Messenger activity of Sendai virus ribonucleoprotein in a cell free protein-synthesizing system

VIROLOGY

42,50&521

Messenger

(1970)

Activity a Cell

Free

A. G. BUKRINSKAYA, D. I. Ivanovsky

Institute

of Sendai

Virus

Ribonucleoprotein

Protein-Synthesizing

System

A. PH. BYKOVSKY,

.4ND

of Virology and .V. Ph. Gamaleya Institute USSR

Academy

of Medical Accepted

Sciences,

June

in

Moscow,

V. M. ZHDANOV of Epidemiology USSR

and Microbiology,

16, 1970

The messenger function of Sendai virus ribonucleoprotein (RNP) was examined in a cell-free system using ribosomes from Ehrlich tumor and chick embryo cells. RNP obtained by treatment of virions with 0.5% sodium deoxycholate, preincubated with ribonuclease, and purified by banding in CsCl density gradient (p = 1.31 g/cm3), was able to associate with ribosomes, forming complexes that sedimented at 160 S, 180 S, and, predominantly, at > 300 S when centrifuged in sucrose gradients. The buoyant density of the complexes in CsCl was 1.43 g/cm3 while the complexes formed between viral RNA and ribosomes had a buoyant density of 1.57 g/cm3. Dissociation of the complexes with EDTA and RNase released the RNP with buoyant density of 1.35 and 1.31 g/cm3, respectively. The complexes incorporated labeled amino acids, the incorporation being inhibited by puromycin. The plateau in amino acid incorporation was reached when 40 Gg of RNA in RNP per 500 fig of ribosomes was used. INTRODUCTION

cavity of lo-day-old embryonated eggs 32P04(500 PC1 per egg) or 14C-labeledalgal Evidence has been presented in the prehydrolyzate (200 &i per egg) 1 hour after vious report that parental Sendai virus riboinfection. nucleoprotein (RNP) in the cytoplasm of Virus purification. The allantoic fluid was infected Ehrlich tumor cells is associated clarified at 3000 g and the virus was colwith ribosomes, forming RNP-ribosome lected by centrifugation at 30,000 g for 40 complexes with buoyant density 1.45 g/cm3 min. The virus pellets were suspended in a (Bukrinskaya et al., 1969). Since these 0.01 M phosphate buffer, pH 7.4, layered complexes incorporate labeled amino acids, onto a 60 to 15% sucrose gradient, and the possibility arises that virus RNP is centrifuged at 30,000 9 for 2 hours. The band capable of directing protein synthesis. This containing the virus was collected and possibility was explored in the present dialyzed overnight against.phosphate buffer, investigation using a cell-free proteinpH 7.4. synthesizing system with ribosomes isolated Isolation of virus RXA ad RNP. The from Ehrlich tumor and chick embryo cells. virus preparation was treated with riboThe results presented here show that virus nuclease (10 pg/ml for 15 min at 37”) to RNP possesses messengeractivity, forming digest associated exogenous RNA of cellular polysomes with buoyant density 1.43 g/cm3. origin (Kingsbury and Darlington, 1968)) and virus RNA was extracted by the sodium MATERIALS AND METHODS dodecyl sulfate (SDS)-phenol method as Virus. The 960 strain of Sendai virus was previously described (Barry and Bukrinsgrown in chick embryos as described (Barry kaya, 1968). To isolate virus RNP, the virus and Bukrinskaya, 1968). The virus was suspension was made to 0.5% in sodium labeled by inoculating into the allantoic deoxycholatuts and then treated with ribo508

