Transcription in vaccinia virus cores

34

Biochimica et Biophysica A eta. 782 (1984) 34 40 Elsevier

BBA 91343

T R A N S C R I P T I O N IN VACCINIA VIRUS CORES T H E RNA P R O D U C E D DURING T H E F I R S T R O U N D OF T R A N S C R I P T I O N D O E S N O T DIFFER F R O M RNA TRANSCRIBED IN S U B S E Q U E N T R O U N D S PIROSKA HUVOS Department of Microbiology, Duke University Medical Center, Durham, N C 2 7710 ( U. S.A ~) (Received September 5th, 1983) (Revised manuscript received January 30th, 1984)

Key words: Viral transcription," Vaccinia virus," Transcript analysis; Restriction enzyme analysis

RNA was synthesized in vitro in vaccinia virus cores for times sufficiently short that only the first round of transcription took place. This RNA was compared to RNA synthesized for longer times (which is mainly comprised of reinitiated RNA) by hybridization to fragments of vaccinia virus DNA obtained with several different restriction endonucleases. Under the conditions studied, the composition of mRNA produced during the first round of transcription was the same as that produced in subsequent (reinitiated) rounds. Thus, there is no special arrangement of polymerase molecules within the cores that would allow, in the first round of transcription, for the synthesis of mRNAs distinct from those made in subsequent rounds of transcription. Thus, attachment of polymerase molecules to DNA is likely to occur during preincubation in a way similar to that during reinitiation.

Introduction Vaccinia is a large DNA virus (genomic complexity 123.106 daltons) which replicates in the cytoplasm. Enzymes necessary for the synthesis and modification of mRNA are contained in the virus particles and can be activated in vitro in virus cores. (There are 150-200 RNA polymerase molecules per core particle; Ref. 1). The mRNA synthesized in vitro has been studied in detail. The sedimentation coefficient of the RNA synthesized in cores is about 10-12 S, like that of early mRNAs transcribed in infected cells. This size is such that about 200 different m R N A species could be encoded in the whole genome. The major translation products of mRNA synthesized in vitro have been identified as 'early proteins' [2,3]. This means that most of the mRNAs transcribed in vitro correspond to early mRNAs 0167-4781/84/$03.00 © 1984 Elsevier Science Publishers B.V.

transcribed in infected cells. Cabrera et al. [4] showed that RNA transcribed in vitro hybridized to most HindIII fragments of vaccinia DNA, showing that there were no major portions of the genome that were not transcribed. There have been a number of studies on the mode of transcription of the RNA synthesized in vitro. Although there have been reports of very large core-associated transcripts, which would imply the necessity for processing (see Ref. 5), it was subsequently shown, using a coupled transcription-translation system [6], that most major mRNA species are synthesized from individual promoters. Also, a study on some of the immediate early mRNAs by Wittek et al. [7] has shown that RNAs encoded within the inverted terminal repetition are not formed by splicing. During RNA synthesis in vitro by viral cores, the amount of RNA made can be many-fold greater

35 than the DNA, i.e., there is continuous reinitiation, termination and release of RNA [8]. The studies mentioned so far relate to RNA preparations that were primarily reinitiated RNA. The properties of the RNA produced during the first round of transcription are unknown. Such RNA may or may not resemble the RNA made during subsequent rounds of transcription. If RNA polymerase molecules are stored by being attached only to certain genes, then the population of RNA molecules transcribed during the first round may differ significantly from that transcribed during subsequent rounds. If the enzyme molecules are not attached to DNA, and the requirements for initiation are the same in the first and subsequent rounds of transcription, then the population of RNA molecules synthesized during the first round should be the same as those synthesized in subsequent rounds of transcription (reinitiated RNA). Thus, comparison of the products of the first round of transcription and reinitiated RNA may indicate how RNA polymerase molecules are stored within virus particles. The present study describes the results of such a comparison. Materials and Methods

