by haemagglutination. Attempts to isolate DI particles that might be responsible for this effect failed. However, infected cells were shown to contain large amounts of 8oS particles as well as lesser amounts of 160 S, 13o S, 45 S, 14 S and 5 S particles. The proportion of these subviral particles detectable by density gradient sedimentation depended on the ionic strength of the gradient buffer. At high ionic strength I3oS particles were transformed into i6oS particles, and 45 S into 80 S particles. The polypeptide composition of each virus particle was examined. Pulse-chase experiments confirmed that 80 S particles were the predominant virus particles accumulating. No precursor-product relationship could be established for the 8oS particle, although 5S and I4S particles were shown to be precursors of mature virus particles. INTRODUCTION

The infection of HeLa and L cells with bovine enterovirus-I (BEV-I) results in a rapid inhibition of host protein synthesis and gross c.p.e, within 6 h after infection (CordellStewart & Taylor, I973). However, despite the effectiveness of host cell disruption by BEV-I, few infective particles are produced per cell (Taylor et al. 1974). This observation led us to search for defective interfering particles as have been observed in other picornavirus systems (Huang & Baltimore, I97O). In the course of this search, a number of subviral particles were detected and characterized as possible precursors of the mature virion. We noted that aggregation and disaggregation of such particles could be effected by varying the ionic strength of sucrose gradients. METHODS

Virus assays. Plaque assays and growth of cells were as previously described (CordellStewart & Taylor, 1973). Haemagglutination assays were carried out at room temperature by the addition of 2 x IO6 rhesus monkey red blood cells (RBC) to successive 1:2 serial dilutions of BEV-I lysates. Dilutions were performed in disposable microtitre U plates (Cooke EngineeringrCo, Alexandria, Va) containing o'o5 ml of sterilized saline/well. The plates were then sealed * Present address: Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts. "~To whom all correspondence should be addressed.

318

R. T. SU AND M. W. TAYLOR

with cellophane tape and allowed to incubate at room temperature for 3 h at which time haemagglutination end points were recorded. Labelled virus. Radioactively labelled BEV-I was prepared by a modification of the method of Johnston & Martin 097I). L cell monolayers were infected with Ioo BEV-I particles/cell in the presence of 5 #g/ml of actinomycin D at 37 °C- At 2"5 h post-infection, the medium was decanted and replaced with Hanks MEM without any amino acids for 30 rain. Hanks M E M without amino acids, containing 5 % foetal calf serum, 5 #g/ml actinomycin D and 5/zCi/ml [14C]-protein hydrolysate (57 mCi/mAtom carbon), replaced the starvation medium and infected cells were incubated at 37 °C for an additional 4 to 5 h. The crude lysate was fractionated on a pre-formed I5 to 5o % sucrose gradient. The virus peak was determined by infectivity and radioactivity. The pooled fractions containing infectious virus were dialysed against one litre of RSB buffer overnight and banded in a CsC1 solution. Fractions of o.2 ml each were collected and the refractive index and infectivity determined. The measurement of density was facilitated by use of the linear relationship between refractive index and density (lift, Vort, & Vinograd, I96I ). Sedimentation analysis of virus particles. Virus particles were analysed by sucrose gradient sedimentation as adapted from Phillips, Summers & Maizel 0968). Infected cells were labelled with 5/~Ci/ml of [14C]-protein hydrolysate (57 mCi/mAtom) and 2 #Ci/ml of [~H]-uridine (sp. act. = Io #Ci/mmol), in the presence of actinomycin D. The clarified lysate was dialysed for Io h against one litre of RSB buffer containing o'05 M-EDTA, treated with RNase A (20 #g/ml), and loaded on to a I5 to 5o % sucrose gradient in RSB-EDTA buffer. The gradients were centrifuged at 25 ooo rev/min for 3 h in a SW 27. I rotor. Fractions were collected and samples precipitated with cold TCA and collected on millipore discs. The effect of salt concentration on subviral particles. The [~C]-labelled viral lysate, without dialysis, was loaded directly on to a 15 to 5o % sucrose gradient prepared in high salt buffer (o'I5 M-NaCI, o.oI M-tris-HC1, pH 7"2, o.oI M-EDTA). The gradient was fractionated and analysed as before. The pooled fractions containing mature virus and empty capsid were then dialysed separately against low salt buffer (o.ot M-NaC1, o-oT M-tris-HC1, pH 7"2, o.ooi M-MgC12, o'05 M-EDTA; or o.oi M-NaC1, o.oi M-tris-HC1, pH 7"2, o.or M-EDTA) for I6 h, and sedimented in a pre-formed I5 to 5o % sucrose gradient in low salt buffer. Other fractions obtained from sucrose gradients prepared in low salt buffers were dialysed against the high salt buffer and layered on the sucrose gradients in the same buffer. The approximate S value of subviral particles resulting from the change of ionic strength was determined. The relative S values used throughout the experiment were calculated by comparing the distance sedimented with a molecule of standard S value (Martin & Ames, r96 0 . Ribosomal subunits prepared from Sarcoma-18o cells were used as markers. Pulse-chase labelling of virus-specific particles. The precursor-product relationship of virus-specific particles was determined by the method suggested by Jacobson & Baltimore (I968) with some modification. L cells were infected with BEV-I in the presence of actinomycin D, as described previously. At 3 h post-infection, cells were labelled with 25 #Ci/ml of [14C]-protein hydrolysate (57 mCi/mAtom) for I5 min. At the end of the labelling period, the medium was decanted, monolayers were washed twice with warm saline and Io ml of double concentrate Earle's M E M added for 2o or 4o min. The monolayers were then removed with a rubber policeman and disrupted by the addition of 2 ml of TE buffer (o.oot M-tris-HC1, p H 7"4, o.ol M-EDTA). After one cycle of freeze-thaw, samples were centrifuged at Ioooo rev/min for Io min to sediment the cellular debris. The supernatant fluid was dialysed against low salt buffer for 8 to Io h. One part of the sample was loaded