MESSENGER

FUNCTION

OF

SENDAI

VIRUS

RNP

IN

CELL-FREE

SYSTEM

509

nuclease as described. The material was pmole ATP, 0.1 pmole GTP, 10 pmoles twice diluted with 0.01 ll4 phosphate buffer phosphoenolpyruvate, 50 fig pyruvate kinase, 0.2 mg 1005 fraction, 1 pmole each of 18 to decrease the concentration of deoxycholate and to prevent the formation of the unlabeled amino acids excluding leucine and and 0.01 pmole leucine and cesium-deoxycholate precipitate, and 0.5 ml phenylalanine of the material was layered on a 4-ml phenylalanine. The components of the comlinear CsCl density gradient (p = 1.21 to plete system were preincubated for 30 min at 35” before the addition of templates and 1.42 g/cm3) prepared in 0.01 M phosphate buffer, pH 7.4, containing 0.002 M EDTA radioactive amino acids (1eucineJH and 2 &i/ml). The same and 0.33 mg/ml bovine albumin. The phenylalanine-3H, centrifugation was performed in a kISE volume of HB instead of RNA or RNP m-as Superspeed 50 centrifuge bucket rotor 2114 added to control samples. After incubation at 36,000 rpm for 2 hours. A visible band of at 35”, incorporation was terminated by virus RNP at, p = 1.31 g/cm” was carefully precipitating with an equal volume of 10% trichloroacetic acid; the precipitates were collected and resuspended in phosphate buffer. The ribonucleoprotein thus obtained heated with 5% trichloroacetic acid at 90” were had a sedimentation constant of about 210 S for 15 min. Then the precipitates and was resistant to RNase and EDTA placed onto Millipore filters, mashed with treatment (Bukrinskaya and Zhdanov, 5% trichloroacetic acid, dried, placed into 1970). Only one protein was revealed in this vials containing toluene (PPO + POPOP) radioactivity was counted in a preparation by means of electrophoresis in scintillator; polyacrylamide gel (Zhdanov, unpublished Packard TriCarb scintillation spectrometer. data). Sucrose and CsCl density grcdients. After Freshly extracted virus RNA and RNP 30 min incubation at 35”, l-ml samples of were used in all experiments. the incubation mixtures were chilled in an Chick embryo and Ehrlich ascites tumor cell ice bath and layered onto 17..ml linear ribosomes. Chick embryos 9 days old were gradient of 15-60% sucrose prepared in decapitated, rinsed in Hanks’ solution, and 0.01 M phosphate buffer, pH 7.4, containing minced. Ehrlich tumor cells were obtained 0.003 M MgCl, and 0.01% Macaloid. The on day 7 after inoculation into mice and gradients were centrifuged in a AiSE Superwashed with Hanks’ solution. The cells were speed 50 centrifuge bucket rotor 59,590 at allowed to swell in RSB (0.01 M Tris-HCl, 25,000 rpm for 2 hours at 2”. Fractions were pH 7.4; 0.01 M KCI; 0.0015 M XfgCL) for collected and continuously monitored for 20 min and were then broken with 15-25 absorbance at 260 nm, and radioactivity in strokes of a Dounce homogenizer. The aliquots was determined in acid-insoluble 13,000 g supernatant was layered on succesmaterial as previously described (Bukrinssive layers of 0.5-2.0 M sucrose prepared in kaya et al., 1969). 0.01 M phosphate buffer containing 0.003 Appropriate fractions of sucrose gradients M XgCl, and centrifuged for 2 hours at were selected, diluted with 0.01 M phosphate buffer containing 0.003 M MgC13 , fixed with 140,000 n. A polysomal pellet was suspended to 2 mg/ml in HB buffer (0.05 M Tris HCI, 4% formaldehyde, and centrifuged in prepH 7.4; 0.01 M KCl; 0.005 M MgC12) with formed CsCl gradients. To analyze the 0.25 M sucrose and stored frozen at -70”. incubation mixtures in CsCl gradient, they To obtain 3H-labeled ribosomes, 40 PC1 of were first centrifuged at 120,000 g for 1 hour, uridine-3H was inoculated into the abdominal the pellets were resuspended in 0.01 M cavity of mice on day 5 after tumor inoculaphosphate buffer containing 0.003 M MgC12 tion, the cells were taken on day 7, and and fixed with 4% formaldehyde. CsCl ribosomes were prepared as described. gradients were centrifuged, fractionated, Cell-free protein-synthesizing system. The and absorbance at 260 nm, refractive incomplete incubation mixture contained 0.25 dices and radioactivity was determined as M sucrose and, in 1 ml: 50 pmoles Tris-HCl described before (Bukrinskaya et al., 1969). buffer, pH 7.4, lFi0 pmoles KU, 5 pmoles Electron microscopy. A drop of a sample nlgClz ) 6 pmoles 2-mercaptoethanol, 1 was applied to a Formvar-coated grid,