Vaccinia virus (strain WR) was grown and purified as described by Joklik [9] and by Holowczak and Joklik [10]. The virus isolated from 25-40% linear sucrose density gradients in 1 mM Tris-HC1, pH 9.0, was pelleted and resuspended in 1 mM Tris-HC1, pH 9.0, at a concentration of about 1.2 m g / m l . Cores were prepared as described by Nevins and Joklik [2]. The conditions of core RNA synthesis in vitro were as described by Nevins and Joklik [2], but no macaloid was used. The components of the reaction mixture were: 50 mM Tris-HCl, pH 8.5; 45 mM 2-mercaptoethanol; 1.25-10 A260/ml vaccinia virus cores; 50 mM KC1; 5 mM MgC12; 3 mM MnClz; 4 mM ATP, 2 mM G T P and CTP, 5-50 ~M UTP. RNA that was used for hybridization was labelled with [et-32 P]UTP to a specific activity of (4-40)-106 c p m / ~ g UTP. Preincubation prior to initiation of transcription was carried out for 10 min at 37°C in a mixture identical to that described above for core RNA synthesis but lacking either UTP (when the reaction was started by

addition of UTP) or divalent cations (when the reaction was started by addition of MgC12 and MnC12). Purification of core RNA by digestion with DNAase and extraction of cores with phenol was essentially as described by Cabrera et al. [4]. Isolation of viral DNA was achieved by digesting virus particles suspended in 10 mM Tris-HCl (pH 7.8)/20 mM NaC1/1 mM CaC12/0.5% SDS for 2 h at 37°C with proteinase K (1 m g / m l ) , extracting with phenol and precipitating with ethanol. Restriction endonuclease digestion: restriction endonucleases were obtained from New England Biolabs. The buffers used for incubation were those recommended by New England Biolabs. Gel electrophoresis of DNA fragments: restriction endonuclease fragments of DNA were separated on 0.8 or 1.2% agarose slab gels (16 x 16 x 0.3 cm). Electrophoresis was in Tris-phosphate buffer containing EDTA [11]. After electrophoresis the gels were stained for 30 min with ethidium bromide (0.5 /tg/ml). Molecular weights were estimated by comparison of electrophoretic mobilities with those of phage X or ~ X 174 RF DNA restriction fragments produced with HindIII or HaeIII (New England Biolabs). Transfer of DNA fragments from gels to nitrocellulose membrane-paper (Schleicher-Schuell) was carried out according to Southern [12]. Hybridization of RNA (or nick-translated DNA) to nitrocellulose filters: strips of about 5 mm width were cut from the nitrocellulose sheets to which DNA fragments had been transferred and preincubated for 6 h or overnight in Denhardt [13] solution (0.02% polyvinyl pyrrolidine, 0.02% Ficoll, 0.2% bovine serum albumin) containing 4 x SSC ( l x S S C = 0 . 1 5 M NaC1/0.015 M sodium citrate). They were then covered (after being rolled up) with about 0.75 ml of hybridization mixture (4 x SSC, 1.4 x Denhardt solution, 0.4% SDS and 1 mM EDTA) containing the sample to be analyzed. DNA on the filters was in overall excess over the probe: the amount of DNA on the filter was estimated to be 0.4-0.6 /~g, and the amount of RNA used for hybridization ranged from 0.04 to 0.2/~g. Nick-translation of vaccinia DNA was per-

36

formed essentially as described by Maniatis et al. [14]. The specific activity of nick-translated D N A was (1.6-8.0). 106 c p m / # g D N A and about 1 • 105 cpm were used for hybridization. The size of R N A synthesized by cores in vitro was estimated by sucrose density gradient analysis essentially as described by Kates and Beeson [8] using H e L a ribosomal R N A as standard (a gift from Dr. L.W. Cashdollar).

