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Transcription of the genome of pseudorabies virus (A herpesvirus) is strictly controlled
VIROLOGY
97, 316-327 (1979)
Transcription
of the Genome of Pseudorabies is Strictly Controlled1
LARRY FELDMAN, Department
Virus (A Herpesvirus)
FRAZER J. RIXON,2 JONG-HO JEAN,3TAMAR AND ALBERT S. KAPLAN4
of Microbiology,
Vanderbilt
University
School of Medicine, Nashville,
BEN-PORAT, Tennessee 572.92
Accepted May 16, 1979
Rabbit kidney cells infected with pseudorabies virus in the presence of cycloheximide synthesize RNA complementary only to a restricted part of the viral genome. This immediate-early (IE) RNA is complementary to 10% of the viral genome as determined by hybridization in solution. Blot hybridization showed that IE RNA accumulating in the cells during a 5-hr labeling period hybridizes only to the ends of the inverted repeat regions of the viral DNA. IE RNA synthesized during a 13-min labeling period in intact cells or a Z-min labeling period in nuclear monolayers also hybridized to these regions of the genome only, indicating that an unstable class of viral RNA is not made and subsequently degraded in cycloheximide-treated, infected cells. Synthesis of IE RNA is detectable by 30 min postinfection during the normal course of infection and continues until about 50 min postinfection. Early RNA is first transcribed at 40 min postinfection and transcription continues thereafter. A switch in the transcriptional program from the synthesis of IE RNA to that of early RNA has been also demonstrated following removal of cycloheximide. Late RNA labeled from 6-9 hr postinfection or RNA synthesized in nuclear monolayers during a 5-min pulse at 6 hr postinfection yields practically identical blot hybridization patterns, indicating that extensive selective degradation of some viral RNA transcripts does not occur at late times after infection. These results show that controls operate at the transcriptional level in Pr virusinfected RK cells.
It consists of a terminal sequence (MW, 9.9 x 106) which is inverted internally at The studies described in this paper deal the other side of a short sequence (MW, with the controls that may exist at the level 6.0 x 106). The remainder of the molecule of transcription of the genome of pseudo- forms the so-called long, unique sequence rabies (Pr) virus in infected cells. Pr virus, (Stevely, 1977; Ben-Porat et al., 1979). swine herpesvirus, interacts with its host It has been generally assumed that host cells in a similar manner to that of herpes cell polymerases are responsible for the simplex virus (HSV), a herpesvirus of man transcription of the incoming herpesvirus (Ben-Porat and Kaplan, 1973). The genome genome, because naked viral DNA is infecof Pr virus is a linear, double-stranded tious (Sheldrick et al., 1973). The extent of molecule of approximately 90 x lo6 daltons (Rubenstein and Kaplan, 1975) and a high transcription of the incoming viral genome G-C content (Ben-Porat and Kaplan, 1962). by the unmodified cell RNA polymerase, however, has been a subject of some disagreement. Kozak and Roizman (1974) have ’ A preliminary report of this work was presented reported that sequences complementary to at the IVth International Congress for Virology, The 50% of the viral DNA; i.e., to one comHague, The Netherlands, p. 502 (1978). plete strand of the HSV genome, are syn2 Present address: Institute of Virology, University thesized in cycloheximide-treated cells. of Glasgow, Glasgow Gil 5JR, Scotland. However, Swanstrom et al. (1975) have de3 Present address: Wistar Institute, Philadelphia, tected transcripts complementary to only a Pa. 19044. smaller fraction of the viral genome under a To whom reprint requests should be addressed. INTRODUCTION
0042-6822/79/120316-12$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
316
TRANSCRIPTION
OF PSEUDORABIES
the same conditions. Similar data were obtained also by Clements et al. (197’7), who showed that RNA transcribed in the presence of cycloheximide did not hybridize to all restriction fragments of the HSV genome. These data suggested that either synthesis of immediate-early (IE) RNA was restricted or that rapid processing of primary transcripts may have occurred in cells in the presence of cycloheximide. Previous results from our laboratory (Rakusanova et al., 1971) showed that 25% of the sequences transcribed at early times postinfection are also transcribed in cycloheximide-treated, infected cells and that IE RNA is, therefore, transcribed from only a small part of the Pr genome. Because of the discrepancies in the results reported by various laboratories that have appeared since then concerning the transcriptional controls of HSV, it seemed of interest to reexamine this problem in greater detail in the Pr virus-rabbit kidney (RK) cell system. The results reported in this paper show (1) that in cells infected in the absence of protein synthesis, the cellular polymerase transcribes a maximum of 12% of the Pr viral genome and that strict transcriptional controls exist in this system; (2) that selective degradation of transcripts does not contribute sigmficantly to the type of transcripts detected in the infected cells.
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KH2P04, 20 m&f MgC&, 1.8 mM CaCl, pH 7.0. Enzymes and chemicats. Restriction endonucleases KpnI and BamHI were obtained from New England Biolabs, Inc. DNase I and RNase A were purchased from Worthington Biochemical Corporation. [3H]Uridine (25 Ci/mmol) was purchased from SchwarziMann and inorganic 32P(carrier-free) was purchased from ICN. Restriction enzyme digestion and gel electrophoresis offragments. Digestion and
agarose gel electrophoresis of Pr viral DNA was carried out as described by Rixon and Ben-Porat (1979). Filter strips to which restriction fragments of Pr DNA were fixed were prepared by the method of Southern (1975). Labeling of RNA with 32P. Primary RK cells were preincubated in EDS S PO, for 3 days. The cells were then infected with Pr virus (m.o.i., 50-100 PFU/cell). After a lhr adsorption period, the inoculum was removed and replaced with EDS s PO, and the cells were labeled by incubating them in EDS s PO, + (200 &i/ml) 32P0,, as indicated in each experiment. In some experiments (in which the RNA synthesized from the time of infection was to be labeled) the cells were preincubated for 16 hr before infection in EDS SPO, + (100 $X/ml) 32P0,. Preparation
of nuclear
monolayers.