Bovine enterovirus morphogenesis

319

on a I5 to 50 % sucrose gradient and centrifuged at 25000 rev/min for 3 h. The other part of the same sample was centrifuged in a 5 to 15 % sucrose gradient at 4o ooo rev/min for 6 h in an SW 41 rotor. Gradients were fractionated and assayed for acid-insoluble materials. Gel electrophoresis of virus-specific polypeptides. The use of Io % potyacrylamide gel electrophoresis to separate virus-specific polypeptides was performed by the methods suggested by Laemmli (197o) and Raft et al. (197I). Viral and subviral particles obtained from sucrose gradients and CsC1 gradients were pooled and dialysed overnight against the electrophoretic running buffer containing o.2 % SDS. The samples were then concentrated in polyethylene glycol and pelleted at 5oooo rev/ rain for 3 h at 4 °C in a SW 65 rotor. However, the dialysed subviral particles having low S values were concentrated directly by lyophilization and resuspended into o.z to o'5 ml of sample buffer. Samples containing 1% 2-mercaptoethanol, 2 °//o SDS, O'OOI ~o bromophenol blue were boiled for 2 min. After mixing with a small amount of glycerol, the samples were loaded on to gels which were electrophoresed for 7 to 8 h at 5 mA per gel and then fixed in 7 % acetic acid. Each gel was frozen in a dry ice-ethanol bath, sectioned into I mm slices and placed in scintillation vials. Each slice was dissolved by the addition of o.2 ml of 15 % H~O2, and incubated at 60 °C for 12 h. After the vials were cooled to room temperature, 2 ml of Beckman solvent and Io ml of scintillation fluid was added. The vials were kept in the dark at least 24 h before counting to eliminate non-specific fluorescence. RESULTS

Virus yield Although bovine enterovirus-I (BEV-I) is extremely cytopathogenic to cells in cultures of different origin, the number of infective virus particles produced per cell is relatively low. This low effective viral yield is accompanied by the production of a large number of noninfective particles particularly empty capsids (8oS; Fig. I). Utilizing haemagglutination with Rhesus monkey blood cells, the number of virus particles per haemagglutination unit was 3000 in the normal lysate (Table I). Because of the large number of virus particles detected by haemagglutination the existence of defective particles (DI) as observed in poliovirus preparations was investigated (Huang & Baltimore, 197o; Huang, 1973). These particles have been reported to interfere with the multiplication of infectious virions. Although poliovirus DI particles contain the same virus structural proteins and behave antigenically as the mature virion, they are characterized by a lower density than complete virions in CsC1 due to the presence of defective viral RNA (Cole et al. 1971). Sucrose gradient purified viral lysates, of material larger than 80 S, give only one peak with a density of 1.34 g/ml on CsC1 gradients. This result suggests that there are no DI particles present in BEV lysates, the high haemagglutination titre being due to the presence of incomplete virus and/or viral capsid material in the crude lysate. Virus maturation BEV-I infected L cells were labelled either with [3H]-uridine (2 #Ci/ml) or [l~C]-protein hydrolysate (5/~Ci/ml), in the presence of actinomycin D. Cells were ruptured 8 h after infection and prepared for sucrose gradient analysis in low-salt buffer (RSB-EDTA). The profile obtained on a I5 to 50 % sucrose gradient in RSB-EDTA buffer is illustrated in Fig. 1. Using ribosomal subunits obtained from Sarcoma-I8O, the sedimentation value for the