BUKRINSKAYA,

510

BYKOVSKY, TABLE

aH-AMINO

ACID

INCORPORATION COMPARED

ACTIVITY WITH

AND ZHDANOV 1 RNP AS

OF UNLABELED AND 32P-L~~~~~~ OTHER TEMPLATES

Counts/min per 0.5 mg of ribosomes isolated from: Ehrlich tumor cells

Incubation

mixture

alone

+ POlY U

+ + + + + +

virus virus virus virus virus virus

RNA RNP RNP, RNP, RNP RNP

+ puromycin (100 rg) + ribonuclease (5 pg) without ATP without 100s fraction

360 1023 935 1544 310 477 -

3282 3160 1774 -

Chick embryo cells

242 1130 1029 1284 321 331 192 161

a The incubation mixtures were preincubated for 30 min at 35” before the addition of templates. 32P-labeled RNP was added to the samples containing Ehrlich tumor cell ribosomes, and unlabeled RNP to that containing chick embryo cell ribosomes. Leucine-*H and phenylalanine-3H (2 &i/ml of each) were added simultaneously with templates. After 30 min incubation, the samples were treated as described under Materials and Methods and radioactivity was counted.

stained with uranyl acetate, and viewed in the electron microscope JEM 7. Materials. 32P04, 14C-algalhydrolyzate (1 mCi/ml) and uridine-3H (318 mCi/mmole) were obtained from Leningrad Radioisotope Centre. LeucineX (10 mCi/mmole), leu‘cine-3H (8,2 mCi/mmole), and phenylalanine-3H (10 mCi/mmole) were purchased from Radioisotope Centre, Amersham, England. Phosphoenolpyruvate kinase and polyuridylic acid were obtained from Calbiothem, USA. Bovine pancreatic ribonuclease was purchased from Worthington Biochemical Co., USA. Puromycin was purchased from Nutritional Biochemical Corp., USA.

arations stimulate 3H amino acid incorporation into proteins, the enhancement of amino acid incorporation directed by RNP being somewhat greater than that directed by RNAs. The incorporation induced by RNP was inhibited when ATP or 100 S fraction were omitted or puromycin (100 pg/ml) was added before incubation. When ribonuclease (5 rg/ml) was added before incubation, the amount of radioactivity due to RNP and to the incorporated amino acids was significcantly reduced while RNP before incubation was completely resistant to ribonuclease (3120 cpm before and 3010 cpm after RNase treatment), Therefore, the initial RNP RESULTS becomespartly sensitive to the enzyme durStimulation of Amino Acid Incorporation by ing the translation. Scndai ‘Virus RNP The incorporation of 3H-amino acids was Incubation mixt,ures containing either shown to proceed about linearly for 1 hour Ehrlich tumor or chick embryo cell ribosomes of incubation with all three templates (Fig. 1). were divided into equal volumes; one portion was used as a control to measureendogenous protein synthesis, and to the other parts Correlation of Incorporation Activity with RNA Concentration polynridylic acid, virus RNA, or virus RNP were added at concentrations of about 200 Specific activity of 32P-labeledRNA was estimated by the relation of 32P-radioactivity pg of RNA per 500 pg of ribosomes. RNP was used either unlabeled or 32P-labeled. to RNA concentration determined by ultraAs Table 1 demonstrates, all three prep- violet absorbance at 260 nm. The concentra-

MESSENGER

FUNCTION

OF

SENDAI

vi& RNP

10 -

?