These monolayers were made according to the method of Bell (1974). The cytoplasm of MATERIALS AND METHODS the cells in the culture was removed by washVirus and cell culture. The preparation ing for 1.5 min in a solution of 0.5% NP-40 in of Pr virus and cultivation of RK cells have 0.5% HEPES buffered saline, pH 7.6, conbeen described previously (Kaplan and Vat- taining 1 mM MgCl,. The monolayers were ter, 1959). then washed with 5 ml of PBS and labeled Media and solutions. Denhardt’s Solu- by incubating them in 50 m&f HEPES (pH tion: 0.02% Ficoll, 0.02% polyvinylpyrol7.6), 10 null MgC&, 100 mM NaCl, 0.5 mM lidone, 0.02% bovine serum albumin (Den- CaCl,, 1 mM DTT, 50 fl CTP, 50 fl hardt, 1966) in 2 x SSC. EDS: Eagle’s GTP, 5 mJ4 ATP, and 200-400 $X/ml synthetic medium (Eagle, 1959)plus 3% dia- [a-32P]UTP. Extraction of RNA. 32P0, or [3H]uridinelyzed bovine serum. EDS S PO,: The same as above but without PO,. RSB-2% SDS: labeled cells were harvested by scraping 0.1 M Tris, pH 7.4, 0.01 M KCl, 0.0015 M them into RSB-2% SDS and adding imMgCl, (Warner et al., 1963), plus 2% sodium mediately an equal volume of phenol:chlorolauryl sulfate. SSC: 0.15 M NaCl, 0.015 M form:isoamyl alcohol (25:24:1) preheated to sodium citrate, pH 7.4. S, Buffer: 0.001 M 60”. The samples were shaken for 2 min at ZnS047Hz0, 5% glycerol, 0.1 M NaCl, 60” and 10 min at room temperature, pre0.03 M Na-acetate, pH 4.6. PBS: 0.136 M cipitated by the addition of 2 vol of 95% NaCl, 2.6 mM KCl, 8 mM Na2HP04, 1 mM ethanol, and stored overnight at -20”. The
FELDMAN
318
samples were then centrifuged at 14,000 rpm for 1 hr, the pellets were resuspended in RSB-2% SDS, were heated to 60” for 2 min, and were digested for 3 hr at 37” with nuclease-free Pronase (Calbiochem) at a final concentration of 2.5 mg/ml. The samples were extracted three to four times with phenol:chloroform:isoamyl alcohol. The RNA in the final aqueous phase was precipitated with ethanol, and the dried pellets were resuspended in 1 ml PBS. The samples were digested with DNase I (40 mg/ml at 37” for 20 min). The reaction was stopped by the addition of SDS (final concentration, 1%); the samples were diluted with 4 ml of RSB-1% SDS containing 25 pglml salmon sperm DNA; and the RNA was extracted twice with phenol:chloroform:isoamyl alcohol at room temperature. After precipitation with alcohol, the RNA was sedimented and washed with ethanol before resuspension in hybridization solution. Hybridization of viral RNA to nitrocellulose$Eters containing DNA. Filter strips
to which DNA fragments generated by KpnI or BamHI had been fixed were preincubated for 24 hr at 45” in Denhardt’s solution containing 50% formamide, 2 x SSC, 100 pg/ml salmon sperm DNA, and 50 pg/ ml yeast RNA. The samples were further incubated for 4 days at 45” in the same solution containing the labeled RNA samples. The filters were washed at 60” in 3 x SSC for 1 hr, digested with. RNase A (10 pgiml) in 2 x SSC for 2 hr at room temperature, and washed again several times at 60” in 3 x SSC. The filters were then air dried and exposed for autoradiography on X-omat film (Kodak). The amount of 32P-labeled RNA hybridized to the different bands of DNA was measured by scanning the autoradiograms (after different exposure times to ensure linearity of the film) with a Joyce-Loebl microdensitometer. The areas under the peaks were measured with a planimeter. Preparation
of 3H-labeled
viral
probe.
3H-Labeled Pr viral DNA was prepared as described previously (Ben-Porat et al., 1974). The specific activity of the DNA was 5 X IO5 cpm/pg. Hybridization in solution of viral DNA with RNA. Viral DNA (I500 cpmsample)
ET AL.
was incubated with various concentrations of RNA in 0.12 M phosphate buffer at 68” for 16 hr. The samples were diluted with 3 vol of S, buffer and were digested with S, nuclease to determine the fraction of the DNA that had become double stranded. RESULTS
Transcripts of the Pr Viral Genome that Accumulate in Cells Infected in the Presence or Absence of Cycloheximide
We have shown previously by filter hybridization that IE RNA is complementary to only a small part of the Pr genome (Rakusanova et al., 1971) and that approximately 25% as many sequences accumulate in the cycloheximide-treated, infected cells as in untreated cells at early stages of infection. However, because hybridization in solution (with excess RNA) to a labeled viral DNA probe is a more sensitive test than filter hybridization and is more likely to detect relatively rare transcripts, we determined by this method whether the 1
I 1 so 1600 CONCENTRATION
I 2400 OF RNA
, 3200 (,ug/ml)
I
FIG. 1. Hybridization in solution of unlabeled RNA to a labeled viral DNA probe. IE RNA was extracted from cells infected in the presence of cycloheximide at 5 hr postinfection; late RNA was extracted from infected cells in the absence of cycloheximide at 5 hr postinfection. The RNA was hybridized for 16 hr to a 3H-labeled Pr viral DNA probe (5 x 10’ epmlml) at 68” in 0.18 1M PO, buffer. The amount of viral DNA which had hybridized and had become insensitive to digestion with S, nuclease was determined. Hybridization of mock-infected RNA is represented by open triangles, IE RNA by closed circles, and late RNA by open circles.
TRANSCRIPTION
OF PSEUDORABIES
presence of transcripts complementary to a larger fraction of the viral genome can be detected in cycloheximide-treated, infected cells. Figure 1 shows the results of a representative experiment. Viral RNA that had accumulated by 5 hr postinfection during the course of a normal lytic infection hybridized to approximately 50% of the viral DNA probe. On the other hand, RNA that had accumulated in the infected, cycloheximide-treated cells during the same period of time (IE RNA), hybridized to approximately 10% of the viral DNA probe, even when RNA was present in large excess. Assuming the presence only of asymmetric transcripts at late times after infection, these results indicate that transcripts to the equivalent of one whole strand of DNA are present in the infected cells at late times during the normal course of infection. In cycloheximide-treated, infected cells, on the other hand, transcripts complementary to 20% of the equivalent of one complete strand are present. These results, therefore, confirm our previous finding (by filter hybridization) and show that transcripts only to a limited part of the Pr viral genome are detectable in cycloheximidetreated, infected cells. Mapping of Immediate-Early, Late Transcripts
Early, and
With the availability of restriction enzyme maps of the DNA of Pr virus (Clements et al., personal communication; Rixon and Ben-Porat, 1979), it has become possible to map the transcripts accumulating under various conditions of infection, using the technique of Southern (1975). This teehnique allows one to estimate the relative abundance of transcripts from various areas of the genome. Furthermore, areas of the genome giving rise to rare transcripts should, in principle, also be detectable after long periods of exposure of the auturadiograms. RNA synthesized in cycloheximidetreated cells, as well as at early (O-2 hr) and late (6-9 hr) times postinfection, was labeled with 32P; the labeled RNA was extracted from the cells and hybridized to viral DNA restriction fragments which had been
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319
FIG. 2. Hybridization of “P-labeled RNA to nitrocellulose filters to which Pr DNA digested by KpnI and BamHI had been fixed. IE RNA was labeled from O-5 hr in the presence of cycloheximide; early and late RNA were labeled in the absence of cycloheximide between 0 and 2 hr (early) and 6 to 9 hr (late), respectively. Purified RNA was hybridized to the filters which were washed and exposed for autoradiography, as described under Materials and Methods.
fixed to nitrocellulose filters. Figure 2 shows the autoradiograms of the RNA that had hybridized to KpnI and BamHI DNA digests fixed to filters. The densitometer tracings of the autoradiograms obtained from another, similar experiment are illustrated in Figs. 3 and 4. The regions of the viral genome from which IE, early, and late RNA were transcribed, as well as the relative abundance of these were computed from the transcripts, cumulative data obtained from these results and are summarized in Fig. 5. For the reasons stated in the legend to Fig. 5, we have used only the data obtained with the KpnI digest to map IE RNA. This RNA hybridizes to fragments H and E.
320
FELDMAN
ET AL.
FIG. 3. Densitometer tracings of autoradiograms of IE, early, and late RNA hybridized to KpnI filters. The experiment was performed as described in the legend to Fig. 2. The autoradiograms were scanned with a Joyce-Loebl microdensitometer. (A) IE RNA: cycloheximide-treated, infected cells labeled between O-5 hr after infection. (B) Early RNA labeled between O-2 hr after infection. (C) Late RNA: labeled between 6 and 9 hr after infection.