320

T. SU AND M. W. TAYLOR

to

"I

I

I

I

I

I

I

20

18

60 S

16 x

~!

80

14

15

E z)

X

-s 10 d

160 i

8 v

=. r-

,z

45 S

6

..-

;c

/~[

130 S

4

I

~,,"

2 I

B

5

10

I

15

20

25

30

35

T

Fraction number Fig. I. Sedimentation analysis of virus-specific particles. Infected L cells were labelled with 5 #Ci/ml of P~C]-protein hydrolysate (57 mCi/mAtom) or 2 #Ci/ml of pH]-uridine (sp. act. = io #Ci/mmol) at 3 h after infection in the presence of actinomycin D. Viral lysates were prepared after 8 h and sedimented through a 15 to 5o % sucrose gradient in RSB-EDTA buffer in a SW 27.I rotor at 25ooo rev/min for 3 h at 4 °C. Gradients were fractionated from the bottom and analysed for acid-insoluble material. The relative sedimentation values were estimated as described in Methods. O O, virus particles labelled with [14C]-protein hydrolysate; 0 - - - 0 , virus labelled with [3H]uridine. Table L Calculation of virus particles and the specific infectivity of BEV- I grown

in L cells, by haemagglutination of Rhesus monkey red blood cells Input virus* (p.f.u.) 2 × IO 7

Rhesus monkey RBC 2 × 106

Haemagglutination titre 215

No. of particles 6"4 ×

I O 1°

Specific]" infectivity I : 3000

* Input virus was determined by plaque assays. "~ Specific infectivity is calculated as number of p.f.u, versus number of particles. mature virion was calculated to be approx. 160 S. Slower sedimenting particles with approximate sedimentation values of I3oS, 8oS, and 45S were also detected on the gradient. W h e n [14C]- and [aH]-labelled samples were co-sedimented in a sucrose gradient, virus-specific R N A was present in both the I 6 o S and I3oS particle, but not in the 8oS and 45S particles. The 8oS particle probably corresponds to the empty viral capsid found in poliovirus infected cells (Maizel, Phillips & Summer, I967). However, the proportion of 8oS: I6oS particles in BEV is very m u c h greater than that reported for other viral systems, being 2: I. The 13o S particle, which could be equivalent to the defective interfering particle reported for other systems, was non-infective. However, the R N A extracted f r o m the I 3 o S particles had the same sedimentation value as virion R N A , arguing against this being a defective (DI) particle. Fernandez-Tomas & Baltimore 0 9 7 3 ) have reported the isolation o f a I25 S

Bovine enterovirus morphogenesis 20

I

i

i

VP.

i

X

i

321

i

|VP3

V zl II

"6 .'E_ lO

U

© VP~

£ ~

5

,

0 I

I

o X

8O

40 60 Fraction number !

I

I

I

I

90 I

[

I

(h) 12 t

I

10

VP~ VPo~

.o 6-

,a

4 ~

? I

10

20

I

5

i

40 50 60 70 Fraction number

30

I

I

I

I

I

I

I

I

I

80

90

100

I

I

(")

I

)< VP

74

VP3

O "~ 3

O

VP°

~

~4

~, 2

?

l0

20

30

40 50 60 70 Fraction number

80

90

100

Fig. 2. Polypeptide profile of purified [14C]-amino acid labelled virion, and subviral particles. Electrophoresis was performed on IO ~ acrylamide gel at 5 mA/gel for 8 fi. The fractionation and the measurement of radioactivity were determined as described in Methods. (a) Disrupted virions, (b) Empty capsid (8oS), (c) 'provirion' (I 3oS).

