RNA synthesis in vaccinia-infected L cells: Inhibition of ribosome formation and maturation

VIROLOGY

46, 730-744 (1971)

RNA Synthesis

in Vaccinia-Infected Formation

ELAINE Department

R. JEFFERTS

of Microbiology,

L Cells: and

inhibition

of Ribosome

Maturation JOHN A. HOLOWCZAK

AND

Rutgers Medical

School, New Brunswick,

Accepted July

New Jersey

08909

.%,1971

The shutoff of cellular ribosome formation following infection by vac~iniavir~ was studied in L mouse flbroblasts. The synthesis of precursor rRNA, its met,hylation, and its conversion first to nuclear, and finally to cytoplasmic ribosomal particles was followed. The datashow that by 2 hr after infection, the processing of precursor rRNA molecules and the rate of maturation of ribonueleoprotein particles was slowed. Three hours after infection little or no synthesis of new ribosomes was detected. The appearance of newly synthesized protein in cytoplasmic ribosomes was compared to that of new rRNA at various times after infection. The synthesis of new ribosomal protein was clearly depressed before that of new rRNA. Experiments in which cells were infected with amino acid-W labeled virions showed that peptides derived from the infecting virions become associated with the nucleus and the nucleolus. It is postulated that the observed inhibition of cellular ribosomal synthesis may be mediated by such peptides.

disease virus, and it is sugg~ted that methylation of the nuclear RNA is interrupted (Brown et al., 1966; Vande Woude et al.,

INTROJECTION

Many viral infections cause marked inhibition of host cell RNA metabolism. Among the major DNA viruses, only the small papova viruses have been report,ed not to interfere with cellular RNA synthesis (Benjamin, 1966). The adenoviruses, with approximately a 7-fold larger genome, have been shown to cause a suppression of new ribosome formation after infection (Hodge and Scharff, 1968; Raskas et ab., 1970). The still larger Herpes ~~~~Ze~virus has been shown to inhibit both synthesis and processing of ribosomal precursor RNAs (Hay et at., 1965; Flanagan, 1967; Wagner and Roizman, 1969). Among the RNA viruses, the effect of poliovirus infection on cellular RNA metabolism has been studied in greatest detail (Attardi and Smith, 1962; Darnell et al., 1967). Synthesis of 45 S precursor RNA is inhibited; the cleavage of this precursor RNA is delayed; and the 50 S ribosomal subunit is nob processed in the nucleus but, accumulates. Similar results have been obtained in cells infected with foot-and-mouth

1969).

Vaccinia virus, with a DNA genome of 160 to 180 X lo6 daltons, also markedly affects host cell RNA metabolism. Previous studies in HeLa cells have shown that, by 4 hr after infection, newIy synthesized rRNA cannot be detected in cytoplasmic extracts. The pattern of nueIear RNA synthesis remains similar to that of contro1 ceils for more than 7 hr, after which the synthesis of 45 S RNA synthesis appears to be prefere~~~,ially blocked (Becker and Joklik, 1964; Salzman et al., 1964). Similar findings have been reported in HeLa cells infected with Shope fibroma virus (Shope, 1932; Ewt.on and Hodes, 1967). In contrast, abortive infection of BHK21 cells with adenovirus 12, has been shown to stimulate rRNAl synthesis (RaSka et al., 1971). 1 Abbreviations used: EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecyl sulfate; sorbital palmitate; polyoxyethylene Tween,

730

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The kinc,tics of rRNA synthesis and processing has been studied in HeLa cells (for recent rev&s seeDarnell, 1968; Attardi and Amaldi, 1970) and in L cells (Rake and Graham, 1964; Perry, 1964; Perry and Kellev, 1966, 1968). It is known that vaccinia infection of L cells results in a more rapid shutoff of host furwt.ions and produces a pattern of viral mR=\‘A synthesis significantly different’ from that of infected HrLa cells (Oda and Joklik, 1967). The cause of this difference is unknown. We have undertaken a detailed study of the inhibit’ion of RNA synthesis in L cells in an attempt to clarify the nature of this virus-w11 interaction and the manner in which the virus can influence the expression of host)genetic information.