Fragment H encompasses a segment of but, in addition, also contains a segment of DNA of 5.5 x 10s daltons which is located DNA of 0.8 x lo6 daltons originating from at the extreme end of the inverted repeat. the long unique region. Because the amounts Fragment E contains the same sequence of RNA that bind to fragments H and E are
TRANSCRIPTIONOFPSEUDORABIESVIRUSGENOME
321
C
A
FIG. 4. Densitometer tracings of autoradiograms of IE, early, and late RNA hybridized to BumHI fragments. This experiment is similar to the one described in Fig. 3 except that the RNA was hybridized to &mnHI fragments.
the same, it is unlikely that the DNA in fragment E, which is part of the long unique region, is transcribed. IE RNA could be transcribed either from one or from both sides of the inverted repeats (RNA transcribed from one side would also hybridize to the other). Thus,
approximately 6 or 12% of the viral DNA is transcribed; the sequence complexity of the DNA from which IE RNA is transcribed represents, however, approximately 7% of that of the total viral genome. [Because Pr DNA has a molecular weight of 90 x lo6 and the inverted repeat has a molec-
FELDMAN
322
ET AL.
I.E. RNA
-
EARLY RNA (O-2h)
(6-Qhl
I
I
0.1
I
I
I
0.2
0.3
0.4
0.5
MAP
UNITS
1
0.6
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0.7
0.8
a9
1.0
FIG. 5. Summary of the patterns obtained by hybridizing IE, early, and late RNA to filters containing electrophoretically separated fragments of KpnI and BarnHI digested DNA. The relative amounts of 3*P-labeled DNA bound to each fragment were determined using a planimeter and the percentage of the total RNA bound to the filter present in each DNA fragment was determined. This percentage is expressed as a histogram according to the map position of each fragment. The locations of the cleavage sites for KpnI (open triangles) and BamHI (closed triangles) restriction enzymes are shown on the bottom of the graph. We have not used the IE data obtained with the BumHI DNA fragments for the following reasons: BarnHI fragment 13 has a molecular weight of 1.4 x 106,and fragment 8 has a molecular weight of 3.2 x 106.Half the sequences which can hybridize to fragment 13 will also hybridize to band 8’ (see restriction map at bottom of figure), which comigrates with band 8. We would, therefore, expect about ‘/sas much RNA to hybridize to fragment 13 as to fragment 8 if all of fragment 13had been transcribed at the same rate as fragment 8. However, less than Yloas much RNA annealed to fragment 13 than to fragment 8. Similarly, if the whole of the KpnI fragment H were transcribed, one would expect a segment of DNA with a MW of 0.9 x lo6 in fragment 5 to be transcribed. If the whole of this region were transcribed at the same rate as fragment 8, approximately 28% as much RNA would hybridize to BanzHI fragment 5 as to fragment 8. However, less than HOas much RNA hybridized to fragment 5 as to fragment 8. The relatively smaller amounts of RNA hybridizing to fragments 13 and 5 than to fragment 8 could be due to a slower rate of transcription of the sequences in these fragments than those in fragment 8; it is, however, more likely that only a fraction of fragments 13 and 5 are transcribed and that the whole area is transcribed as one abundance class. Because of this uncertainty, we have not incorporated the data obtained tram the BamHI DNA digest with IE RNA in the summary in Fig. 5.
TRANSCRIPTION
OF PSEUDORABIES
VIRUS GENOME
323
ular weight of 9.9 x lo6 (Ben-Porat et al., 19’79) the sequence complexity of the genome is that of a molecule of 80 x lo6 daltons]. Figure 5 shows also that early RNA hybridizes to virtually all the KpnI and BumHI fragments with the exception of the middle of the inverted repeat. Transcripts originating from the junction between the repeats and the short unique sequence accumulate most abundantly at early times. At late times, transcripts complementary to all the restriction fragments that we have tested are synthesized. The relative abundance of transcripts of different regions of the genome varied. Blot Hybridization
of Pulse-Labeled RNA
In the experiments described above, the stable viral RNA, i.e., the RNA that had accumulated in the infected cells during a relatively long labeling period, was analyzed either in solution or by blot hybridization. In cycloheximide-treated infected cells, the presence of transcripts complementary to a relatively small part of the Pr genome was detected. Because a larger part of the viral genome might have been transcribed but rapidly degraded thereafter, we analyzed the RNA synthesized during short labeling periods. This procedure should allow one to detect the synthesis of unstable RNA sequelices if degradation is not instantaneous. The following two types of experiments were performed: (1) RNA synthesized during a short pulse with [3H]uridine by intact cycloheximide-treated cells was analyzed (Fig. 6); (2) RNA synthesized during a short pulse with (a-32P)-labeled UTP by nuclear monolayers prepared from cycloheximidetreated infected cells was analyzed (Fig. 7). A labeling period of 13 min was the shortest period allowing a sufficient amount of uridine to be incorporated into RNA for analysis (Fig. 6). The hybridization pattern of IE RNA synthesized during this relatively short pulse (Fig. 6A) was identical to that of IE RNA synthesized during a 60-min labeling period (Fig. 6B). In both cases, RNA complementary to KpnI bands E and H only was detected. (The position of the
FIG. 6. Hybridization of RNA pulse-labeled with [3H]uridine to filters to which restriction fragments generated by digestion with KpnI had been fixed. Cycloheximide-treated, infected cells were labeled for 13 min with [3H]uridine (250 /.&/ml) at 4 hr postinfeetion (A), or labeled with [3H]uridine (25 &i/ml) for 60 min at 4 hr postinfection (B). RNA was also labeled in infected cells not treated with cycloheximide between 0 and 5 hr with [3H]uridine (10 &i/ml) (C). After hybridization, the strips were washed as described under Materials and Methods, cut into 2-mm slices, scintillation fluid was added, and the radioactivity was measured.
DNA fragments on the filter strips are indicated in Fig. 6C, which shows the annealing pattern of RNA synthesized from O-5 hr postinfection during the course of a normal infection.) Thus, unstable sequences transcribed from a large part of the viral genome could not be detected in cycloheximide-treated infected cells after a 13min labeling period. To shorten the pulse time further, nuclear monolayers in which the pools of nucleotides equilibrate rapidly and in which RNA can be labeled using 32P-labeled triphosphates of high specific activity (Bell, 1974) were used. By this means, we were able to analyze RNA synthesized during a period as short as 2 min (Fig. 7). IE RNA synthesized during a 2- or a 5-min pulse (Figs. 7a and b) hybridized to KpnI frag-
324
FELDMAN
ET AL.
genome present after a short pulse in nuclear monolayers and present in intact cells after a shorter labeling period are the same, it is unlikely that extensive selective degradation of some transcripts occurs at late stages of the infective process. Transition from the Synthesis of Immediate-Early RNA to Early RNA To show that the regions specifying for the RNA transcribed in cycloheximidetreated cells are also those which are transcribed first during the normal course of infection, we labeled the infected cells in the absence of cycloheximide at early times of the infective process. The hybridization patterns of RNA labeled with 32P0, accumulating in the infected cells up to 80 min postinfection are shown in Fig. 8. Up to 30 min postinfection, RNA complementary to KpnI fragments E and H only (i.e., IE RNA) were detectable. Thus, as expected, IE RNA is the first to be transcribed during the normal infective process. The exclusive synthesis of IE RNA at early times after infection indicates that the accumulation of IE RNA in infected cells, in which protein synthesis is inhibited, is not an artifact resulting from drug treatment. By 40 min postinfection, RNA complementary to fragments A, I and J were also detectable; by 90 min postinfection, RNA complementary to fragments I and J were still predominant and RNA complementary to most of the other DNA Kpn fragments were also detectable. These sequences of events were confirmed by the hybridization of the RNA to BamHI DNA fragments, In Fig. 9 the accumulation of RNA complementary to KpnI fragments E, H, and J is summarized. The main point to emerge from these data is that the amount of RNA complementary to fragments E and H increased up to 50 min postinfection and then leveled off. The amount of RNA complementary to other restriction fragments as exemplified by that complementary to fragment J, on the other hand, started to be synthesized around 40 min postinfection and continued being synthesized up to at least 80 min. A similar conclusion may also be drawn from the results of experiments in which the
Sequential
FIG. ‘7. Hybridization of RNA pulse-labeled in nuclear monolayers to KpnI fragments. Nuclear monolayers of cycloheximide treated or untreated, infected cells were prepared and pulse-labeled for 2 min with 400 &i/ml of [SlP]UTP or 5 min with 200 &X/ml of [3*P]UTP. The RNA was extracted and hybridized to KpnI DNA filters. The filters were washed, exposed as described under Materials and Methods, and the autoradiograms developed and scanned after appropriate exposure times. (A) IE RNA, 2-min pulse; (B) IE RNA, 5-min pulse; (C) late RNA (6 hr postinfection), 5-min pulse.