1 -

JO’

. 60’

Time o) in~utk,t~~~ FIG. 1. The time dependence of leucine-3H and phenylalanineJH incorporation into proteins primed by polyuridylic acid, and Sendai virus RNA and RNP. The concentration of all three templates was about 200 ag of RNA per 500 rg of chick embryo cell ribosomes; the concentration of labeled amino acids, 2 &i/ml of each. The final volume of incubation mixture was 0.5 ml.

tion of RNA in RNP was estimated by relation of 32P-radioactivity to specific activity of RNA isolated from the samevirus suspension. One experiment where 32Plabeled RNA and RNP with specific activity 17 cpm per microgram of RNA and 500 pg of chick embryo cell ribosomesper sample were used is shown in Table 2. It is seen that an increase of RNA concentration from 30 to 200 pg does not induce a significant stimulation of amino acid incorporation. A plateau in amino acid incorporation is also observed when 40 pg of RNA in RNP is added. It may be seen again that the incorporation activity of similar concentrations of RNA in RNP is 1.5 times higher than that of deproteinized RNA. Analysis of Polyribosomes in Sucrose- and CsCl-Density Gradients To analyze polyribosomes formed in vitro, 3H-labeled Ehrlich ascites cell ribosomes were used. The incubation mixture containing labeled ribosomes was divided into two

VIRUS

RNP

IN

CELL-FREE

SYSTEM

511

parts. One part was used as a control, and leucineJ4C was added to this sample for 5 min to determine the extent of endogenous protein synthesis. 14C-labeled RNP was added to the other sample. After 30-min incubation the samples were chilled and centrifuged in sucrosegradients; aliquots of fractions were counted. No significant amounts of labeled ribosomes and incorporated amino acids were revealed in the polyribosomal region when the control samplewas centrifuged (Fig. 2A). The considerable amount of ribosomal label at > 300 S, 210 S, and 180 S coinciding with input virus radioactivity was found after RNP-containing sample centrifugation (Fig. 2B). The value of > 300 S polyribosomes is not specified more accurately as far as they occupy the lower parts of 15-60% sucrose gradient where the calculations of sedimentation coefficients using the 80 S ribosome marker give underestimated values. Buoyant density analysis of the peak polyribosome fraction at 180 S (7 and 8 fractions) showed that the principal component bands at 1.43 g/cm3 and a minor peak bands at 1.35 g/cm3 (Fig. 2C). To determine whether this polyribosomal TABLE

2

STIMULATION OF 3H-AMINO ACID INCORPORATION BY DIFFERENT CONCENTRATIONS OF 32P-LABELED VIRUS RNA AND RNPa “P-radioactivity h-4 Control RNA

RNP

RNA

0 523 1250 3750 32 125 714 899 2985 6034

Concentration ofcFgy

30 73 220 2 7 42 52 175 355

3H-radioactivity (cpm) 284 1016 1053 1121 301 568 1417 1474 1839 1652

a The templates were added to the preincubated samples containing chick embryo cell ribosomes simultaneously with leucine-3H and phenylalanineaH (2 #X/ml of each). After 30 min incubation, the samples were treated, and radioactivity was counted as described.