) I, CISLLS

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ters and dried. Whm 14C-labeledprccursor~ were emplo\.rd, samples were count et1 in :I. Nuclear Clqicago low background cwuntr~r. Samples conbaining tritium were wuntctl in a Packard wintillation counter. Cell jractionatz’on. Cytoplasmic extract :: 0 t L cells were propared by the met,hod of Penman (1966) Ivith slight modifications. Incorporadon of radioactivity \\-a~ stopprd by the addition of an equal volume of ice cold Earlc’t; saline (Earlc, 1943). The cells were immrdiately collected by centrifugat,ion at 500 ~1for 5 min., washed twice n-i th Earl& saline, swollen in hypotonic RSB bufTc>r, (0.01 M NaCl, 0.0015 M MgCl,, 0.001 JI Tris . HCl, pH 7.4), diluted 1: 2 with dist>illcd water and then disrupted in :I glass homogenizer with pestIt: c~learancc c%librated (0 break virtually all the wlls, hut lws tlw1 MATERIALS AN11 METHODS 1 70 of t 1~~1 nwlri ad determined by phaw Cells and virus. L cells and HeLa S3 cells microscop!, and the rolcwseof lab&~~ nwlwr DNA. ruclei were collected by cacbntrifug:iwre propagated as described previously (Oda and ,Joklik, 1967). Stock preparations tion at 900 q for :3 min, resuspcndctl in 2 ml of vaccinia virus, st,rain WR, ranging in RSB, and 0.3 ml of a solution consist,ing of titer from 2 to 4 X 1011EB/ml, purified as 1 part 10”; DOC:2 parts 10”; T\vc>cn 40 described by Joklik (1962), were employed was added. The preparat’ion ww sl~:dx~I vigorously for 3 sw 011 a Vortex mixer, and as the inoculum. Infection of cells. Cells were infected at a the nucl(~i pcllrt ed as before (Traub cl al., multiplicity of 500 EB/cell as described by 1964). The wmbinod supernatants cons:ti.Becker and ,Joklik (1964). The cells mere tuted the cytoplasmic fraction. Nucleoplasm n-as obtained by lysing the then adjusted with regular medium to a nuclei in HS buffer (0.5 JI NaCI, 0.05 MgCl,, densit,y of 8 X lo5 cells/ml. Control cells were mock-infected by the same procedure. 0.01 ,V Tris, pH 7.4) and treating with deoxyrihonucloase I (Worthington BiochemAll times are expressedas time after dilution. Radioactive chemicals. Uridine-2-14C (55 ical) as described by Penman (1969). xuclcoli wre prepared by the Triton s&i,/pmole), uridine-S-3H (30 pCi/pmole), 100 (Sigma Chemical Co.) and DOC fracvaline-“H (2% pCi/pmole), L-methioninetionation method of Perry and J
732

JEFF~RTS

AND

0.01 M EDTA, 0.01 TriseHCl, pH 7.4), and the suspension was clarified by centrifugation at 12,100 g for 5 min. The resulting supernatant was analyzed by velocity gradient centrifugation using the conditions indicated in the accompanying figures. This method resulted in quantitative conversion of 74 S ribosomes to subunits. These and any preexisting free subunits in the cytoplasm were recovered as ribonucleoprotein particles which sedimented at 50 S and 30 S and which contained only 28 S and 18 S RNA, respectively (Vaughan et al., 1967; Girard et al., 1965). Subunits so prepared have been shown to contain all the structural proteins of native ribosomes (Warner, 1966). Pur@cation of RNA. In some experiments RNA was released from the ribonucleoprotein complex by the addition of SDS (final concentration, 1%) to the extract from cytoplasm or nucleoplasm. The resulting sample was analyzed in 15-30% (~~/w) sucrose gradients prepared in SDS buffer [O.l M NaCI, 0.001 M EDTA, 0.01 M Tris, 0.5% SDS, pH 7.41 (Penman, 1969). Cytoplasmic RNA was purified by a modification of the method of Farnham and Dubin (1965). To samples rendered 2 % in respect to SDS and 0.2 M in respect to Na acetate, pH 5.1, a l/10 volume of Bentonite was added and the mixture was incubated at 3’7” for 20 min. The samples were then shaken for 20 min three successive times with equal volumes of water-satiated, re~st~led phenol. The phenol phase was washed with a 3/4 volume of NaCl-Na acetatd buffer (NaCl, 0.05 M; Na acetate 0.01 .M, pH 5.1). The aqueous phases were combined, made 0.2 M in respect to Na acetate, and 2 volumes of cold ethanol were added to precipitate the RNA. The precipitated RNA was redissolved in NaCl-Na acetate buffer, MgClz (1O-3 M) was added and t,he sample was treated with DNase (10 Icg/ml) for 20 min at room temperat~e and reprecipitated. Nucleoiar RNA was purified by Penman’s method (1969). The phenol-chloroform extraction was followed by three chloroform extractions when radioactive uridine was used as precursor; the entire procedure was carried out twice when the precursor was labeled methionine. NaCl,

HOLOW~ZAK

Conditions for

pulse-chase experiments.