ments H and E only. This kind of restricted hybridization occurred also with BamHI DNA fragments (data not shown). Thus, in the presence of cycloheximide, transcripts only to the ends of the inverted repeats can be detected after a labeling period of 2 min in nuclear monolayers or after a labeling period of 13 min in intact cells. Thus, transcription of a large part of the genome, followed by selective degradation of parts of the transcribed sequences, is not the reason that only transcripts to a restricted part of the viral genome can be detected in cycloheximide-treated infected cells. We conclude that controls operate at the level of transcription of the Pr virus genome. The data in Fig. ?C show also that RNA synthesized during a 5-min pulse of nuclear monolayers at 6 hr postinfection in the absence of cycloheximide, has virtually an identical pattern of hybridization as does RNA synthesized between 6 and 9 hr in intact, infected cells (see Fig. 2). Because the abundance of the various labeled transcripts complementary to different regions of the
TRANSCRIPTION
OF PSEUDORABIES
VIRUS GENOME
325
tion of IE RNA virtually ceases when protein synthesis is allowed to resume, indicating that the promoters for these regions may not be recognized efficiently by the cellular polymerase, once protein synthesis in the infected cells is allowed to occur. Also, only a relatively small part of the viral genome is transcribed after removal of cycloheximide and only the regions which are predominantly transcribed at early stages accumulate. We do not know why under these conditions further transcription of the viral genome is blocked. As reported previously (Jean et al., 1974), the rate of transcription of the viral genome is low and infectious virus is not produced by cells treated with cycloheximide during the first few hours of infection. DISCUSSION
The experiments described in this paper are part of a study the ultimate aim of which is to identify the nuclear precursor to cytoplasmic mRNA and to determine the viral FIG. 8. Hybridization of RNA accumulating in the and cellular functions involved in the traninfected cells at early times after infection to filters to scription and processing of Pr viral RNA. In this paper, we attempt to sort out the conwhich DNA fragments generated by digestion with KpnI had been fixed. Cultures of RK cells were prein- trols operating at the level of transcription cubated for 16 hr in EDS S PO, containing 3zP (266 of the viral genome in the infected cells. pCi/ml). The labeled cells were infected at a m.o.i. of 199 PFU/cell with Pr virus grown in EDS S PO,. Cells were harvested at lo-min intervals. The RNA was purified and hybridized to filters to which restriction fragments of Pr DNA generated by KpnI had been fixed as described under Materials and Methods. The filters were washed, exposed, and the autoradiograms were scanned. The time after infection (in minutes) when the cells were harvested, is indicated at the top of each panel.
type of RNA synthesized in infected cells after removal of cycloheximide is analyzed. The results of this experiment are summarized in Fig. 10. As expected from the results described earlier, in the presence of cycloheximide, RNA complementary to KpnI fragments E and H only are synthesized; after removal of cycloheximide there is a dramatic switch from the transcription of KpnI DNA fragments E and H to the transcription of the early region of the DNA, predominantly I and J. It is interesting that the transcrip-
TIMI lmin.1 FIG. 9. Accumulation during the early stages of infection of viral transcripts complementary to different KpnI restriction fragments. The area under some of the peaks in the scans of autoradiograms obtained from an experiment similar to the one illustrated in Fig. 8 was determined and plotted. All strips were scanned with the same microdensitometer wedge so that the areas under the peaks in the different filter strips could be compared to one another.
326
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FIG. 10. Transition from the synthesis of IE RNA to that of early RNA after removal of cycloheximide kom infected cells treated with cycloheximide during the early stages of infection. All cultures were infected in the presence of eycloheximide and incubated with the drug up to 5 hr after infection. (C) IE RNA labeled in the presence of cycloheximide from O-5 hr; (B) RNA labeled in cyclaheximide treated cells from O-5 hr as well as labeled after removal of cycloheximide from 5-9 hr; (A) RNA labeled from 5-9 hr, aker removal of cycloheximide.
Because there is no evidence to show that herpes virions carry an RNA polymerase, the assumption has been made that the first transcripts from Pr viral DNA are synthesized by an unmodified host RNA polymerase. Indeed, studies using inhibitors have implicated RNA polymerase II in the transcription of the HSV genome (Alwine et at., 1974; Ben-Zeev and Becker, 1973). The question whether the unmodified RNA polymerase is capable of transcribing the entire incoming virion genome or whether the enzyme recognizes one or only a few promoters and is thereafter modified by a protein synthesized in the infected cells (or is replaced by a viral coded RNA polymerase) is controversial (see Introduction). The results in this paper show that IE RNA synthesized in Pr virus-infected RK cells is complementary to the DNA of approximately half the sequences of the inverted repeats. This DNA represents approximately 12% of the total viral genome
ET AL.
but has a sequence complexity of approximately 7% of that of the total complexity of the viral genome. By hybridization in solution (Fig. l), we have found that IE RNA is complementary to 10% of the total DNA sequences or to the equivalent of 20% of one complete strand of DNA. Thus, unless both strands of the fragments to which IE RNA is complementary are transcribed, the results obtained by hybridization in solution represent an overestimation by almost a factor of 2. It is unlikely that minor, undetected transcripts are synthesized in cycloheximide-treated, infected cells because we have not detected the presence of such transcripts even when the autoradiograms have been greatly overexposed. However, we cannot eliminate completely the possibility that other regions of the genome are transcribed at a very low rate. The possibility that transcripts to some regions of the genome are synthesized but are rapidly degraded thereafter has been eliminated, however, since identical transcripts are detected after a short (2 min) pulse and after longer labeling periods. Our data show the exclusive synthesis of IE RNA during very early stages (up to 30 min postinfection) during the normal course of infection (i.e., in the absence of inhibitors of protein synthesis). Thereafter, the rate of synthesis of this RNA decreases with a concomitant increase in the transcription of “early” sequences. A switch from the synthesis of IE RNA to early RNA occurs also after removal of cycloheximide from cells infected in the presence of the drug. The synthesis of RNA hybridizing to DNA fragments from which IE RNA is transcribed is reduced considerably after removal of cycloheximide, indicating that the cellular polymerase may have been modified and may have lost its affinity for the promoter of these regions of the genome. The data discussed in this paper show that stringent transcriptional controls operate in Pr virus-infected cells. However, processing of RNA in animal cells has by now been widely documented (Berget et al., 1977; Chow et al., 1977; Aloni et al., 1977). It seems likely that, in addition to these transcriptional controls, controls operate at the level of selection of tran-
TRANSCRIPTION
OF PSEUDORABIES
scribed sequencesthat are transported to the cytoplasm to act as messenger RNA. Our data, however, indicate that extensive degradation of IE RNA or RNA transcribed at late times of the infective process does not occur because the hybridization pattern and abundance classes of RNA complementary to different regions of the Pr genome (as determined by hybridization to the 30 djfferent restriction fragments which are generated from the Pr genome by KpnI and BamHI) synthesized during a 2- or 5-min pulse is the same as that found after a long 3- to 5-hr labeling period. Thus, the abundance classes of transcripts of various regions of the genome that we have observed are generated at the level of the rate of transcription of various regions of the genome rather than through selective degradation of some transcripts. After this manuscript had been completed, Watson and Clements (1978) reported that a temperature-sensitive mutant of HSV-1 makes only IE RNA at the nonpermissive temperature. This is the first direct evidence of a viral protein modifying the transcription of a herpesvirus genome. ACKNOWLEDGMENTS This work was supported by Grant AI-10947 from National Institutes of Health. Larry Feldman was supported by Training Grant 5-T32GM-07319. REFERENCES ALONI, Y., DHAR, R., LAMB, O., HOROWITZ, M., and KHOURY, G. (19’77).Novel mechanism for RNA maturation. The leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences.
