preparations, support the hypothesis that there are at least two 13S species: (i) given the .... Duesberg, P. H., and P. K.Vogt. 1973. Gel electropho- rem of avian ...
0022-538X/78/0025-0453$02.00/0 OF VIROLOGY, Jan. 1978, p. 453468 Copyright a 1978 American Society for Microbiology
Vol. 25, No. 1
JOURNAL
Printed in U.S.A.
Effect of Protein Synthesis Inhibitors on Viral mRNA's Synthesized Early in Adenovirus Type 2 Infection FAYE EGGERDING AND HESCHEL J. RASKAS Department of Pathology and Department of Microbiology and Immunology, Division ofBiology and Biomedical Sciences, Washington University, School of Medicine, St. Louis, Missouri 63110 Received for publication 26 May 1977
Viral mRNA species synthesized early in adenovirus type 2 infection in the presence of cycloheximide were compared with those synthesized in the absence of drug or in the presence of the DNA synthesis inhibitor 1-l-D-arabinofuranosylcytosine. Cycloheximide caused approximately a 10-fold stimulation in the accumulation of [3H]uridine into early viral mRNA species. The only exception was a 24S mRNA transcribed from the transforming end of the genome; in the presence of cycloheximide, accumulation of this mRNA species was stimulated no more than 2-fold. Treatment with cycloheximide also resulted in the accumulation of polyadenylated RNAs transcribed from EcoRI-C that are heterogeneous and smaller than the 20S mRNA. Other translation inhibitors were shown to have similar effects, suggesting that inhibition of protein synthesis early after infection induces alterations in the metabolism of specific RNA sequences.
Before the onset of viral DNA synthesis in KB cells infected with adenovirus type 2, only a limited portion of the viral genome is transcribed into functional mRNA (10, 15, 16, 31, 33). Using viral DNA fragments, the early viral transcripts have been mapped to specific regions of the adenovirus 2 genome. Liquid hybridizations of unlabeled early mRNA to labeled separated strands of adenovirus 2 DNA fragments have shown that four distinct regions, two from each DNA strand, specify early mRNA (30, 31, 33). Hybridization of radioactive early cytoplasmic RNA fractionated by size identified seven or eight cytoplasmic RNA species encoded by the four early gene blocks (8, 12, 34). The analyses of early viral RNA were facilitated by the use of inhibitors of either DNA or protein synthesis. Such inhibitors are effective in preventing the switch from the early to the late phase of infection. The transition from early to late gene expression parallels the onset of DNA replication (4, 17), and DNA replication is contingent upon earlylviral protein synthesis (19). Cycloheximide has been particularly useful in analyzing early transcription; the cytoplasmic RNAs synthesized in the presence of this inhibitor are enriched approximately 10-fold in viral sequences as compared with RNAs synthesized in the presence of 1-l-D-arabinofuranosylcytosine (ara-C) (9, 28). The increased hybridization has facilitated analysis of early cytoplamic mRNA species. However, studies in other viral systems (1, 2, 6, 20, 21) have demonstrated that disruption of protein synthesis can affect viral
gene expression. Consequently, we reinvestigated the effect of cycloheximide on viral RNAs synthesized early after adenovirus 2 infection. RNAs synthesized in the absence or presence of drug were compared by size analysis. First, the 3H-RNAs were fractionated by electrophoresis in polyacrylamide gels containing 98% formamide. The size distribution of viral RNAs transcribed from various regions of the genome was then determined by hybridization to DNA fragments produced by cleavage with endo R EcoRI (Table 1). The early cytoplasmic RNA from the right 42% of the genome includes transcripts of one gene block from the r strand and two regions from the 1 strand. The r-strand mRNA's include transcripts from map positions 76 to 86, sequences contained in EcoRI fragments D and E (30). The I-strand RNAs include molecules specified by EcoRI fragments B and C and mapping, approximately, at positions 66 to 73 and 93 to 99, respectively (30). The size distributions of viral RNAs specified by EcoRI fragments B and D were identical whether prepared from cultures infected in the absence of drug or in the presence of cycloheximide or ara-C (data not shown). As previously reported, a 19S RNA was specified by EcoRI-B, and 19S and 138 RNAs were detected by hybridization to EcoRI-D (12, 34). However, considerably more viral RNA was detected in cyclohexidetreated preparations (Table 1). Normaliing the amount of 3H-labeled viral RNA to cell number, the quantity of labeled RNA species encoded -
453
454
NOTES
TABLE 1. Effect of cycloheximide on the accumulation of viral cytoplasmic mRNA's early after infectiona