18S

45

L

o x

Results Cores were allowed to synthesize R N A for different times, so that either complete transcripts could not be formed or at least six rounds of transcription would be completed. In order to plan the time course of the experiments, the chain-elongation rate was measured. This was done by using sucrose density gradients to estimate the size of R N A synthesized by cores after short pulses. Representative data are shown in Fig. 1 and a summary of data and calculations is given in Table I. As can be seen in Fig. 1, R N A synthesized during short pulses did not contain aggregates. Also, after each pulse well-defined single R N A peaks were found, showing that the R N A was of low heterogeneity. For example, it is clear that the R N A synthesized during a 1.5-min pulse at 5 # M U T P with a peak s value of 6.2 does not contain any full-length RNA. Thus, initiation proceeds sufficiently quickly compared to the rates of elongation under the conditions studied for the R N A population to be relatively homogeneous. Rates of elongation are shown in Table I. Our data compare well with those of Kates and Beeson, who found that at 70 # M U T P 10 nucleotides were incorporated per s. We found that 16 nucleotides were incorporated per s at 50/~M U T P under the same conditions and that 3.4 nucleotides were incorporated per s at 5 /~M U T P (Table I). The size of R N A synthesized in vitro is between 10 and 12 S (see Ref. 8, and Table I) or about 850 nucleotides long; the synthesis of a molecule of this size therefore takes about 4.15 min at 5 # M U T P and 53 s at 50 # M UTP. Using these data, cores were permitted to synthesize R N A for 3 min at 5 /~M U T P (to synthesize R N A chains about 600 nucleotides long); or for 30 min, which is sufficient for about seven

E £a. t9

1

ct I-S) e --I "D

FRACTION NUMBER

Fig. 1. Sucrose density gradient centrifugation of vaccinia virus core RNA synthesized at various concentrations of UTP. The relevant parts of the gradients are shown which contain the RNA peaks synthesized in cores in vitro. No counts were detected in regions heavier than 18 S. Cores were preincubated at 37°C for 10 min as described in Methods, and the reaction was started by the addition of UTP. Samples were withdrawn at the indicated times and were mixed with EDTA and SDS in final concentrations of 0.01 M and 0.5%. Aliquots of the samples were layered on 5-20% w / w linear sucrose gradients in 0.01 M sodium acetate buffer (pH 5.1)/0.01 M EDTA/0.1% SDS and centrifuged for 16 hours at 25000 rpm at 20°C in the Beckman SW 41 rotor. Samples were preincubated with 10% trichloroacetic acid and counted on nitrocellulose filters. The right-hand ordinate shows cpm values for the RNA standard, the left-hand ordinate for the RNA samples synthesized in cores: × - × , RNA standard; O ©, RNA synthesized in cores at 5 ~tM UTP for 1.5 min; • •, RNA synthesized at 50 #M UTP for 22 s; r, z~, RNA synthesized at 50/LM UTP for 45 s.

rounds of transcription, so that the newly synthesized R N A should contain at least 85% reinitiated chains. Both R N A preparations were hybridized to EcoRI, HpaII, BglII and HindIII restriction endonuclease fragments of vaccinia virus DNA. For c o m p a r i s o n the h y b r i d i z a t i o n p a t t e r n to nick-translated D N A is also shown (Fig. 2).

37 TABLE I C H A R A C T E R I Z A T I O N O F R N A S Y N T H E S I Z E D BY CORES AT D I F F E R E N T U T P C O N C E N T R A T I O N S : RATES OF m R N A E L O N G A T I O N A N D A M O U N T OF R N A SYNTHESIZED The conditions of core R N A synthesis were as described in Materials and Methods. The reaction was started by addition of [3H]UTP. The molecular weights were estimated from a IOg SEo,w versus log molecular weight relationship obtained by Kurland [22]. Incorporation of nucleotides was linear for 20-30 rain under these conditions. [UTP] during

Time of

's' value

Calculated M r of product

No. of nucleotides

Nucleotide/s synthesized

nmol U T P incorporated/

No. of nucleotides

synthesis (/~ M)

synthesis

of product

( × 10- s )

in product

(i.e. elongation rate)