Cells labeled with radioactive uridine were “chased” by t’he addition of actinomycin D (Calbiochem) at, a concentration of 0.1 pg/ ml (Dubin, 1967). Valine-3H was chased by adding 100 times the normal concentration of cold valine. RESULTS

The erect of I~~ect~o~ with ~acc~~~a Virus on ~ncorpor~~on of Radioactive Precursors into Ribosomal Subunits.

L cells were infected as described; at various times 4 X 10’ cells were removed and labeled for 60 min with uridine-14C. Control cells were mock-infected and similarly labeled. From the cytoplasmic extracts prepared at the end of each labeling period, ribosomal subunit,s were isolated as described in Materials and Methods and analyzed on WEB gradients. The results are described in Fig. 1. Control cells could be shown to have a peak of radioactivity coinciding with the 30 S optical density peak. After a 1 hr labeling period there was slight incorporation into the 50 S subunit. This pattern was constant for all periods of labeling in individual samples when pulsed as described (Scherrer and Darnell, 1962). In infected cells the pattern was quite different. At 1 hr, radioactivity under the 30 S peak was evident, but there was substantially more incorporated into heterogeneous RNA sedimenting between 7 and 26 S. By 3 hr, the size of this disperse RNA had reached a maximum; from this point on, both its size and quantity decreased, beeoming almost negligible at 9 hr. A concomitant decrease in total optical density of the subunits isolated from infected cells at times late after infection was seen. This has been attributed to Ieakage of cytopl~m from L cells infected at high m~tiplicities for 4 hr or more (Oda and Joklik, 1967). A similar phenomenon has been reported in HEp-2 cells infected wit,h herpes simplex virus (Wagner and Roizman, 1969). Ribosomal subunits were similarly prepared after labeling infected cells with thymidine-3H (1 MCi: 10’ cells). Label was added at 2.5 hr after infection, the time at

RNA SYNTHESIS

IN \‘ACCINI&INFECTED

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FIG. 1. Incorporation of radioactive uridine into the ribosomal subunits of control and infected cells. Ribosomal subunits were prepared from samples of 4 X 10’ cells labeled for 60 min with 14C-uridine (1 pCi:lO* cells) as described in Materials and Methods. Samples were layered on 28 ml of l&30% (w/w) sucrose gradients prepared in NEB buffer and centrifuged for 16 hr, 25 K, at 8°C in a Spinco SW 25.1 rotor. (A) Control cells. (B) Infected cells, labeled O-l hr pi. (C) Infected, %3 hr. (D) Infected, G-5 hr. (E) Infected, 8-9 hr. O-O, Cpm, laC; --, A~G,,,,~ .

which viral DNA synthesis is maximal (Fig. 2C). Only a small amount of labeled heterodisperse material with no prominent peaks was recovered (Fig. 2A and B). In order to show that the large amount of heterogeneous RNA sedimenting behind the 30 S peak in Fig. 1, B-E, was viral specific mRNA, ribosomes were prepared from cells pulsed for 12 min with uridine-14C. It has been shown that’ only viral messenger and tRNA are labeled within 15 min (Joklik, 1968). Alternate fractions were hydrolyzed in 1 N NaOH for 2 hr at, 37”, neutralized with HCl, and

precipitated with TCA (Dubin et al., 1963) to det)ermine whether there was any incorporation into DNA (Fig. 2D). In addition, the RNA from this region was isolated, purified, and analyzed on 15-30 % SDS gradients as shown in Fig. 2E. From these experiments it was concluded t,hat the labeled material was in fact RNA, that little or no precursor was incorporated into DNA and that this RNA was largely viral mRNA released from the polysomes under the conditions used to prepare bhe ribosomal subunits. Hybridization studies are now in progress to charac-