22

VIR 30

322

R. T. SU AND M. W. TAYLOR I

I

I

I

16 ~-

I

I

i

80 s

? 14 ×

~; l0

Virion "5

6

~'

4

t

I

--a

2

5

10

15

20

25

30

35

T

F r a c t i o n i1 l l i l l b e r

Fig. 3. Sedimentation analysis of virus-specific particles. The [laC]-labelled viral lysate was sedimented on a I5 to 5o ~/, sucrose gradient in high salt buffer (o'I5 M-NaC1, o'oI u-tris-HC1, pH 7'2, o'oi M-EDTA) at 25ooo rev/min for 3 h in a SW 27.I rotor. The fractionation and the measurement of radioactivity were determined as described in Methods.

particle from poliovirus infected HeLa cells. This particle (the 'provirion') contains a 35 S viral genome and behaves as a precursor of the mature virion. This may be equivalent to the 13o S BEV particle described here. It is well established that picornaviruses contain multiple species of proteins (Summer, Maizel & Darnell, I965). The polypeptide composition of purified BEV-I virus and empty capsid was examined by Io % polyacrylamide gel electrophoresis. A typical profile (Fig. z a) shows that the mature virus has three major proteins, VP1, VP2, VP3 and a minor protein VP4. However, the gel pattern for the empty capsid, 8oS, shows only three polypeptides, VP0, VP1, and VP3 (Fig. 2b). It has been shown that the large polypeptide, VPo of poliovirus is a precursor molecule of peptides VP 2 and VP4 (Jacobson & Baltimore, I968). Gel electrophoresis of the proteins of the ~3oS particle (Fig. 2 c), shows that, in addition to VP~, VP2, VP 3 and VP~, a small amount of VP0 is present. The tool. wt. of the protein components were determined by comparing their mobilities on SDS-polyacrylamide gels with those of standard proteins (Shapiro, Vinuela, & Maizel, I967). The approximate mol. wt. are VPo, 38ooo; VPa, 34ooo; VP~, 280oo; VP3, 25ooo and VP4, I I ooo. The molar ratios of the polypeptides in the mature virion were measured by comparing the proportion of the radioactivity of each peak to the protein mass. VPx, VP2 and VPa were present in approx, equimolar proportion, while VP 4 is present in only half the amount. A similar result has been reported for bovine enterovirus (VG-5-27) by Johnston & Martin 0 9 7 0 .

Bovine enterovirus morphogenesis i

I

I

I

323

I

~)

7 7

X

-

160 S

.=_ 6 5 43-

o

2-

,

1

\

10

I

20

I

i/x.

30 40 Fraction number !

I

I

50

I

I

(h)

,q

)
~

80 S

Virion

~ ,.2.,

Virion 4

Z

10

20

30

l0

20

30

10

20

30

Fraction number Fig. 7. Pulse-chase labelling of virus-specific particles. L cells were infected with ~oo p.f.u, virus{cell, in the presence of actinomycin D. At 3 h post-infection, cells were pulse labelled with 25 #Ci of [x4C]protein hydrolysate (57 mCi/mAtom) for I5 min, then chased with the addition of 2 × Hanks MEM for 2o and 40 min. Viral lysates were dialysed against RSB-EDTA buffer overnight and sedimented through 15 to 5o % sucrose gradients in a SW 27.t rotor at 25ooo rev/min for 3 h, The fractionation and the measurement of radioactivity were determined as described in Methods. (a) Infected cells pulse labelled for 15 rain at 3 h post-infection, (b) At the end of the labelling period, infected cells were chased with 2 × Hanks MEM for 2o min. (c) Infected cells were chased with 2 × Hanks M E M for 40 rain.

326

R. T. S U A N D

l 5 S (a)!

I

20 -,' X

1

M. W . T A Y L O R I

I

I

i

I

(c)-

16

.~

14

"5

12

5S 141 14S

--~' 10 © _o

8

m

14S 0 ~e-

6

=~

4

,.&,

t 10

20

30

10 Fl'actiol]

20

30

10

I

I

20

30

llumb~r

Fig. 8. Pulse-chase labelling o f virus-specific particles. T h e s a m e p r e p a r a t i o n used in the previous experiment was sedimented t h r o u g h 5 to I5 % sucrose gradients in a S W 4t r o t o r at 40oo0 r e v / m i n for 6 h. T h e fractionation a n d the m e a s u r e m e n t o f radioactivity were determined as described in M e t h o d s . (a) Infected cells were pulse labelled for 15 m i n with p4C]-amino acids at 3 h after infection. (b) At the end of the labelling period, cells were chased with 2 × H a n k s M E M for 2o rain. (¢) Cells were chased for 40 min.