30 min per 260 core

polymerized per core per s

28 18 4 4 4.4 6.2 7.7 10 10.8

14.89 6.77 0.46 0.46 0.54 1.01 1.48 2.37 2.72

4515 2051 140 140 165 306 450 718 824

0.5 5 50

12 45 90 22 45 90

min s s s s s

0.19 3.6 3.4 20.4 15.9

0.06 1.5

10.8

6.7 1.66.10 2

1.2.103

Fig. 2. Hybridization of [32p]UTP-labelled R N A synthesized in 3 or 30 min by vaccinia virus cores to various vaccinia virus D N A restriction endonuclease fragments. Cores were preincubated at 37°C for 10 min in a reaction mixture lacking Mg 2+ and M n 2+ and the reaction was started by adding these. The concentrations of cores and of U T P were 2.5 A26o per ml and 5 / t M , respectively. The time of synthesis was (1) 30 min and (2) 3 rain. The a m o u n t of R N A synthesized was (1) 8.8.10-10 and (2) 6.1.10-11 tool U T P per A260 virus. The specific activity of the R N A s was (1) 4.106 clam and (2) 20.106 cpm per lag UTP; 200000 c p m were used for hybridization. Hybridization was to fragments of vaccinia D N A produced by (A) EcoRI, (B) HpaII, (C) BglII and (D) HindIII. A pattern of hybridization with nick-translated D N A is presented in lane 3 for all restriction endonucleases. Arrowheads indicate regions of the D N A that were either not transcribed at all or only transcribed to a very small extent by cores in vitro. Molecular weight values and designation of HindIII restriction fragments are taken from Mackett and Archard [20].

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It can be seen that the hybridization patterns of the RNA populations synthesized for 3 and 30 min are strikingly similar. This similarity cannot be due to contamination of 'first-round' RNA by 'second-round' RNA. Even if elongation rates were 50% higher than the values estimated from Fig. 1 the average size of the majority of the RNA synthesized during the first 3 min would be fulllength, i.e., 850 nucleotides long, with less than 10% contamination by 'second-round' RNA. It is also apparent that the pattern of hybridization with RNA used as the probe is different from the pattern obtained with nick-translated DNA. With all restriction nucleases there are two to four bands that are visible in the nick-translated pattern, but not visible when hybridized to RNA. For example, among the restriction fragments produced by EcoR1, bands D, L and M and another low molecular weight band are unexpressed (see arrowheads in Fig. 2a). The composite molecular weight of these bands is about 1 1 , 1 0 6, a size which could code for about 22 molecules of 850 nucleotides long. Similarly, on the Hpa|I and BglII patterns (see Fig. 2b,c) four bands marked by arrowheads are unexpressed or only slightly expressed. The composite molecular weights of these bands are 9 . 5 " 1 0 6 and 9.1-10 6 , respectively, which could code for 18-19 m R N A molecules of average length. The value of 20 unexpressed genes out of the possible 200 total is rather low, when the similarity of in vitro synthesized RNA and RNA made early in infection is considered. About half of the genes are expressed early in infection [15,16]. The low proportion of bands that are not detectable after hybridization with RNA is probably due to: (1) close interspersion of early and late genes [4,17]; and (2) minor differences between early messengers and in vitro produced RNA [3,16,18]. Consistent with the first is the fact that in the case of HindIII fragments the composite molecular weight of the unexpressed bands J + L is only 5 . 1 0 6 . Because the average molecular weight of HindIII fragments is higher than that of those produced by the other three restriction endonucleases, fewer bands can be expected to contain early (or in vitro expressed) genes only. What is noteworthy from the point of view of comparing the two populations of RNA is that all restriction bands that cannot be detected after

I 4.4

g

2.9

2.5

1.55 1.38 1.02

2

I.M

3

DIRECTION OF" E L E C T R O P H O R E S I S

:)