512

BUKRINSKAYA,

BYKOVSKY,

3

10

15

AND

ZHDANOV

5

10

20

fraction

5

IO fraction

I5

15

20

25

fraction

FIG. 2. Formation of polyribosome complexes. Incubation mixture containing 3H-labeled Ehrlich tumor cell ribosomes was divided into two parts. To the first part used as a control leucine-i4C (2pCi/ml) virus RNP was added was added 25 min after incubation for 5 min (A), to the second part, i4C-labeled (B). After 30-min incubation, the samples were chilled, layered on 15-607, sucrose gradients and centrishows the position of monoribosomes as revealed by fuged at 25,000 rpm for 2 hours at 2’. The arrow optical density. Aliquots of fractions were counted. Three-fourths of the radioactivity peak in 7 and 8 fractions from sucrose gradient shown in Fig. 2B were fixed with formaldehyde and centrifuged in C&l at 35,000 rpm for 16 hours at 4-S” (C). FIG. 3. Sedimentation constants and buoyant densities of complexes containing incorporated labeled amino acids. Complete system containing in the volume of 2 ml about 2 mg of chick embryo cell ribosomes was incubated for 30 min with SaP-labeled virus RNP containing 500 rg of RNA. Leucine-3H and phenylalanineJH (2 &i/ml of each) were added in 25 min for 5 min (a) and in 15 min for 15 min (b). The samples were chilled and centrifuged in sucrose gradients as described in Fig. 2. The radioa.ctivity in aliquots was counted, and the sucrose fractions 1 from (a), 11,13, and 15 from (b) were twice diluted with 0.01 M phosphate buffer containing 0.003 M MgC12, fixed with formaldehyde, and centrifuged in CsCl (a, b). Sucrose gradients. (c-f) CsCl gradients.

ci pellet:

32P= 1891&/mitt 3H = 580 ds/min

FZactinn frac+ion

5

IO

15

Fraction

20

25

90

5

fraction

Fraction

Fraction FIG. 3 513

d

0 , 1.6

25

30

j’

15

514

BUKRINSKAYA,

a

BYKOVSKY,

AND

ZHDANOV 1.6

‘A

b

RN-ase

1

c

EDTA \

1.6 I

FIG. 4. Release of 14C-labeled RNP from the 1.43 complexes by RNase and EDTA. The samples containing, in 1 ml, 1 mg of chick embryo cell ribosomes were incubated with W-labeled virus RNP. Leucine-3H, phenylalanine-3H were added in 15 minutes. After 30-min incubation, the samples were sedimented and pellets were resuspended as described under Materials and Methods and then divided into three equal parts, one part (a) was left unchanged, the second part (b) was treated with RNase (10 pg/ ml at 20’ for 30 min), and the third part (c) was made 0.02 M in respect to EDTA. The material was fixed and banded in CsCl gradient.

complex is a functional one, 3H-amino acids were added for 5 min (Fig. 3a) or for 15 min (Fig. 3b) to the incubation mixtures containing unlabeled ribosomes and 32P-labeled RNP, and the samples were sedimented on sucrose gradients. The distribution of radioactivity was similar to that shown in Fig. 2B: the peaks of 3H and %P radioactivity at 160 and 180 S and a shoulder at 210 S were found in both gradients, the peaks coinciding with ultraviolet absorbing material. The considerable amounts of radioactivity were found in the pellets and in someexperiments in the first fraction from the bottom of the tube (Fig. 3b). The fractions containing radioactivity were further analyzed on a CsCl gradient. Fig. 3c shows that all the isotope in the first fraction of a sucrose gradient shown in Fig. 3a is localized in a single sharp peak with buoyant density of 1.43 g/cm3. The same component with 32P and 3H radioactivity

coincided

was revealed

in the 180 S

and 160 S region. (Fig. 3e, f). The 210 S region contained 3zP-radioactivit y in the form of unmodified RNP with

buoyant density of 1.31 g/cm3 (Fig. 3d) while the 180 and 160 S regions contained 32P-radioactivity banded at 1.35 and 1.38 g/cm3, respectively (Fig. 3e, f). Since the last two components were not associated with significant amounts of labeled ribosomes they appearedto represent two forms of modified partially deproteinized RNP. The experiments with RNase and EDTA treatment confirm this suggestion. Dissociation of the 1.&l Complex by RNase and EDTA To compare the position of RNP protein and nucleic label, RNPs labeled with 14Calgal hydrolyzate or 32P04were used in these experiments. After 15 min incubation, 3Hamino acids were added to the samples for 15 min, then the samples were chilled and sedimented at 120,000 g; pellets were resuspended, divided into equal parts, treated with RNase (10 pg/ml at 20” for 30 min) and EDTA (0.02 M), and centrifuged in CsCl gradients. As Fig. 4a and 5a, c show, the both RNPs yield in CsCl four peaks at p = 1.43, 1.38,