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3690. BELL, D. (1974). DNA synthesis in “nuclear monolayers” from BSC-1 cells infected with herpes virus. Nature
{London)
245, 505-505.
BEN-P• RAT, T., DEMARCHI, J. M., and KAPLAN, A. S. (1974). Characterization of defective interfering viral particles present in a population of pseudorabies virions. Virology 60. 29-37. BEN-P• RAT, T., and KAPLAN, A. S. (1962). The chemical composition of herpes simplex and pseudorabies viruses. Virology 16, 261-266. BEN-P• RAT, T., RIXON, F. J., and BLANKENSHIP, M. (1979). Analysis of the structure of the genome of pseudorabies virus. Virology 95, 285-294. BERGET, S. M., MDORE,C., and SHARP, P. A. (1977).
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Spliced segments at the 5’ terminus of Ad 2 late mRNA. Proc. Nut. Acad. Sci. USA 74,3171-3175. GROW, L., GELrK?As,R., BROKE=, T., and ROBERTS, R. (1977). An amazing sequence arrangement at the 5’ ends of adenovirus mRNA. Cell I,?, 1-8. CLEEMENTS,B., WATSON,R., and WILKIE, N. (1977). Temporal regulation of herpes simplex type I transcription: Location of transcripts on the viral genome. Cell 12, 275-285. DENHARDT, D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biockem. Biophys. Res. Commun. 23, 641-646. JEAN, J. H., BEN-P• RAT, T., and KAPLAN, A. S. (1974). Early functions of the genome of herpesvirus. III. Inhibition of transcription of the viral genome in cells treated with cycloheximide early during the infective process. Virology 59, 516-523. KAPLAN, A. S., and VATTER, A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. ViroEogy 7, 394-407.
KOZAK, M., and ROIZMAN, B. (1974). Regulation of herpesvirus macromolecular synthesis: Nuclear retentian of nontranslated viral RNA sequences. Proc. Nat. Acad. Sci. USA 71, 4322-4326. RAXUSANOVA,T., BEN-P• RAT, T., HIMENO, M., and KAPLAN, A. S. (1971). Early functions of the genome of herpesvirus. I, Characterization of the RNA synthesized in cycloheximide-treated, infected cells. Virology 46, 877-890. RrxoN, F. J,, and BEN-Po%~, ‘I’. (1979). Structural evolution of the DNA of pseudorabies defective viral particles. Virology 97, 151-163. RUBENSTEIN, A, S., and KAPLAN, A. S. (1975). Electron microscopic studies of the DNA of defective and standard pseudorabies virions. Virology 66, 385392. SHELDRICK, P., LAITHIER, M., LANDa, D., and RHYNER, M. (1973). Infectious DNA from herpes simplex virus: Infectivity of double stranded and single stranded molecules. Proc. Nat. Aead. Sci. USA 70, 3621-3625. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mot. Biol. 98, X13-517. STEVELY, W. S. (1977). Inverted repetition in the chromosomes of pseudorabies virus. J. Viral. 22, 232-234. SWANSTROM,R. E., PIVO, K., and WAGNER, E. K. (1975). Restricted transcription of the herpes simplex virus genome occurring early after infection and in the presence of metabolic inhibitors. Virolog7~ 66, 140- 150. VOGT, V. (1973). Purification and further properties of single-strand specific m&ease from Aspergitlus oryzae. Eur. J. Biochem. 33, 192-200. WARNER, J. P., KNOPF, P. M., and RccH, A. (196.3). A multiple ribosamal structure in protein synthesis. Ptoc. Nat. Acad. Sei. USA 49, 122-129.
97, 316-327 (1979)
Transcription
of the Genome of Pseudorabies is Strictly Controlled1
LARRY FELDMAN, Department
Virus (A Herpesvirus)
FRAZER J. RIXON,2 JONG-HO JEAN,3TAMAR AND ALBERT S. KAPLAN4
of Microbiology,
Vanderbilt
University
School of Medicine, Nashville,
BEN-PORAT, Tennessee 572.92
Accepted May 16, 1979
Rabbit kidney cells infected with pseudorabies virus in the presence of cycloheximide synthesize RNA complementary only to a restricted part of the viral genome. This immediate-early (IE) RNA is complementary to 10% of the viral genome as determined by hybridization in solution. Blot hybridization showed that IE RNA accumulating in the cells during a 5-hr labeling period hybridizes only to the ends of the inverted repeat regions of the viral DNA. IE RNA synthesized during a 13-min labeling period in intact cells or a Z-min labeling period in nuclear monolayers also hybridized to these regions of the genome only, indicating that an unstable class of viral RNA is not made and subsequently degraded in cycloheximide-treated, infected cells. Synthesis of IE RNA is detectable by 30 min postinfection during the normal course of infection and continues until about 50 min postinfection. Early RNA is first transcribed at 40 min postinfection and transcription continues thereafter. A switch in the transcriptional program from the synthesis of IE RNA to that of early RNA has been also demonstrated following removal of cycloheximide. Late RNA labeled from 6-9 hr postinfection or RNA synthesized in nuclear monolayers during a 5-min pulse at 6 hr postinfection yields practically identical blot hybridization patterns, indicating that extensive selective degradation of some viral RNA transcripts does not occur at late times after infection. These results show that controls operate at the transcriptional level in Pr virusinfected RK cells.
It consists of a terminal sequence (MW, 9.9 x 106) which is inverted internally at The studies described in this paper deal the other side of a short sequence (MW, with the controls that may exist at the level 6.0 x 106). The remainder of the molecule of transcription of the genome of pseudo- forms the so-called long, unique sequence rabies (Pr) virus in infected cells. Pr virus, (Stevely, 1977; Ben-Porat et al., 1979). swine herpesvirus, interacts with its host It has been generally assumed that host cells in a similar manner to that of herpes cell polymerases are responsible for the simplex virus (HSV), a herpesvirus of man transcription of the incoming herpesvirus (Ben-Porat and Kaplan, 1973). The genome genome, because naked viral DNA is infecof Pr virus is a linear, double-stranded tious (Sheldrick et al., 1973). The extent of molecule of approximately 90 x lo6 daltons (Rubenstein and Kaplan, 1975) and a high transcription of the incoming viral genome G-C content (Ben-Porat and Kaplan, 1962). by the unmodified cell RNA polymerase, however, has been a subject of some disagreement. Kozak and Roizman (1974) have ’ A preliminary report of this work was presented reported that sequences complementary to at the IVth International Congress for Virology, The 50% of the viral DNA; i.e., to one comHague, The Netherlands, p. 502 (1978). plete strand of the HSV genome, are syn2 Present address: Institute of Virology, University thesized in cycloheximide-treated cells. of Glasgow, Glasgow Gil 5JR, Scotland. However, Swanstrom et al. (1975) have de3 Present address: Wistar Institute, Philadelphia, tected transcripts complementary to only a Pa. 19044. smaller fraction of the viral genome under a To whom reprint requests should be addressed. INTRODUCTION
0042-6822/79/120316-12$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
316
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the same conditions. Similar data were obtained also by Clements et al. (197’7), who showed that RNA transcribed in the presence of cycloheximide did not hybridize to all restriction fragments of the HSV genome. These data suggested that either synthesis of immediate-early (IE) RNA was restricted or that rapid processing of primary transcripts may have occurred in cells in the presence of cycloheximide. Previous results from our laboratory (Rakusanova et al., 1971) showed that 25% of the sequences transcribed at early times postinfection are also transcribed in cycloheximide-treated, infected cells and that IE RNA is, therefore, transcribed from only a small part of the Pr genome. Because of the discrepancies in the results reported by various laboratories that have appeared since then concerning the transcriptional controls of HSV, it seemed of interest to reexamine this problem in greater detail in the Pr virus-rabbit kidney (RK) cell system. The results reported in this paper show (1) that in cells infected in the absence of protein synthesis, the cellular polymerase transcribes a maximum of 12% of the Pr viral genome and that strict transcriptional controls exist in this system; (2) that selective degradation of transcripts does not contribute sigmficantly to the type of transcripts detected in the infected cells.