Becker & Darnell 0964) that very little label appears in the mature virion of poliovirus during a I5 min pulse suggesting in fact that these subunits are precursors of mature virion. These data also indicate that whereas the 5 S component has a short half life and is being utilized to form subviral particles, 8o S empty capsids continue to accumulate. After 4o min chase with cold amino acids, the empty capsid accumulates more radioactivity than any other subviral particle. DISCUSSION

The work reported in this paper suggests that the inefficient production of BEV-I infective virus is possibly due to the accumulation of a variety of subviral particles. Six classes of particles of viral origin are found on sucrose gradients (I6oS; I3oS; 8oS; 45S, I4S; and 5S). That the I6oS through 45S are of viral origin has been confirmed by polypeptide analysis. The I6oS particle is the final product of virus infection, being the infective particle. It contains viral polypeptides VP1, VP2, VPa, and VP4. These viral capsid proteins are approx. the same size as those reported for BEV-serotype VG-5-27 (Martin, Johnston & Clements, I97O). That the I3oS may be a precursor of the i6oS is suggested by the effect of salt concentration. In low salt, both I6oS particles and a small amount of the t3oS particle are present. However, by raising the salt concentration of the gradient, the 13o S particle is converted into I6oS. While this work was in progress, Hoey & Martin (I974) reported the isolation of a I45 S particle from BEV (VG-5-27). Both the particle isolated by these workers, and the I3oS

Bovine enterovirus morpkogenesis

327

particle reported here appear to be identical. Both particles contain normal size viral RNA, all four viral polypeptides, a small amount of VP0, and are sensitive to CsC1. They are not defective interfering particles. Fernandez-Tomas & Baltimore (I973) have reported a I25S particle in poliovirus preparation. They have provided evidence that this particle was a precursor in the maturation pathway of poliovirus. However, this particle is not identical to the I3oS particle of BEV, since it contains VP0, VP1 and VP3 and unlike the BEV t3oS is sensitive to EDTA. However, these results may in fact reflect basic differences between BEVq, and poliovirus. The fact that a I3oS -+ I6oS transition occurs in high salts argues for it being a precursor. The 8oS component is assumed to be the empty capsid, as described for other picornaviruses. It contains the three polypeptides VP0, VP1 and VP~. The 45 S component contains the same polypeptides, and can be aggregated into the 8o S component by raising the salt concentration. The smallest structural unit of picornavirus origin noted by us is the 5 S component. The decrease in radioactivity of the 5 S fraction during pulse labelling experiments suggests that the 5 S component might be a common precursor for various viral particles. It has been shown that viral capsid protein requires about 2o to 30 rain to be incorporated into virus particles (Penman et aL I964). Ghendon, Yakobsen & Mikhejeva 0972) and Perlin & Phillips (I973) have shown for poliovirus that the 5S component could be a precursor of the 14S component. Our data shows a slight increase in the I4S component with time. Perlin & Phillips 0973) have reported the assembly of 14S particles into 73 S empty capsids. These results suggest that there is an intracellular pool of capsid precursor proteins from which the virion is derived. The level of I4S in our experiment remained constant after a 2o and 4o min chase, which could be explained by replacement of I4S by 5 S material. The pulse chase experiment confirms the original observation that 8o S empty particles are the predominent particles made during virus infection. From these experiments it is impossible to decide whether the small number of I6oS and I3oS particles formed during this period arise from 8oS particles as suggested by Jacobson & Baltimore 0968) or from a 5 S, or ~4S precursor as suggested by Ghendon et al. 0972). Although a number of experiments were done, in no case could we find evidence for 8oS being a precursor of 13o or I6oS. Further experimentation using longer pulses might elucidate this point. The mechanism of morphogenesis of BEV-I is similar to that proposed for poliovirus (Perlin & Phillips, 1973). The primary difference is the finding of a 13o S particle, that might be a provirion, the large amount of 8o S that accumulates and never matures during the infective process, and the ability of BEV subviral particles to aggregate (or self-assemble) in gradients of different ionic concentrations. The reason for the accumulation of empty procapsids, which gives rise to inefficient infection is not known. Whether these particles are true precursors of mature virus awaits further experimentation. This work was supported by Public Health Service grant CA 10417 from the National Cancer Institute. REFERENCES AGOL, V. L, LIPSKAYA, G. YU., TOLSKAYA, E. A., VOROSHILOVA, M. K. & ROMANOVA, L. L (I970). Defect in poliovirus m a t u r a t i o n u n d e r h y p o t o n i c conditions. Virology 4 t , 533-540. COLE, C. N., SMOLER, D., WIMMER, E. & BALTIMORE, D. (197D. Defective interfering particles o f poliovirus. I. Isolation a n d physical properties. Journal of Virology 7, 478-485. CORDELL-STEWART, B. & TAYLOR, M. W. 0973)- Effects o f viral double-stranded R N A o n protein synthesis in intact cells. Journal of Virology xx, 232-237.