734

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FIG. 2. DNA synthesis in infected Cek3, the distribution of DNA in gradient8 used to analyze ribosomal aubunits, and the analysis of pulse-labeled RNA obtained from the ribosomal fraction of infected cells. (A and B) Samples of 4 X lo7 control and infected cells were labeled from 2.5-3.5 hr after infection with 2.7 pCi 3H-thymidine. Ribosomal subunits were prepared and analyzed on 23 ml of 15-30% NEB gradient8 by centrifuging for 15 hr at 25 K in an SW 25.1 rotor. (A) Control. (B) Infected. O-0, Cpm 3H; --, A~P,~~~. (C) Identical samples of 2 X 10’ infected cells were labeled for lo-min periods at the times indicated with 4 pCi 3H-thymidine. They were then chilled, cytoplasm was prepared, and acid-precipitable counts were determined. (D) A sample of 4 X 107 infected cells was labeled with 8 PCi I%-uridine from 1.52.5 hr. Ribosomal subunit8 were prepared as described in Material8 and Methods except that the ribosomal pellet was suspended in 0.5 ml of NEB buffer adjusted to contain 2 X 1V mole of EDTA. The sample was then layered on an 11.6 ml 1530% NEB gradient and centrifuged for 5 hr at 40 K in a Spinco SW 41 rotor. The odd-numbered fractions were hydrolyzed with alkali as described in Material8 and Methods, and all gradient fractions then TCA precipitated. O---C, Cpm r4C; e-0, alkali-resistant counts. (E) 4 X lo7 cells were labeled with 40 j&i 3H-uridine at 2.5 hr p.i. At the end of 12 min, ribosomes were prepared; the RNA was purified from the ribosomal pellet as described in Materials and Methods. The sample was layered on a 16307$ (w/w) sucrose SDS gradient and centrifuged at 20 K for 18 hr, 25’C, in an SW25.1 rotor.

more fully these RNA speciesand to confirm the results of the pulse-label studies described here.

1966; Scherrer et al., 1963), in control and infected cells. Actinomycin D at a concentration of 0.1 pg/ml was used to block rRNA synthesis (Perry e2al., 1964; Dubin, 1967). E$ect of Viral Infection on the Flow of Labeled The rRNA precursors labeled in the nucleus RNA and Protein into Ribosomes in before addition of actinomycin D were effecPulse-ChaseExperiments tively chasedinto the cytoplasm under these The kinetics of formation and processing conditions (Girard et al., 1964). At the times of RNA molecules was analyzed, using indicated (Figs. 3 and 4), uridineJ4C was methods described previously (Warner et al., added to control and infected cult,ures; and

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FRACTION Fra. 3. Distribution of radioactiviby in ribosomal subunits after a 1-hr labeling period followed by a 2-hr chase with actinomycin D. Cultures containing 2.4 X lo* cells were mock-infected or infected as described, and 2.4 @Zi ‘Guridine was added 3 hr p.i. Act,inomycin D, 0.1 fig/ml, was added 1 hr later (4 hr p.i.). Aliquots containing 8 X lo7 cells were removed at the times indicated, and ribosomal subunits wereprepared from one half of each sample. The material was layered on %-ml NEB gradients and cenfrifuped for IF hr at 25 K, 8’C, in an SW 25.1 rotor. (A) Control cells. 4 hr. (B) Cont,rol, 6 hr. (C) Infected, 4 hr. (I>) Infected, 6 hr. O-----O, Cpm I%; --, Az60nn2 .

after a I-hr period of labeling, duplicate samples were removed and actinomycin D added to the remaining cells. Two subsequent samples were taken at intervals of 1 hr. The samples from each time point were divided; from the cytoplasmic extract of the first half, ribosomal subunits were prepared as described and analyzed. The cytoplasm from t,he other half was made 1% in respect to SDS and the RNA analyzed on a 27 ml sucrose-SDS gradient. In Figs. 3 and 4 are

shown the result’s of such an experiment. fn control cells, there was, as expected (SC~PT~PI et aE., 1963; Warner et al., 1966), a flow of radioact’ivity first into the small subunit and t,hen into the larger. The two subunits w(‘re labeled to about t’he same extent after a 2-hr chase w&h actinomycin D (Fig. 3B). A similar pattern was seen in SDS-ireatr4 cytoplasm: the 18 S RNA became significantly labeled in a 1-hr pulse with label qpearing in 28 S RNA as the chase prr,c*c~edt4

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FIG. 4. Incorporation of labeled precursor into the cytoplasmic RNA of control and infected cells pulsed for 1 hr and then chased with actinomycin for 2 hr. The cytoplasmic extract of the second half of the samples of Fig. 3 was made 1% in respect to SDS and layered on 27 ml 153O’?/e (w/w) sucrose-SDS gradients, Centrifugation was for 16 hr, 21 K, at 25’ in a Spinco SW 25.1 rotor. (A) Control cells, 4 hr. (3) Control, 6 hr. (C) Infected, 4 hr. (Df Infected, 6 hr. O-O, Cpm I%; --, &nnm .