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GENOME
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KH2P04, 20 m&f MgC&, 1.8 mM CaCl, pH 7.0. Enzymes and chemicats. Restriction endonucleases KpnI and BamHI were obtained from New England Biolabs, Inc. DNase I and RNase A were purchased from Worthington Biochemical Corporation. [3H]Uridine (25 Ci/mmol) was purchased from SchwarziMann and inorganic 32P(carrier-free) was purchased from ICN. Restriction enzyme digestion and gel electrophoresis offragments. Digestion and
agarose gel electrophoresis of Pr viral DNA was carried out as described by Rixon and Ben-Porat (1979). Filter strips to which restriction fragments of Pr DNA were fixed were prepared by the method of Southern (1975). Labeling of RNA with 32P. Primary RK cells were preincubated in EDS S PO, for 3 days. The cells were then infected with Pr virus (m.o.i., 50-100 PFU/cell). After a lhr adsorption period, the inoculum was removed and replaced with EDS s PO, and the cells were labeled by incubating them in EDS s PO, + (200 &i/ml) 32P0,, as indicated in each experiment. In some experiments (in which the RNA synthesized from the time of infection was to be labeled) the cells were preincubated for 16 hr before infection in EDS SPO, + (100 $X/ml) 32P0,. Preparation
of nuclear
monolayers.
These monolayers were made according to the method of Bell (1974). The cytoplasm of MATERIALS AND METHODS the cells in the culture was removed by washVirus and cell culture. The preparation ing for 1.5 min in a solution of 0.5% NP-40 in of Pr virus and cultivation of RK cells have 0.5% HEPES buffered saline, pH 7.6, conbeen described previously (Kaplan and Vat- taining 1 mM MgCl,. The monolayers were ter, 1959). then washed with 5 ml of PBS and labeled Media and solutions. Denhardt’s Solu- by incubating them in 50 m&f HEPES (pH tion: 0.02% Ficoll, 0.02% polyvinylpyrol7.6), 10 null MgC&, 100 mM NaCl, 0.5 mM lidone, 0.02% bovine serum albumin (Den- CaCl,, 1 mM DTT, 50 fl CTP, 50 fl hardt, 1966) in 2 x SSC. EDS: Eagle’s GTP, 5 mJ4 ATP, and 200-400 $X/ml synthetic medium (Eagle, 1959)plus 3% dia- [a-32P]UTP. Extraction of RNA. 32P0, or [3H]uridinelyzed bovine serum. EDS S PO,: The same as above but without PO,. RSB-2% SDS: labeled cells were harvested by scraping 0.1 M Tris, pH 7.4, 0.01 M KCl, 0.0015 M them into RSB-2% SDS and adding imMgCl, (Warner et al., 1963), plus 2% sodium mediately an equal volume of phenol:chlorolauryl sulfate. SSC: 0.15 M NaCl, 0.015 M form:isoamyl alcohol (25:24:1) preheated to sodium citrate, pH 7.4. S, Buffer: 0.001 M 60”. The samples were shaken for 2 min at ZnS047Hz0, 5% glycerol, 0.1 M NaCl, 60” and 10 min at room temperature, pre0.03 M Na-acetate, pH 4.6. PBS: 0.136 M cipitated by the addition of 2 vol of 95% NaCl, 2.6 mM KCl, 8 mM Na2HP04, 1 mM ethanol, and stored overnight at -20”. The
FELDMAN
318
samples were then centrifuged at 14,000 rpm for 1 hr, the pellets were resuspended in RSB-2% SDS, were heated to 60” for 2 min, and were digested for 3 hr at 37” with nuclease-free Pronase (Calbiochem) at a final concentration of 2.5 mg/ml. The samples were extracted three to four times with phenol:chloroform:isoamyl alcohol. The RNA in the final aqueous phase was precipitated with ethanol, and the dried pellets were resuspended in 1 ml PBS. The samples were digested with DNase I (40 mg/ml at 37” for 20 min). The reaction was stopped by the addition of SDS (final concentration, 1%); the samples were diluted with 4 ml of RSB-1% SDS containing 25 pglml salmon sperm DNA; and the RNA was extracted twice with phenol:chloroform:isoamyl alcohol at room temperature. After precipitation with alcohol, the RNA was sedimented and washed with ethanol before resuspension in hybridization solution. Hybridization of viral RNA to nitrocellulose$Eters containing DNA. Filter strips
to which DNA fragments generated by KpnI or BamHI had been fixed were preincubated for 24 hr at 45” in Denhardt’s solution containing 50% formamide, 2 x SSC, 100 pg/ml salmon sperm DNA, and 50 pg/ ml yeast RNA. The samples were further incubated for 4 days at 45” in the same solution containing the labeled RNA samples. The filters were washed at 60” in 3 x SSC for 1 hr, digested with. RNase A (10 pgiml) in 2 x SSC for 2 hr at room temperature, and washed again several times at 60” in 3 x SSC. The filters were then air dried and exposed for autoradiography on X-omat film (Kodak). The amount of 32P-labeled RNA hybridized to the different bands of DNA was measured by scanning the autoradiograms (after different exposure times to ensure linearity of the film) with a Joyce-Loebl microdensitometer. The areas under the peaks were measured with a planimeter. Preparation
of 3H-labeled
viral
probe.
3H-Labeled Pr viral DNA was prepared as described previously (Ben-Porat et al., 1974). The specific activity of the DNA was 5 X IO5 cpm/pg. Hybridization in solution of viral DNA with RNA. Viral DNA (I500 cpmsample)
ET AL.
was incubated with various concentrations of RNA in 0.12 M phosphate buffer at 68” for 16 hr. The samples were diluted with 3 vol of S, buffer and were digested with S, nuclease to determine the fraction of the DNA that had become double stranded. RESULTS
Transcripts of the Pr Viral Genome that Accumulate in Cells Infected in the Presence or Absence of Cycloheximide
We have shown previously by filter hybridization that IE RNA is complementary to only a small part of the Pr genome (Rakusanova et al., 1971) and that approximately 25% as many sequences accumulate in the cycloheximide-treated, infected cells as in untreated cells at early stages of infection. However, because hybridization in solution (with excess RNA) to a labeled viral DNA probe is a more sensitive test than filter hybridization and is more likely to detect relatively rare transcripts, we determined by this method whether the 1
I 1 so 1600 CONCENTRATION
I 2400 OF RNA
, 3200 (,ug/ml)
I
FIG. 1. Hybridization in solution of unlabeled RNA to a labeled viral DNA probe. IE RNA was extracted from cells infected in the presence of cycloheximide at 5 hr postinfection; late RNA was extracted from infected cells in the absence of cycloheximide at 5 hr postinfection. The RNA was hybridized for 16 hr to a 3H-labeled Pr viral DNA probe (5 x 10’ epmlml) at 68” in 0.18 1M PO, buffer. The amount of viral DNA which had hybridized and had become insensitive to digestion with S, nuclease was determined. Hybridization of mock-infected RNA is represented by open triangles, IE RNA by closed circles, and late RNA by open circles.