J. VIROL.
arations, it is clear that the different profile of EcoRI-A transcripts obtained from cycloheximide RNA results from an approximate 10-fold Viral cpm hybridized/10O increase in the 13S and 11S size classes as comendo . RNA infected cells treated with: pared with a maximum 2-fold increase in the Pom size R EcoRI amount of 24S RNA (Fig. 2 and Table 1). By No class tion fragment ara-C Cycloheximide drug comparison with the results obtained with EcoRI-B, -C, and -D transcripts, it is clear that 0-58.5 248 1,250 1,350 2,600 A the EcoRI-A 24S species is the only cytoplasmic 13S 2,250 2,600 20,200 13,400 B 58.5-70.7 19S 2,500 2,220 RNA whose accumulation is not increased sig19S 1,400 1,200 75.9-83.4 9,100 D nificantly by cycloheximide treatment. The size 89.7-100 20S 1,500 1,500 18,000 C distribution of EcoRI-A transcripts in polyriboa After a l-h virus adsorption period, a culture of KB cells somes was also analyzed (Fig. 2D). The relative was diluted into three 100-ml portions. One portion received amounts of radioactivity in the 24S and 13S no drug treatment, a second was treated with ara-C (20 pg/ peaks were similar to those observed in cytoml) and the third portion received cycloheximide (25 pg/ml). plaic preparations labeled without drug or in Cultures were labeled from 3 to 5 h postinfection with 20 pCi of ['H]uridine per ml. At 5 h, the cultures were harvested; the presence of ara-C and were distinctly differcytoplasmic polyadenylic acid-containing RNA was isolated ent from the profiles obtained from cyclohexiand fractionated by size as described in the legend to Fig. 1. mide-treated cultures. The size-fractionated RNAs were annealed simultaneously to membranes containing RI fragments of adenovirus 2 DNA The 24S and also the 13S polysomal RNAs derived from 0.5 pg of whole genome DNA. Map positions of encoded by EcoRI-A were further analyzed by the endo R * EcoRI fragments were determined by Mulder et to smaller DNA fragments. hybridization al. (26). Hybridization conditions are given in the legend to Hindm fragments G and C, which include the Fig. 1. The number of virus-specific counts per minute in sequences from map positions 0 to 7.5 and 7.5 each size class was calculated. to 17.0, respectively, were used as hybridization by EcoRI-B and -D was 5- to 10-fold more in probes (data not shown). The 24S RNA anthe presence of cycloheximide than the amounts nealed approximately equally to both fragments. detected in preparations not treated with drug In contrast, the 13S RNA annealed five- to sevenfold more to HindHI-G than to the C fragor treated with ara-C. A discrete 20S viral RNA species encoded by ment, as previously reported for cytoplasmic EcoRI-C DNA was present in polyadenylated RNAs (5, 7, 8). Simila experiments were percytoplasmic RNA from both untreated and ara- formed with fragments produced by cleavage C-treated cultures (Fig. 1). In the case of polya- with endo R * Sma I (data not shown). The 13S denylated RNA from cycloheximide-treated RNA annealed to fragments J (map positions 0 cells, a 20S peak of hybridization was discernible to 3) and E (3 to 10.7); the 24S size class annealed in addition to a substantial amount of smaller primarily to fragment E and also to fragment J. heterogeneous material. As for transcripts from Fragment F (11.3 to 18) did not anneal a reprofragments B and D, the 20S [3H]RNA specified ducible peak of RNA. To determine if the effect of cycloheximide by EcoRI-C annealed approximately 10-fold more in the preparations from cycloheximide- on early transcripts was a general effect due to treated cultures as compared with the two other the inhibition of protein synthesis, analogous labeling conditions (Table 1). studies were performed with three other transOnly a small portion of EcoRI-A specifies lational inhibitors, pactamycin (5 x 10-7 M), early mRNA; liquid hybridization studies have puromycin (10- M), and emetine (10-6 M). The identified the early mRNA's as transcripts from inhibitors were added after virus adsorption was map positions 1 to 11 of the r strand (30). RNAs completed; cultures were labeled with [3H]uriprepared from cultures infected in the presence dine from3 to 5 h after infection. Polyadenylated of cycloheximide showed qualitative and quan- RNA fractionated by size was hybridized simultitative differences in the size distribution of taneously to EcoRI-A and EcoRI-C DNAs (Fig. EcoRI-A transcripts (Fig. 2). RNAs labeled 3). The profiles of RNA specified by these two without drug or in the presence of ara-C con- fragments were essentially the same for all intained two major peaks, migrating as 24S and hibitors. Data for the inhibitor emetine is not 13S RNAs. RNAs synthesized in the presence shown, but the results were the same. In this of cycloheiide included a major 13S peak and series of experiments the amount of smaller hetrelatively smaller amounts of 11S and 24S erogeneous material hybridizing to EcoRI-C DNA was prominent. As a control, RNA from RNAs. Comparing the RNAs synthesized in the pres- celLs treated with pactamycin was hybridized to ence of cycloheximide with the other two prep- the other EcoRI fragments. The patterns of
NOTES
VOL. 25, 1978
hybridization seen with EcoRI fragments B, D, E, and F were as observed with cycloheximide (data not shown). The stimulatory effect of cycloheximide on early viral RNA synthesis has been described previously by several laboratories (9, 28, 35). The present study has analyzed the effects of cycloheximide and other protein synthesis inhibitors on the cytoplasmic viral RNA species transcribed from various regions of the genome. With the exception of a 24S size class transcribed from EcoRI-A, all other size classes accumulated 5- to 10-fold more [3H]RNA in the presence of cycloheximide as compared with RNAs synthe-
A.
285 18S
12 10 I
-
8 6 x
E aLU
-
4
2
0 Lu
3 Ccco = 4
z
a
-0
S. 8
6 4I 2p
455
-
k C.
8C 6C
4C
2C
i
-
20 30 SLICE NUMBER 10
FIG. 1. Size fractionation and hybridization of 3Hlabeled early cytoplasmic RNA specified by EcoRIC DNA. To prepare cytoplasmic RNAs, culures were infected with adenovirus 2 at an input multiplicity of 100 PFU per cell (12, 32). The infected culture was divided into three equal portions; one portion was treated with 25 pg of cycloheximide (Calbiochem) per ml, a second portion was treated with 20 pg of ara-C (Sigma Chemical Co.) per ml, and the third portion was incubated in the absence of drug. At 3 h after infection, cultures were exposed to 20 ,Ci of [5,6-3HI uridine (40 Ci/mmol; New England Nuclear Corp.)
per ml; cultures were harvested at 5 h. Cytoplasmic extracts were prepared by suspending washed cells in isotonic buffer (0.15 M NaCl-0.01 M Tris-hydrochloride (pH 7.4)-0.0015 M MgC12, containing 1% diethyl pyrocarbonate) followed by addition of 0.05 to 0.1% Nonidet P-40 (SheU Chemical Co.). Nuclei
were removed, cytoplasmic extracts were clarified by centriftigation at 12,X00 x g for 20 min, and RNA was extracted as previously described by Craig and Raskas (9). Since the virus-specified RNAs synthesized early in infection are polyadenylated (10, 24), RNA preparations were fractionated by oligodeoxythymidylic acid-cellulose chromatography (3). Polyadenylated pHJRNA (1.2 x 106 cpm) synthesized in the absence of drug (A) and in the presence of ara-C (B) and polyadenylated pHJRNA (2.3 x 10C cpm) from cycloheximide-treated cultures (C) were fractionated on 3.5% formamide-polyacrylamide gels (11, 13). Each gel contained pH]RNA from the same number (5 x 107) of infected cells. Electrophoresis was at room temperature for 9 to 12 h at 1.8 to 2.0 mA per gel RNA was eluted from each 2 mM gel slice by incubation in 300 ,u of 6x SSC (lx SSC is 0.15 M NaCl-0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate at 66°C for 36 h. "4C-labeled cytoplasmic RNA from uninfected KB cells was added to a small portion of the pHlRNA sample and fractionated in parallel with the test samples to provide reference positions for 28S and 1SS rRNA. Eluted RNA was hybridized to filters containing 0.5,ag equivalents of EcoRI-C DNA prepared as described previously (11, 34). A 1-,mg equivalent of fragment DNA is defined as the amount of fragment DNA derived from 1 mg of whole genome DNA. To eliminate any possibility of fragment contamination, all DNA fragments were purified by a second round of electrophoresis. Before use in RNA-DNA hybridizations, all filers were pretested by monitoring the efficiency of hybrid formation with 32P-labeled adenovirus 2 DNA (18). RNA-DNA hybridization conditions were 6x SSC and 0.1% sodium dodecyl sulfate for 36 h at 66C (25). To confirm that hybridization of RNA was performed in DNA excess, a second round of hybridization was performed to anneal remaining viral RNA. For aU samples tested, at least 80% of the viral transcripts were annealed in the initial hybridization.