(Fig. 4A, B). In extracts prepared from cells 4 hr and later aft,er infection, ribosomal subunits were never labeled (Fig. 3C, D) ; and cytoplasmic RNA was seen as a rather broad peak with an average sedimentation coefficient of about 13 S (Fig. 4C, D). Synthesis of ribosomal protein was studied by me~uring the incorporation of valine-3H into t,he ribosomal subunits (Warner, 1966). Cultures were infected or mock-infected and adjusted to 8 X lo6 cells/ml with regular medium. At 1.5 hr and 4 hr, cells were centrifuged, suspended in 5 ml of valine-free medium, and label added (50 $&‘S X lo7 cells). After 10 min, the cells were readjusted to 8 X 106/ml and chased for 2 hr with 100 times the normal concentration of cold valine. Ribosomal subunits were then prepared as described. The amount of radioactive ribosomal protein appearing in the subunits was decreased in cells labeled at 1.5 hr; and,

as shown in Fig. 5, declined even further when label was added at 4 hr after infection. 5?heeject of Vufxiniu ~nfe~i~ on Pre~rsor Synthesis in the Nucleolus The identification of the 45 S rapidly labeled RNA of the mammalian cell nucleolus as the precnrsor to both 28 and 18 S rRNA has been established for some time (Girard ct al., 1964; Scherrer et al., 1963; Zimmerman and Holler, 1967; Greenberg and Penman, 1966; Weinberg and Penman, 1970). Accordingly, we examined t,he synthesis, methylation, and processing of nucleolar RNA t,o determine what the effect of poxvirus infection would be on these conversions and on the methylation of the molecules involved in this process. Cultures of 1.2 X lo* L cells, infected or mock-infected, were collected by centrifugation at 2 hr p.i., resuspended in 30 ml of

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FIG. 5. The incorporation of valine-3H into ribosomal protein in control and infected cells. Samples of 4 X lo7 cells, mock-infected or infected, were dilut,ed into regular medium. At 1.5 hr and at 1 hr I).i. the cells were collected by centrifugation, resuspended in 5 ml of valine-free medium, and pulsed for 10 rnin with 50 &i valine-3H. The cells were immediately diluted to a concentration of 8 X lo5 cells/ml, and 100X the normal concent,ration of cold valine was added. Two hours after addition of the label, the cells were harvested, and ribosomal subunit,s prepared as described in Materials and Methods. Samples were analyzed as described in the legend to Fig. 1. (A) Control. (B) Infected, labeled at, 1.5 hr. (C) Infected, labeled at, 4 hr. --, A26Unrn O---O, Cpm 3H.

methionine-free medium, and labeled with 5 &i of methionine-methyl-l% and 60 6Ci of uridine-3H. An addibional infected culture was similarly treated at, 5 hr p.i. After 10 min of incorporation, one-third of each sample was removed; the remainder was adjusted to 8 X lo5 cells/ml with regular medium and actinomycin added (0.1 .ug/ml). Samples of each culture were removed at 25 min and at 40 min. Each sample was immediately chilled and nucleoli prepared by Perry’s detergent fractionation procedure as described. The nucleolar RNA from half of each sample was purified and analyzed as shown in Fig. 6. At 2 hr after infection, the amount of label incorporated into 45 S RNA was approximately equal in control and infected cells. Methylation and cleavage of this specieswas also observed, but both the level of methylation and the rate of cleavage appeared to be reduced in the infected cells. At, 40 min after labeling there was only half as much 32 S in infected nucleoli as in the control; and by Ti hr after infection, incorporation of label into 45 S RNA could not be detected. The assembly of pre-rRNA and protein into new ribosomal subunits occurs in the nucleus (Warner and Soeiro, 1967; Vaughan et al., 1967). Precursor particles are first

detected in the nucleolus, then pass to the nucleoplasm, where further maturation occurs. To show that ribosomal particles could be recovered from the nucleoplasm of L cells, an extract of cont,rol cells labeled for 90 mm with uridine-3H was prepared and analyzed. Unlabeled cytoplasmic extract from cont,rol cells was adjusted to the ionic strength of HS buffer and added to the experimental samples to provide optical density markers (Fig. 7). Half of each of the fractions under the peaks indicated was pooled, made 1 %I in respect to SDS, and cent)rifuged with cytoplasm prepared from 2 X 10’ HeLa cells also adjusted to a final concentration of 1 ‘X SDS. As can be seen in Fig. 8, except for a small amount of the heterogeneously sedimenting RNA characteristic of nucleoplasm (Warner et al., 1966), only 28 S RNA was present in the 50 S particle while the small 30 S peak contained only 1X S RNA. To determine whether vaccinia virus influences the mat uration of ribosomal precursors, nucleoplasm was prepared from infected and mock-in fected cells labeled for 50 min or 90 min with uridine-3H and analyzed as described in Fig. 7. When infected cells were pulsed from 30 to 80 min after infection the amount of radioactivity recovered in the ribosomal pre-