MWSENGER

FUNCTION

OF SENDAI

VIRUS

RNP IN CELL-FREE

SYSTEM

515

b

5

io

15

20

25

3raction d

EDTA

9 ‘511

p i-

Fraction 5. Release of 3Wlabeled RNP from the 1.43 complex by RNase and EDTA. The experiment was carried out as described in Fig. 4 except that 32P-labeled RNP was used. The resuspended pellets were divided into two equal parts, one part (a, c) was left unchanged and the other part was treated with RNase (b) and EDTA (d) as described in Fig. 4. The samples were fixed and banded in CsCl gradient. FIG.

1.35, and 1.31, the peak at p = 1.43 coinciding with a major portion of newly synthesized protein, and other components coinciding with minor peaks of incorporated amino acids. The 1.43 complex dissociates after RNase digestion, and the incorporated amino acids are now revealed in the position of mono-

ribosomesat p = 1.55 and 1.51. (Perry and Kelley, 1966; Bukrinskaya et al., 1970) A small amount of 14C-radioactivity was also observed at p = 1.55 in many similar experiments although the principal component containing 14Cand 32Pvirus label was found at p = 1.31 (Figs. 4b, rib). The 1.38 and 1.35 RNP components shown in Fig. 4:~

516

BUKRINSKAYA,

BYKOVSKY,

AND

ZHDANOV

remain practically unchanged after RNase treatment. Similarly, the only component which disaDDears after EDTA treatment is the 1.43 c&ponent. The amount of RNP in the 1.35 component markedly increases while the p = 1.31 component remains unchanged (Fig. 4c, 5d).

t-31

Absence of RNP ModiJications in Incubation Mixture with Ribosomes Eliminated

(_____--yIILyyIIIIO 5

As an additional control of specificity of RNP modifications the incubation mixture containing all components excluding ribosomes was incubated with 14C-labeled RNP for 30 min. As Fig. 6 shows, only one 14Ccontaining component at p = 1.31 g/cm3 is recovered under these conditions. 15

fraction FIG.

6. Buoyant density of RNP after incubation in mixture without ribosomes. W-labeled RNP was added to 1 ml of incubation mixture containing all components (see Materials and Methods) excluding ribosomes. After 30 min incubation, the sample was sedimented and the pellet was resuspended, fixed, and banded in CsCl.

Analysis in C.&l of RNA-Containing bation Mixture

Icun-

The incubation mixture containing Ehrlich tumor cell ribosomes was divided into two parts, one part left as a control of endogenous protein synthesis and to the other part about 400 pg of Sendai virus RNA was

b

Fraction FIG. 7. Buoyant density of incorporated amino acids in virus RNA-containing tion mixture containing about 2 mg of chick embryo cell ribosomes was divided into part was left as a control (a), and to the other part 400 rg of Sendai virus RNA was and phenylalanine-3H (2 &X/ml) was added to the both parts in 15 min incubation. tion the samples were sedimented and the pellets were resuspended, fixed, and banded

samples. An incubatwo equal parts. One added (b). LeucineAfter 30 min incubain CsCl.