456
NOTES C.
B
A.
2SS1
J. VIROL.
ZS OS
28S 185
D.
28S 185 0
E
10
i
E
m
80
10
10 20 30
10 20 30 10 2030 SLICE NUMBER
60 -,, I 40 Z 20 2 tn
degradation or aberrant processing occurs. This finding explains the heterogeneous size distribution of RNAs from EcoRI-C that was previously reported (34). Lewis et al. (23) identified 19K and l1K polypeptides synthesized from EcoRI-C-specific RNA purified from cells treated with cycloheximide for much longer times (17 h). However the 20S mRNA specified by EcoRI-C contains sufficient genetic information to code for a polypeptide with a molecular weight of 80,000 to 90,000. If the mRNA used in these experiments was similarly affected
10 20 30
FIG. 2. Size fractionation and hybridization of early cytoplasmic and polysomal RNAs specified by EcoRI-A DNA. Polyadenylic acid-containing cytoplasmic RNA was isolated from cultures labeled with 3H]uridine from 3 to 5 h after infection. Polysomal polyadenylic acid-contauwng RNA was isolated from cultures infected in the presence of ara-C and labeled with pHJuridine from 3 to 6 h after infection. To isolate polyribosomes, the cytoplasmic fraction was layered on top of a 10 to 45% sucrose density gradient formed on top of a 5-ml cushion of 60% sucrose and centrifuged at 4°C for 150 to 180 min at 24,000 rpm in an SW27 rotor (24, 27). Sucrose solutions were prepared in isotonic buffer. Polysome-containing fractions were pooled, and the RNA was either extracted directly or dissociated from polysomes with EDTA (22, 29). Greater than 98% of the viral RNA was released by EDTA treatment, confirming that these molecules were part ofpolyribosome structures. Cytoplasmic pH]RNA from 10& ceUs treated with no drug (1.3 x 10 cpm) (A), ara-C (1.7 x 108 cpm) (B), or cycloheximide (2.4 x 10 cpm) (C), and polysomal pH]RNA from 108 cells (5 x 108 cpm) (D) were fractionated by formamide-polyacrylamide gel electrophoresis for 10 h at 2 mA per gel. pH]RNA elated from each gel fraction was hybridized to filters containing 1-pg equivalents of EcoRI-A DNA. Arrows indicate the positions of 28S and 18S marker rRNA.
sized in the absence of drug or in the presence of ara-C. This increased accumulation may be caused by one or more of the following factors: (i) increased stability of early mRNA; (ii) more efficient processing and transport of RNA from the nucleus to the cytoplasm; (iii) stimulated transciption of the viral genome; or, perhaps, (iv) changes in UTP pools in the presence of cycloheximide. Transcripts from two early gene regions, map positions 1 to 11 and 93 to 99, are affected in a unique fashion by the presence of protein synthesis inhibitors. In the case of the RNA transcribed from 93 to 99, the 1 strand of EcoRI-C, treatment with inhibitors results in the accumulation of large amounts of polyadenylated RNA heterogeneous in size and smaller than the 20S mRNA (Fig 1 and 3), suggesting that
A.