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IO 15 20 25 5 IO 15 20 25 5 IO 15 20 25 FRACTION FIG. 6. Synthesis, methyl&ion, and processing of nucleolar RNA of control and infected cells. Samples of 1.2 X lo* L cells were labeled with 60 &?i uridine-3H and 5 pCi methionine-methyl_% at 2 hr or at 5 hr p.i. After 10 min of incorporation, an aliquot containing 4 X lo7 cells was removed and actinomycin D, 0.1 fig/ml, was added to the remainder. At 25 min and at 40 min after addition of label 4 X lo7 cells were harvested, nucleoli prepared by the detergent fractionation method, and RNA purified as described in Materials and Methods. One half of the purified RNA from each time point was analyzed in a 27 ml 15-30y0 (w/w) SDS gradient centrifuged for 13.5 hr, 22O in a Spinco SW25.1 rotor. (A) Control, 2 hr, 10 min. (b) Control, 2 hr 25 min. (C) Control, 2 hr40 min. (D) Infected, 2 hr 10 min. (E) Infected, 2 hr 25 min. (F)Infected, 2 hr 40 min. (G) Infected, 5 hr 10 min. O-0, Cpm 1%; a---@ Cpm 3H; ----a A 26chln.

from the nucleoplasm was greater than that recovered from mock-infected cells pulsed for the same interval (Fig. 9). When infected cells were pulsed from 60 to 110 min after infection the amount of radioactivity recovered was quite comparable to that from a control culture labeled for the same period (Fig. 9). This may reflect an immediate slowing down of ribosome maturation, while the observed inhibition of 45 S RNA synthesis reported above becomes an important factor later in infection. The reported accumulation of 50 S particles after poliovirus infection (Darnell et al., 1967), was not observed in this syst,em. cursor

Decline in SpeciJic Activity of RibosomesUsing Radioactive Amino Acids and Radioactive Uridine as Label A comparison of the relative decrease in the appearance of radioactivity in cytoplasmic ribosomes was made using amino acids-

3H to label protein and uridine-3H to label RNA. Identical cultures of infected cells were established. To one culture L-amino acid-3H mixture (0.1 &i/ml) was added; to the other, uridine-3H (0.08 pCi/ml). At hourly intervals 4 X 10’ cells were harvested from each culture and ribosomes prepared and analyzed as described. Figure 10 shows the results of such an experiment. Cont,rol cultures labeled in this manner were also analyzed and the results described previously by Warner (1966) could be reproduced. In infected cells the synthesis of new ribosomal proteins was almost immediately depressed, while the synthesis of new rRNA was not curtailed until about 3 hr after infection. This is supported by the experiments described previously (see Figs. 4 and 5). The data suggest that the ability of new ribosomal protein species to become associated with the rRNA is blocked before the synthesis of new rRNA precursors.

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thy infwt ing virions became associatc:d\\-it II the cc>11 nuc~leusand thci nucleolus. .-(Isimil:u distribution of radioactivity from infwt ing virions has been found in HPL;~ (~~11~ :mcl serondq- cult mw of (*Ilick (mbry( 1 fibrtjblasts (Jrffcrts and Holowczak, ~ulpul)li&~tI rwults) infwtc,d :lI the sarn(’ mulr iI)lic*it\.. The phenomenon tlwrcforr owurs it1 :t wriety of host wlls :tnd ma\- 1)~ ~ignifiwnt iu understsnding tlrc’ ShutOfi Of h(J$l I’f’ll $llll(‘tions. l)I,SCUY3ION 6/

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FIG. 7. Itibosomal precursor particles in the nucleoplasm of control L cells. A culture of 6 X 107 L cells was mock-infected and diluted into medium containing 50 &i-uridine-3H. After 90 min, 0.1 pg/ml actinomycin D was added. At the end of 2 hr, t,he sample was chilled and nucleoplasm prepared as described in Matserials and Methods. Cytoplasmic extract from 2 X 10’ unlabeled L cells was brought to the ionic strength of HS buffer, and added to the sample for an optical density marker. The samples were analyzed by ccntrifugation for 18.5 hr at 24 K, 5’, in a SW 25.1 rotor in a 28 ml 15-3075 (w/w) sucrose gradient prepared in HS buffer. One half of each fraction in the regions labeled A, B, and C was pooled and analyzed as described in Fig. 8. Counts have been normalized to refer to the total sample. o------o, cpm “H- -- -A *60,irn. Associatioll

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To determine whether parental viral peptides become associated with cellular st)ructures other t,han the cytoplasm, cells were infected at a multiplicity of 1000 EB/cell with amino acid-14C-labeled virus. At the times indicated in Table 1, samplescontaining 4 X 10’ cells were removed and fractionated by a modificat,ion of t,he sonication method of Muramatsu and Busch (1964) as described in t)he legend to Table 1. The results indicated that peptides derived from