TRANSCRIPTION
OF PSEUDORABIES
presence of transcripts complementary to a larger fraction of the viral genome can be detected in cycloheximide-treated, infected cells. Figure 1 shows the results of a representative experiment. Viral RNA that had accumulated by 5 hr postinfection during the course of a normal lytic infection hybridized to approximately 50% of the viral DNA probe. On the other hand, RNA that had accumulated in the infected, cycloheximide-treated cells during the same period of time (IE RNA), hybridized to approximately 10% of the viral DNA probe, even when RNA was present in large excess. Assuming the presence only of asymmetric transcripts at late times after infection, these results indicate that transcripts to the equivalent of one whole strand of DNA are present in the infected cells at late times during the normal course of infection. In cycloheximide-treated, infected cells, on the other hand, transcripts complementary to 20% of the equivalent of one complete strand are present. These results, therefore, confirm our previous finding (by filter hybridization) and show that transcripts only to a limited part of the Pr viral genome are detectable in cycloheximidetreated, infected cells. Mapping of Immediate-Early, Late Transcripts
Early, and
With the availability of restriction enzyme maps of the DNA of Pr virus (Clements et al., personal communication; Rixon and Ben-Porat, 1979), it has become possible to map the transcripts accumulating under various conditions of infection, using the technique of Southern (1975). This teehnique allows one to estimate the relative abundance of transcripts from various areas of the genome. Furthermore, areas of the genome giving rise to rare transcripts should, in principle, also be detectable after long periods of exposure of the auturadiograms. RNA synthesized in cycloheximidetreated cells, as well as at early (O-2 hr) and late (6-9 hr) times postinfection, was labeled with 32P; the labeled RNA was extracted from the cells and hybridized to viral DNA restriction fragments which had been
VIRUS
GENOME
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FIG. 2. Hybridization of “P-labeled RNA to nitrocellulose filters to which Pr DNA digested by KpnI and BamHI had been fixed. IE RNA was labeled from O-5 hr in the presence of cycloheximide; early and late RNA were labeled in the absence of cycloheximide between 0 and 2 hr (early) and 6 to 9 hr (late), respectively. Purified RNA was hybridized to the filters which were washed and exposed for autoradiography, as described under Materials and Methods.
fixed to nitrocellulose filters. Figure 2 shows the autoradiograms of the RNA that had hybridized to KpnI and BamHI DNA digests fixed to filters. The densitometer tracings of the autoradiograms obtained from another, similar experiment are illustrated in Figs. 3 and 4. The regions of the viral genome from which IE, early, and late RNA were transcribed, as well as the relative abundance of these were computed from the transcripts, cumulative data obtained from these results and are summarized in Fig. 5. For the reasons stated in the legend to Fig. 5, we have used only the data obtained with the KpnI digest to map IE RNA. This RNA hybridizes to fragments H and E.
320
FELDMAN
ET AL.
FIG. 3. Densitometer tracings of autoradiograms of IE, early, and late RNA hybridized to KpnI filters. The experiment was performed as described in the legend to Fig. 2. The autoradiograms were scanned with a Joyce-Loebl microdensitometer. (A) IE RNA: cycloheximide-treated, infected cells labeled between O-5 hr after infection. (B) Early RNA labeled between O-2 hr after infection. (C) Late RNA: labeled between 6 and 9 hr after infection.
Fragment H encompasses a segment of but, in addition, also contains a segment of DNA of 5.5 x 10s daltons which is located DNA of 0.8 x lo6 daltons originating from at the extreme end of the inverted repeat. the long unique region. Because the amounts Fragment E contains the same sequence of RNA that bind to fragments H and E are
TRANSCRIPTIONOFPSEUDORABIESVIRUSGENOME
321
C
A
FIG. 4. Densitometer tracings of autoradiograms of IE, early, and late RNA hybridized to BumHI fragments. This experiment is similar to the one described in Fig. 3 except that the RNA was hybridized to &mnHI fragments.
the same, it is unlikely that the DNA in fragment E, which is part of the long unique region, is transcribed. IE RNA could be transcribed either from one or from both sides of the inverted repeats (RNA transcribed from one side would also hybridize to the other). Thus,
approximately 6 or 12% of the viral DNA is transcribed; the sequence complexity of the DNA from which IE RNA is transcribed represents, however, approximately 7% of that of the total viral genome. [Because Pr DNA has a molecular weight of 90 x lo6 and the inverted repeat has a molec-
FELDMAN
322
ET AL.
I.E. RNA
-
EARLY RNA (O-2h)
(6-Qhl
I
I
0.1
I
I
I
0.2
0.3
0.4
0.5
MAP
UNITS
1
0.6
I
0.7
0.8
a9
1.0
FIG. 5. Summary of the patterns obtained by hybridizing IE, early, and late RNA to filters containing electrophoretically separated fragments of KpnI and BarnHI digested DNA. The relative amounts of 3*P-labeled DNA bound to each fragment were determined using a planimeter and the percentage of the total RNA bound to the filter present in each DNA fragment was determined. This percentage is expressed as a histogram according to the map position of each fragment. The locations of the cleavage sites for KpnI (open triangles) and BamHI (closed triangles) restriction enzymes are shown on the bottom of the graph. We have not used the IE data obtained with the BumHI DNA fragments for the following reasons: BarnHI fragment 13 has a molecular weight of 1.4 x 106,and fragment 8 has a molecular weight of 3.2 x 106.Half the sequences which can hybridize to fragment 13 will also hybridize to band 8’ (see restriction map at bottom of figure), which comigrates with band 8. We would, therefore, expect about ‘/sas much RNA to hybridize to fragment 13 as to fragment 8 if all of fragment 13had been transcribed at the same rate as fragment 8. However, less than Yloas much RNA annealed to fragment 13 than to fragment 8. Similarly, if the whole of the KpnI fragment H were transcribed, one would expect a segment of DNA with a MW of 0.9 x lo6 in fragment 5 to be transcribed. If the whole of this region were transcribed at the same rate as fragment 8, approximately 28% as much RNA would hybridize to BanzHI fragment 5 as to fragment 8. However, less than HOas much RNA hybridized to fragment 5 as to fragment 8. The relatively smaller amounts of RNA hybridizing to fragments 13 and 5 than to fragment 8 could be due to a slower rate of transcription of the sequences in these fragments than those in fragment 8; it is, however, more likely that only a fraction of fragments 13 and 5 are transcribed and that the whole area is transcribed as one abundance class. Because of this uncertainty, we have not incorporated the data obtained tram the BamHI DNA digest with IE RNA in the summary in Fig. 5.