MESSKNGER

FUNCTION

OF

SENDA

added. 3H-amino acids were added in 15 min incubation. After 30 min incubation, the samples were sedimented, and the pellets were resuspended, fixed, and banded in CsCl. Figure 7a shows that OD curve in the control sample yields peaks at p = 1.55 represent’ing presumably ribosomes (Perry and Kelley, 1966)) at 1.49 and at lesser densities. No significant incorporation of labeled amino acids is seen, showing that endogenous synthesis is practically absent. An additional peak of optical density at p = 1.57 g/cm3 coinciding with the peak of radioactivity is observed in RNA-containing sample (Fig. 7b) ; this shows that the labeled

FIG. 8. Electron dient stained with M phosphate buffer

\‘IRUS

RNP

IX

CELL-FREE

SYSTEM

517

amino acids are incorporated into a complex which bands at higher density than ribosomes. Observations with the Electron Microscope The fractions of the CsCl gradients shown in Fig. 4a from the density bands at p = 1.55, 1.43, and 1.35 g/cm3 were investigated in the electron microscope. As Fig. 8a shows, the band at p = 1.55 consists of particles that correspond in size to ribosomes. The = 1.33 band contains few pieces of Larental nucleocapsid with a width of 170 A which are usually continuous with helices about 80-100 A and 25 A wide (Fig. Sb).

micrographs of a material from the bands at p = 1.55 (a) and 1.35 (b) of CsCl uranyl acetate. A drop from the gradient fraction was diluted about 5.fold with cont,aining 0.001 M MgC12 and examined. (a) X 300,000, (b) X 340,000.

gra0.01

518

BUKRINSKAYA,

BYKOVSKY,

AND ZHDANOV

FIG. 8b

Figure 9 shows a material from the p = 1.43 band. It is seen that this band contains the large aggregates of ribosomes (PR) in close proximity to the fragments of parent@ nucleocapsid (RNP-1) and helices 80-100 A wide (RNP-2) (Fig. 9a). Figure 9b, c shows that clumps of ribosomes are usually observed on the ends of these RNP structures. Threads of 25 A wide (RNP-3) were also seen in this band, and in some cases their evident connection with polyribosomes was observed. DISCUSSION

The experiments described here provide evidence that Sendai virus RNP can direct protein synthesis in vitro. The stimulation of amino acid incorporation into proteins appears to reflect translation since (a) the

incorporation is inhibited when ATP or the SlOO fraction are omitted from the incubation mixture, (b) when puromycin is present during incubation, and (c) the incorporation depends upon RNP concentration. Some conclusion about the structure of virus polyribosomes can be drawn from the data presented. It is evident that the complexes containing input virus radioactivity and ribosomes which incorporate labeled amino acids and dissociate after EDTA treatment are formed in vitro; these complexes appear to represent virus polyribosomes. Their low buoyant density (p = 1.43 g/cm3) suggeststhat they contain much excessprotein as compared to cellular polyribosomes, and this protein is likely the RNP protein. This assumption is supported by the following evidence: (1)

MI:SSENGI:R

FUNCTION

OF

SENDAI

FIG. 9. Electron micrographs of a material uranyl acetate. (a) X 67,500; (b, c) X 340,000.

from

L4C-1abeled RNP protein is discovered in the 1.43 complex; (2) virus RNP with buoyant density of 1.35 g/cm3 is released when the complex dissociates after EDTA treatment; (3) electron microscopic investigation of the 1.43 fraction of CsCl gradient shows the

J-IKUS

RNP

IN

the p = 1.43 band

CELL-FREE

of CsCl

SYSTEM

gradient

stained

519

with

agglomerates of ribosomes associated, predominantly, with modified RNP; (4) theoretical calculations for the density of virus polyribosomes (Perry and Kelley, 1966) with RNP participating in their formation give close approximations to the reported density.

520

BUKBINSKAYA,

BYKOVSKY,

FIG.