The infection of wllx in culture \vitll poxviruses results in an inhibition of’ Ilost mw romolecular synthesis. DKA (Jungwirt h and Launer, 1968; Hanafusa, 196Oa,b, 1961, 1962) and protein (Shatkin, 1963; Joklik and Merigan, 1966; Moss, 1968) as w-cl1as host mRNA and rRNA synthesis arc all inhibited (Becker and Joklik, 1964; Salzman rt al., 1964). When virus is inactivated 1,~ physic+al agents such as ultraviolet1 light or hcant,,it may still actively inhibit, thcsc prowuses (Jungwirth and Launer, 1968; nloes, 19ti?i). Further, in the presence of wtinomyc+n D, under conditions where viral fllIl~%i~JIlS arc not expressed, inhibition of host protein synthesis caanowur (Shat’kin, 1963). I’hcw observations have been interpreted w indic~ating t.hat, moleculw which :w part of the infecting virions arc’ actiw in thr inllil)itor> process. Recent studios (Woodson, 1967) have shown that, someviral transcription V;UI occur after infection lvitli U\‘-irr:idiat ed virus. Thus, two types of viral specific molecules can be implicated in the inhibition of host synt,hesis. Early mRn’A molecules t’ranscribed from th(> viral DNA while it is still encapsulated within the cor(J may tw translated into products which :w involwd in the inhibition (Kate? and A[c~.lusl:ln. 1960a,b). Alternatively, these cnrl>- spwiw may function more directly. Thtwt HS,1 specieshave bren dr)monstrated, in p:Lrt , I o be double-stranded molecules (Co11)>- (‘1 ~1.. 1971). Recently it has been propw’trcl 11l:lt double-stranded RKA moleculw fuuct ion to inhibit; protein synthesis in I)oliovirusinfected cells (Hunt and I+~luw~f&l. 1~71). It remains to be shown whc~thw :t +itnil:lr

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FRACTION Fro. 8. Determination of the size of the RNA obtained from ribosomalprecursorparticles in L cell nucleoplssm. The pooled fractions from the regions marked A, B, and C in Fig. 7 were made 1% in SDS. Cytoplasmic extract from 2 X 10’ HeLa cells, prepared in a similar manner, was addedto provide optical density markers. The samples were layered on 27 ml SDS gradients and centrifuged for 18.5 hr, 21 K, 22' in a SW 25.1 Spinco rotor. (A) Region A. (B) Region B. (C) Region C. a-0, Cpm aH A mlnm . -2 0.600

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20

25

5

IO

15

20

25

FIG. 9. Incorporation of radioactivity into nucleoplasmicribosomalparticles in control and infected cells. Control and infected cell cultures containing 8 X lo7 cells were labeled for 50-min periods from 30 to 80 min or from 60 to 110 min pi. with 100 &i uridine-aH. At the end of the labeling period, samples were chilled and nucleoplasm prepared. Samples without added marker were layered on 28 ml 153Cx,, (w/w) HS gradients and centrifuged for 18.5 hr, 23 K, 5’, in a Spine0 SW 25.1 rotor. (A) Control, 3040 min p.i. (B) Infected, 30-80 min p.i. (C) Infected, 60-100 min p.i. a----0, Cpm 3H; ~ A260nrn .

phenomenon occurs after poxvirus infection. Other macromolecules which may be involved in the shutdown of host synthesis are viral structural peptides which might be released during uncoating (Joklik, 1966). This is supported by the data presented here, which show that viral structural peptides enter the host cell nucleus and become associated with nucleoli. Experiments are

now in progress to determine the relationship of these peptides to the structural peptides of the mature virion (Holowczak and Joklik, 1967). The analysis of RNA synthesis and the maturation of ribosomal subunits described in this paper support and extend the work of Perry and his co-workers (Perry, 1964; Perry et al., 1964). The steps in maturation

RNA

SYNTHESIS

IN

VACCINIA-INFECTED

I, CELLS

741

/---+3os S”bun’t I

2 HOURS

4

3

5

6

POST DILUTION

FIG. 13. Specific activity of the ribosomal subunits of infect,ed cells during t,he course of infect ion. A large culture of L cells was infected and labeled at time of dilution with 0.08 &i/ml uridine-aH. At hourly intervals, samples of 4 X lo7 cells each were removed and ribosomal subunits were prepared as described in Materials and IMethods. An identical culture was infected, diluted into a mixture of 50% regular medium and 500/d MEM without amino acids, and labeled wit,h 0.1 &i/ml 3H-amino acid mixture. llibosomal subunits were prepared as shown. Samples were analyzed on 28 ml NEB gradient,s as described in Materials and Methods. Specific activity was det,ermined by integrating the area under the optical density peak for each subunit and determining the acid-precipit,able count,s associated with each. O--O, Sp. Act. 30 S subunit, amino acid-3H label; a---@, sp. act. 50 S subunit,, amino acid3H label; a---n, sp. act. 30 S subunit,, uridineJH label; A--A, sp. art. 50 S subunit,, luGline-“H label.