Fig. 3. H y b r i d i z a t i o n of [32p]UTP-labelled R N A synthesized in 3 or 30 min by vaccinia virus cores to Hpall fragments of vaccinia D N A . C o n d i t i o n s of synthesis of R N A in vaccinia virus cores were as described in Fig. 2. A u t o r a d i o g r a m s presented in lanes 1, 2 and 3 in Fig. 2B were scanned on a Q u i c k - S c a n ( H e l e n a Laboratories) d e n s i t o m e t e r interfaced with a digital P D P - 1 1 / 1 0 computer. The m o l e c u l a r weight values × 10 -6 of some b a n d s are shown.

hybridization to reinitiated RNA are not visible after hybridization to RNA produced during the first round either. It is also seen in Fig. 2 that the relative intensities of several bands differ from each other when the hybridization pattern of RNA is compared with the pattern obtained with nick-translated DNA. For easier comparison of the relative densities of the restriction endonuclease bands after hybridization to the two RNA preparations, a densitomeric analysis of Fig. 2b is shown in Fig. 3. Characteristic strong (or weak) band intensities, compared to the nick-translated pattern, of given restriction bands were the same in the two RNA preparations (Fig. 3 and also Fig. 2), showing that the same regions were expressed with the same efficiency in reinitiated RNA and in RNA synthesized during the first round of transcription. Since this was found with all four restriction endonucleases, the conclusion that the same set of genes are expressed during the first and subsequent rounds of transcription seems to be valid. Minor differences, however, may be undetected in the present system. In a separate experiment RNA synthesized for

39 45 s with 50 /tM UTP initiating the reaction (to obtain RNA chains about 700 nucleotides long) and RNA synthesized for 30 rain under the same conditions (to allow for about 34 rounds of transcription) were compared by hybridization to HpalI fragments in the same way as described in Fig. 2 (data not shown). Again, RNA synthesized during the first round of transcription was found to be the same as reinitiated RNA. Discussion

This study on the nature of RNA produced in the first round of transcription was performed to gain an insight into the way polymerase molecules are stored within the cores of vaccinia virus. If, for example, polymerase molecules were attached to D N A by being distributed equally among all promoters (as might be expected from the ratio of approximately one RNA polymerase per gene, see Ref. 1), then the pattern of RNA made in the first round of initiation would be the same as that obtained with nick-translated DNA after hybridization to restriction fragments of vaccinia DNA. If polymerase molecules were attached to a single (or very few) promoters then only a very few restriction bands would hybridize to 'first-round' RNA. These messages could code for the earliest 'early' proteins. As shown in Results (see Fig. 2), under the conditions examined the pattern of RNA produced during the first round of transcription was the same as that of RNA containing at least 85% reinitiated RNA. As argued in the Introduction, this makes it very probable that the polymerase molecules are not stored by being attached to DNA, but that attachment occurs during preincubation in the same way as later during reinitiation steps, with a frequency characteristic of each gene. Another possibility is that RNA polymerase molecules are stored by being distributed among the promoters used in the in vitro transcription reaction, in a way that corresponds to the relative initiation frequencies of the genes in subsequent rounds of transcription. Whichever the case, it is clear that the possibility of introducing an extra regulatory step in the production of RNA by giving rise in the first round of transcription to mRNAs distinct from those transcribed in later