Cycloheximade
28518S 2
3
Pactomycin
Puromycin
128S 18S
28S18S
1I 0 0
S1 1- 02 0 1 2 z4 462855
0
1
03
c2:
SLICE NUMBER B. Cycloheximide
'
;
28518S
Poctomycin 28S 18S
Puromycen
28S518S
wU8 '4Z
10 20
30
10 20 30 SLICE NUMBER
10 20 30
FIG. 3. Effect of several protein synthesis inhibitors on cytoplasmic RNA transcribed from EcoRI fragments A and C. Two hours after infection, a culture was divided into three equal portions (4 x 107 cells each): cycloheximide (25 pg/ml) was added to one, a second portion received puromycin (1 x 10r M), and the third aliquot was treated with pactamyCin (5 X 1O7 M). Each culture was labeled with pHJuridine from 3 to 5 h after infection. Polyadenylic acid-containing cytoplasmic pHJRNA was isolated
and fractionated by electrophoresis. pH]RNA eluted from each gel slice (2 mm) was hybridized simultaneously to filters containing 1-pg equivalents of either EcoRI-A or EcoRI-C DNA. (A) pHJRNA hybridized to EcoRI-A DNA; (B) pH]RNA hybridized to EcoRIC DNA. The arrows show the positions of 288 and 188 rRNA markers.
NOTES
VOL. 25, 1978
by cycloheximide, some of these peptides may be transcripts of partial mRNA chains. Comparing the cytoplasmic RNAs transcribed from EcoRI-A in the presence of cycloheximide with those synthesized in the presence of ara-C or in the absence of drug, several differences are apparent. In the presence of cycloheximide, accumulation of 13S tanscripts in the cytoplasm increases approxately 10-fold, whereas the amount of 24S material increases no more than 2-fold. As can be seen from earlier data (5, 11), RNA synthesized in the presence of cycloheximide contains 24S RNA, but this size class is a relatively minor component because of the increased accumulation of 13S and 11S RNAs. In the absence of inhibitors of protein synthesis, the 24S peak is a major component of the cytoplasmic and polysomal RNAs from the left end of the genome (see Fig. 2). A 22S RNA encoded by this region of the genome has also been identified by liquid hybridization of nonradioactive RNA from ara-C-treated cultures (14). These studies of the effects of inhibitors have yielded further information regarding the sequences specifying the viral RNA species transcribed from the left end of the genome. Previous observations relating to the 13S RNAs used extracts prepared from cycloheximide-treated cultures (7, 8). These observations, which have been confirmed here by using polyribosome preparations, support the hypothesis that there are at least two 13S species: (i) given the fragments that encode these RNAs, a single 13S species is not large enough to span the coding distance; (ii) the species encoded by HindIH-G (O to 7.5) is present in 5- to 10-fold greater amounts than the 13S RNA transcribed from HindI-C (7.5 to 17.0) (5, 7, 8). The 11S RNA that accumulates in the presence of cycloheximide anneals to HindIlI-G but not to Sma I-J (unpublished data). Since much of the coding capacity of the EcoRI-A early gene region is required to code for a 24S mRNA, this RNA species must necessarily contain sequences in common with one or more of the smaller RNAs. We thank Mark Telle and Joe Kelley for technical assist-
ance. The pactamycin used in these experiments was generously provided by George B. Whitfield, Jr., of The UpJohn
Co. This study was supported by Public Health Service (PHS) grant CA16007 from the National Cancer Institute (NCI) and American Cancer Society grant VC-94. F.E. is supported by PHS taining grant GM00897 from the National Institute of General Medical Sciences. Cell culture media were prepared in facilities funded by grants from the National Science Foundation and the NCI. This study was also supported by the following companies: Brown & Williamon Tobacco Corporation; Larus and Brother Co., Inc.; Liggett & Myers, Inc., Lorillard, a Division of Loews Theatres, Inc.; Philip Morris, Inc.; RJ. Reynolds Tobacco Co.; United States Tobacco Co.; and Tobacco Associates, Inc.
457
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