appear to be similar to those already outlined for the process as it occurs in HeLa cells (see Attardi and Amaldi, 1970). The effect of virus infection on this process in L mouse fibroblast,s is more rapid than that previously described for HeLa cells (Becker and Joklik, 1964; Salaman et al., 1964; Oda and Joklik, 1967). This may reflect smaller pool sizes in the L cells so that the effect is more rapidly expressed than it is in the HeLa system. We have been able to show t,hat the transport of rRNA to the cytoplasm has virtually stopped by 3 hr after infection. RadioacGve precursors appear neither in subunits nor ribosomal RNA after t)his time; nor can label be chased into cytoplasmic ribosomal particles. The cleavage of 45 S RNA to 32 S and 18 S RNA is inhibited by 2 hr after infection. Methylat,ion of the 45 S RNA is also reduced as compared to control cells, but, is never absent ; cleavage of this molecule does occur, and thr processing which takes

place appears to bc normal. We havtt interpreted this to indicate that the synthesis and methylation of these molecules was slow%d but, was not aberrant. A eriCcal study of the methylation pattern of these moleculrs is planned to resolve this problem. A careful comparison of the synthrsis of RNA and protein required for the formation of ribosomes suggests that, after infwtion, the rate of synthesis of ribosomal proteins declines before the synthesis of the ribosomal RNA. Since t)hese two processes must be tightly coupled to produce mature ribosomw it may be that the lack of one or mow rihosomal proteins could manifest, itself in the slowing of the maturation process, and ultimately it,s complete aboMion. Again, it should be possible using techniques already described (Warner, 1966) to approach this problem directly and demonstratcb whether one or more ribosomal proteins are exhausted as t,he inhibition proceeds. Alt~w~atiwl?,,

742

JEFFERTS TABLE

AND

1

I)ISTRIB~TION OF RADIOACTIVITY Fn.4cT10Ns AFTER INFECTION 14C-I,.2~~~~~ VIRIONS”

IN

CELLULAR WITH

14C Cpm recovered in fractions at various times post infection

Cellular fraction

Cytoplasm Nucleoplasm Nucleoli

1.0 Hr

1.5 Hr

2.0 Hr

846 83 164

1093 38 81

925 46 80

0 1.2 X 10s cells were infected at a multiplicity of 1000 EB/cell with 14C-amino acid labeled virus (48 cpm/pg viral protein). At 1, 1.5, and 2 hr pi. samples containing 4 X lo7 cells were removed and cell fractions prepared by a modification of the method of Busch (Muramatsu and Busch, 1964). Cytoplasm was prepared as described in Materials and Methods. The washed nuclei were suspended in 2 ml RSB and sonicated 45 set with the micro tip of a Branson sonifier. l’he nucleoli were pelleted by centrifugation at 10,800 g for 5 min. The supernatant constituted the nucleoplasmic extract. The distribution of TCA precipitable radioactivity in each fraction was determined as described in Materials and Methods.

the virion peptides shown here to fractionate with the nucleolus after infection, could complex wit)h rRNA precursors preventing proper assembly. ACKNOWLEDGMENTS We would like to thank Miss Domenica Bucolo for excellent technical assistance. This work was supported by National Cancer Institute research grant CA-11027 and training grant CA-05234 and by a grant-in-aid from the Moy Mel1 Foundation. Part of the work reported in this paper will be included in a dissertation to be submitted by Elaine R. Jefferts in partial fulfillment of the requirements for the Ph.D. degree in Microbiology awarded by the Graduate School of Rutgers University. REFERENCES ATT~RDI, G., and AMALDI, F. (1970). Structure and synthesis of ribosomal RNA. Annu. Rev. Biochem. 39, 183-226. ATTARDI, G., and SMITH, J. (1962). Cold Spring Harbor Symp. Quant. Biol. 27, 271-292. BECKER, Y., and JOKLIK, W. K. (1964). Messenger RNA in cells infected with vaccinia virus. Proc. Tat. Acad. Sci. U. S. 51, 577-585. BENJAMIN, T. L. (1966). Virus-specific RNA in

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I:Th

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VBCCINIB-I~FECTI~:I~

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