328

R.T.

SU AND

M. W . T A Y L O R

FERNANDEZ-TOMAS, C. B. & BALTIMORE, D. (I973). Morphogenesis o f poliovirus. IL D e m o n s t r a t i o n o f a new intermediate, the provirion. Journal of Virology x2, 1122-I 13o. GHENDON, Y., YAKOBSON, E. & MIKHEJEVA, A. (I972). Study o f some stages o f poliovirus morphogenesis in MiO cells. Journal of Virology xo, 261-266. HOEY, E. M. &MARTIN, S. J. (I974)- A possible precursor containing R N A o f a bovine enterovirus. Journal o f General Virology 24, 5I 5-524. HUANG, A. S. 0973). Defective interfering viruses. Annual Review of Microbiology 27, IOI-I I8. HUANG, A. S. & BALTIMORE,D. 0970). Defective viral particles and viral disease processes. Nature, London 226, 325-327. IFFT, J. B., VORT, D. J. & VINOGRAD, J. (I96I). The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge. Journal of Physical Chemistry 65, I 138-I I45. JACOBSON, i . F. & BALTIMORE,D. (I968). Morphogenesis o f poliovirus. I. Association o f viral R N A with coat protein. Journal of Molecular Biology 33, 369-378. JOHNSTON, M. D. & MARTIN, S. S. (197I). Capsid and procapsid proteins o f a bovine enterovirus. Journal of General Virology xI, 71-79. LAEMMLI, V. K. (1970). Cleavage o f structural proteins during the assembly o f the head o f bacteriophage T4. Nature, London 227, 68o-682. MAIZEL, J. V. JUN., PHILLIPS, B. A. & SUMMER, D. F. 0967)- Composition o f artificially produced and naturally occurring empty capsids o f poliovirus type 1. Virology 32, 692-699. MARTIN, a. 6. & AMES, R. N. (I96I). A method for determining the sedimentation behaviour o f enzymes; application to protein mixtures. Journal of Biological Chemistry 236, 1372-I 379MARTIN, S. J., JOHNSTON, M. D. & CLEMENTS, J. A. (I970). Purification and characterization o f bovine enterovirus. Journal of General Virology 7, 1o 3 - t 13PENMAN, S., BECKER,V. a DARNELL, J. E. (1964). A cytoplasmic structure involved in the synthesis and assembly o f poliovirus components. Journal of Molecular Biology 8, 541-555. PERLIN, M. & ~'mLLIPS, a. A. 0973)- In vitro assembly of polioviruses, lII. Assembly o f I4S particles into empty capsids by poliovirus infected HeLa cell membranes. Virology 29, 3t7-329. PHILLIPS, a.A., SUMMERS, D.F. & MAIZEL, J.V. (1968). In vitro assembly o f poliovirus-related particles. Virology 35, 216-226. RAFF, R. A., GREENHOUSE, 6 , GROSS, K.W. & GROOS, P.R. (I97I). Synthesis and storage o f microtubule proteins by sea urchin embryos. Journal of Cell Biology 5o, 516-527. SHA~'IRO, A. L., VlNUELA, E. & MAIZEL, J. V. (1967). Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochemical and Biophysical Research Communications 28, 815-82o. SUMMER,n. V., MAIZEL, J. V. & DARNELL, J. E. (I965). Evidence for virus specific nonspecific noncapsid proteins in poliovirus infected HeLa cells. Proceedings of the National Academy of Sciences of the United States of America 54, 5o5-513. TAYLOR, M. W., SU, R., CORDELL-STEWART,B., MORGAN, S., CRISP, M. & HODES, E. (I974). Bovine enterovirus-1. Characterization, replication, and cytopathogenic effect. Journal of General Virology 23, 173-178. WAITE, M. R. F. & PFEFFERKORN, E.F. (I970). Inhibition o f sindbis virus production by media o f low ionic strength: Intracellular events and requirements for reversal. Journal of Virology 5, 6o-71.

(Received IZ M a y I 9 7 5 )