rounds is not exploited by the virus. The numerous sites of hybridization observed confirm the existence of a large number of promoters in the vaccinia virus genome, in agreement with the monocistronic mode of RNA synthesis [6,7]. Among the early genes two control classes have been distinguished in vaccinia virus, the immediate early and delayed early classes. Transcription of genes in the immediate early category does not depend on the expression of other viral genes, whereas expression of delayed early genes requires one or more immediate early functions. No distinct class of delayed early mRNA was found by Cooper and Moss [19] by analysis of the in vitro translation products of mRNAs obtained from infected cells, but several polypeptides (one of them very prominent) were found to be synthesized in greater amounts with immediate early than with early RNA (which consists mainly of delayed early species). It may have been tempting to speculate that there is an additional control class within the immediate early category, and that a distinct class of RNAs - possibly coding for the immediate early stage specific proteins - would be synthesized during the first round of transcription. The present results show that this is not the case. No detailed transcriptional map can be established for in vitro synthesized RNA from the data presented here, partly because vaccinia virus strain WR has only been mapped with HindlII [20], but not with EcoRI, HpalI or BglII and partly because of the existence of numerous double bands. Our results on the hybridization of the in vitro synthesized RNA to HindlII fragments agree perfectly with the earlier data of Cabrera et al. [4] in that regions of the genome corresponding to all HindlII fragments are transcribed efficiently except for fragments J and L. Some conclusions about the EcoRI transcriptional map can also be drawn, based on the EcoRI map of rabbit poxvirus strain Utrecht (RPV) and on the comparison of the restriction patterns with EcoRI of RPV and of vaccinia virus strain WR [21]. There are a number of differences between the EcoRI patterns of the two viruses though, which make the assignment of some vaccinia WR bands difficult. Most of these differences are obviously due to the variation in length of the terminal fragments (there is a substantial variation in the

40

length of the terminal fragments of the two viruses, as shown in Ref. 20). Based on this comparison, a number of EcoRl bands (fragments I + J and OP) that we find to hybridize extensively to in vitro synthesized RNA overlap with HindlII fragments (fragments N, M, K and F) that have been shown to code predominantly for early mRNAs [17]. Conversely, some of the regions in the EcoRI pattern that are not expressed in vitro (fragment D) overlap with HindlII fragments (L + J) that have been found to hybridize mainly to late mRNAs [17]. Also, we have found a strongly transcribed fragment (E) corresponding to RPV fragment F, which probably maps at the right terminus. In general our results further substantiate the similarity of in vitro made and early mRNA.

Acknowledgment I thank Dr. W.K. Joklik for suggestions and helpful discussions.

References 1 Nevins, J.R. and Joklik, W.K. (1977) J. Biol. Chem. 252, 6930-6938

2 Nevins, J.R. and Joklik, W.K. (1975) Virology 63, 1-14 3 Cooper, J.A. and Moss, B. (1978) Virology 88, 149-165 4 Cabrera, C.V., Esteban, M., McCarron, R., McAllister W.T. and Holowczak, J.A. (1978) Virology 86, 102-114 5 Paoletti, E. (1977) J. Biol. Chem. 252, 872-877 6 Pelham, H.R.B. (1977) Nature (London) 269, 532-534 7 Wittek, R., Cooper, J.A., Barbosa, E. and Moss, B. (1980) Cell 21,487-493 8 Kates, J. and Beeson, J. (1970) J. Mol. Biol. 50, 1-18 9 Joklik, W.K. (1962) Biochim. Biophys. Acta 61,290-301 10 Holowczak, J.A. and Joklik, W.K. (1967) Virology 33, 717-725 11 Loening, U.E. (1969) Biochem. J. 113, 131-138 12 Southern, E.M. (1975) J. Mol. Biol. 98, 503-518 13 Denhardt, D.T. (1966) Biochem. Biophys. Res. Commun. 23, 641-646 14 Maniatis, T., Jeffrey, A. and Kleid, D.G. (1975) Proc. Natl. Acad. Sci. USA 72, 1184-1188 15 Oda, K. and Joklik, W.K. (1967) J. Mol. Biol. 27, 395-419 16 Boone, R.F. and Moss, B. (1978) J. Virol. 26, 554-569 17 Belle Isle, H., Venkatesan, S. and Moss, B. (1981) Virology 112, 306-317 18 Paoletti, E. and Grady, L.J. (1977) J. Virol. 23, 608-615 19 Cooper, J.A. and Moss, B. (1979) Virology 96, 368-380 20 Mackett, M. and Archard, L.C. (1979) J. Gen. Virol. 45, 683-701 21 Schumperli, D., Menna, A., Schwendimann, F., Wittek, R. and Wyler, R. (1980) J. Gen. Virol. 47, 385-398 22 Kurland, C.G. (1960) J. Mol. Biol. 2, 83-91