In accordance with this explanation of low buoyant density, the density of complexes between virus RNA and ribosomes is much higher (p = 1.57 g/cm3). The largest RNP-ribosome complexes are found at high S values (>300 S) and in pellets of sucrose gradients, while smaller ones sediment at 160 S and 180 S. The light complexes might consist of modified RNP

AND

ZHDANOV

9 b, c

lower sedimentation with a considerably coefficient than the initial one and comparatively few ribosomes while the heavy complexes involve slightly modified RNP uncoiled only on their ends; the last complexes may appear also as the result of the aggregation of RNP threads. A striking similarity exists between experiments in vivo and i?Lvitro in distribution of

MESSENGER

FUNCTION

OF

SENDAI

input virus label and newly synthesized proteins in sucrose- and CsCl-density gradients, the RSP alterations after 30 min incubation in vitro corresponding to that from 1 to 3 hours after infection in vivo (Bukrinskaya et al., 1969). It has been shown in experiments in vivo that only unfolded and partly deproteinized RNP with buoyant density of 1.38 g/cm3 associates with ribosomes. The RNP structures which predominantly function i?l vitro are mostly of somewhat, less buoyant density (p = 1.35 g/cm3). It seems to be a reason for lower density of RNP-ribosome complexes in vitro (1.43 instead of 1.45 g/cm3 as in experiments in vivo) Although these studies do not provide direct evidence for the mechanism of virus polyribosome formation and RNA translation, it appears that ribosomes are associated with the fragments of RNP which either contain insignificant amount of protein or have such arrangement of protein subunits that the protein is incapable of protecting RNA from RKase digestion. Indeed, the 1.43 polyribosomes are fully digested by RN&se, and the RNP with buoyant density of 1.31 is recovered after such digestion. It seems unlikely that ribosomes bind to fully deproteinized RNA since a small amount of RNP prot#ein is discovered in association with monoribosomes after RNase digestion. The results of the present study suggest that the deproteinization of RNP in the in vitro protein-synthesizing system is accomplished by ribosomes since it does not occur when ribosomes are eliminated from the incubation mixture. Then the entire sequence of events may be postulated as follows. The input. RW with buoyant density of 1.31 g/cm” \vhen added to the ribosome containing incubation mixture associates by its ends with ribosomes (see electron microgranhsj \ -0-m r---l

VIRUS

RNP

IN

CELL-FREE

SYSTEM

.Ql

which induce its uncoiling and partial deproteinization so that the 1.35 and 1.35 RNP structures are formed. A further deproteinization of RNP by ribosomes leads to the appearance of RNP fragments which bind with ribosomes to form polyribosomes and are capable of translation. In addition, the present study also suggest that the specific protein in these fragments does not interfere with the translation process, and, moreover, provides favored conditions for translation as compared to deproteinized RNA. One of the protein translated by virus RNP in vitro appears to be virus RNA polymmerase, since the incubation mixture after 30 min incubation with RNP is active in polymerase reaction resistant to actinomycin D action (paper in preparation). REFERENCES 1~. D., and BUKRINSKAYA, A. G. (1968). The nucleic acid of Sendai virus and ribonucleic acid synthesis in cells infected by Sendai virus. J. Gen. Viral. Z, i’-79. BUKRINSKAYA, A. G., and ZHDANOV, V. M. (1970). Formation of early virus specific polyribosomes in Sendai virus infected Ehrlich tumor cells. Mol. Biol. 4, 313-323. BUKRINSKAYA, A. G., BYKOVSICY, A. Ph., and ZHDANOV, T-. M. (1969). The participation of Sendai virus ribonucleoprotein in virus-specific polysome formation. Virology 39, 705-720. BUKRINSKAYA, A. G., NIKOLAEVA, 0. G., and GRIGORIEVA, M., (1970). Two types of polyribosomes in Ehrlich ascites tumor cells. Proc. Accd. Sci. USSR. In press. KINGSBURY, D. W., and DARLINGTON, R. W. (1968). Isolation and properties of Newcastle disease virus nucleocapsid. J. Viral. 2, 248-255. PERRY, R. P., and KELLEY, D. E. (1966). Buoyant densities of cytoplasmic ribonucleoprotein particles of mammalian cells: distinctive character of ribosome subunits and the rapidly labeled components. J. Mol. Biol. 16, 255-268. BARRY,