TRANSCRIPTION
OF PSEUDORABIES
VIRUS GENOME
323
ular weight of 9.9 x lo6 (Ben-Porat et al., 19’79) the sequence complexity of the genome is that of a molecule of 80 x lo6 daltons]. Figure 5 shows also that early RNA hybridizes to virtually all the KpnI and BumHI fragments with the exception of the middle of the inverted repeat. Transcripts originating from the junction between the repeats and the short unique sequence accumulate most abundantly at early times. At late times, transcripts complementary to all the restriction fragments that we have tested are synthesized. The relative abundance of transcripts of different regions of the genome varied. Blot Hybridization
of Pulse-Labeled RNA
In the experiments described above, the stable viral RNA, i.e., the RNA that had accumulated in the infected cells during a relatively long labeling period, was analyzed either in solution or by blot hybridization. In cycloheximide-treated infected cells, the presence of transcripts complementary to a relatively small part of the Pr genome was detected. Because a larger part of the viral genome might have been transcribed but rapidly degraded thereafter, we analyzed the RNA synthesized during short labeling periods. This procedure should allow one to detect the synthesis of unstable RNA sequelices if degradation is not instantaneous. The following two types of experiments were performed: (1) RNA synthesized during a short pulse with [3H]uridine by intact cycloheximide-treated cells was analyzed (Fig. 6); (2) RNA synthesized during a short pulse with (a-32P)-labeled UTP by nuclear monolayers prepared from cycloheximidetreated infected cells was analyzed (Fig. 7). A labeling period of 13 min was the shortest period allowing a sufficient amount of uridine to be incorporated into RNA for analysis (Fig. 6). The hybridization pattern of IE RNA synthesized during this relatively short pulse (Fig. 6A) was identical to that of IE RNA synthesized during a 60-min labeling period (Fig. 6B). In both cases, RNA complementary to KpnI bands E and H only was detected. (The position of the
FIG. 6. Hybridization of RNA pulse-labeled with [3H]uridine to filters to which restriction fragments generated by digestion with KpnI had been fixed. Cycloheximide-treated, infected cells were labeled for 13 min with [3H]uridine (250 /.&/ml) at 4 hr postinfeetion (A), or labeled with [3H]uridine (25 &i/ml) for 60 min at 4 hr postinfection (B). RNA was also labeled in infected cells not treated with cycloheximide between 0 and 5 hr with [3H]uridine (10 &i/ml) (C). After hybridization, the strips were washed as described under Materials and Methods, cut into 2-mm slices, scintillation fluid was added, and the radioactivity was measured.
DNA fragments on the filter strips are indicated in Fig. 6C, which shows the annealing pattern of RNA synthesized from O-5 hr postinfection during the course of a normal infection.) Thus, unstable sequences transcribed from a large part of the viral genome could not be detected in cycloheximide-treated infected cells after a 13min labeling period. To shorten the pulse time further, nuclear monolayers in which the pools of nucleotides equilibrate rapidly and in which RNA can be labeled using 32P-labeled triphosphates of high specific activity (Bell, 1974) were used. By this means, we were able to analyze RNA synthesized during a period as short as 2 min (Fig. 7). IE RNA synthesized during a 2- or a 5-min pulse (Figs. 7a and b) hybridized to KpnI frag-
324
FELDMAN
ET AL.
genome present after a short pulse in nuclear monolayers and present in intact cells after a shorter labeling period are the same, it is unlikely that extensive selective degradation of some transcripts occurs at late stages of the infective process. Transition from the Synthesis of Immediate-Early RNA to Early RNA To show that the regions specifying for the RNA transcribed in cycloheximidetreated cells are also those which are transcribed first during the normal course of infection, we labeled the infected cells in the absence of cycloheximide at early times of the infective process. The hybridization patterns of RNA labeled with 32P0, accumulating in the infected cells up to 80 min postinfection are shown in Fig. 8. Up to 30 min postinfection, RNA complementary to KpnI fragments E and H only (i.e., IE RNA) were detectable. Thus, as expected, IE RNA is the first to be transcribed during the normal infective process. The exclusive synthesis of IE RNA at early times after infection indicates that the accumulation of IE RNA in infected cells, in which protein synthesis is inhibited, is not an artifact resulting from drug treatment. By 40 min postinfection, RNA complementary to fragments A, I and J were also detectable; by 90 min postinfection, RNA complementary to fragments I and J were still predominant and RNA complementary to most of the other DNA Kpn fragments were also detectable. These sequences of events were confirmed by the hybridization of the RNA to BamHI DNA fragments, In Fig. 9 the accumulation of RNA complementary to KpnI fragments E, H, and J is summarized. The main point to emerge from these data is that the amount of RNA complementary to fragments E and H increased up to 50 min postinfection and then leveled off. The amount of RNA complementary to other restriction fragments as exemplified by that complementary to fragment J, on the other hand, started to be synthesized around 40 min postinfection and continued being synthesized up to at least 80 min. A similar conclusion may also be drawn from the results of experiments in which the
Sequential
FIG. ‘7. Hybridization of RNA pulse-labeled in nuclear monolayers to KpnI fragments. Nuclear monolayers of cycloheximide treated or untreated, infected cells were prepared and pulse-labeled for 2 min with 400 &i/ml of [SlP]UTP or 5 min with 200 &X/ml of [3*P]UTP. The RNA was extracted and hybridized to KpnI DNA filters. The filters were washed, exposed as described under Materials and Methods, and the autoradiograms developed and scanned after appropriate exposure times. (A) IE RNA, 2-min pulse; (B) IE RNA, 5-min pulse; (C) late RNA (6 hr postinfection), 5-min pulse.
ments H and E only. This kind of restricted hybridization occurred also with BamHI DNA fragments (data not shown). Thus, in the presence of cycloheximide, transcripts only to the ends of the inverted repeats can be detected after a labeling period of 2 min in nuclear monolayers or after a labeling period of 13 min in intact cells. Thus, transcription of a large part of the genome, followed by selective degradation of parts of the transcribed sequences, is not the reason that only transcripts to a restricted part of the viral genome can be detected in cycloheximide-treated infected cells. We conclude that controls operate at the level of transcription of the Pr virus genome. The data in Fig. ?C show also that RNA synthesized during a 5-min pulse of nuclear monolayers at 6 hr postinfection in the absence of cycloheximide, has virtually an identical pattern of hybridization as does RNA synthesized between 6 and 9 hr in intact, infected cells (see Fig. 2). Because the abundance of the various labeled transcripts complementary to different regions of the
TRANSCRIPTION
OF PSEUDORABIES
VIRUS GENOME
325
tion of IE RNA virtually ceases when protein synthesis is allowed to resume, indicating that the promoters for these regions may not be recognized efficiently by the cellular polymerase, once protein synthesis in the infected cells is allowed to occur. Also, only a relatively small part of the viral genome is transcribed after removal of cycloheximide and only the regions which are predominantly transcribed at early stages accumulate. We do not know why under these conditions further transcription of the viral genome is blocked. As reported previously (Jean et al., 1974), the rate of transcription of the viral genome is low and infectious virus is not produced by cells treated with cycloheximide during the first few hours of infection. DISCUSSION
The experiments described in this paper are part of a study the ultimate aim of which is to identify the nuclear precursor to cytoplasmic mRNA and to determine the viral FIG. 8. Hybridization of RNA accumulating in the and cellular functions involved in the traninfected cells at early times after infection to filters to scription and processing of Pr viral RNA. In this paper, we attempt to sort out the conwhich DNA fragments generated by digestion with KpnI had been fixed. Cultures of RK cells were prein- trols operating at the level of transcription cubated for 16 hr in EDS S PO, containing 3zP (266 of the viral genome in the infected cells. pCi/ml). The labeled cells were infected at a m.o.i. of 199 PFU/cell with Pr virus grown in EDS S PO,. Cells were harvested at lo-min intervals. The RNA was purified and hybridized to filters to which restriction fragments of Pr DNA generated by KpnI had been fixed as described under Materials and Methods. The filters were washed, exposed, and the autoradiograms were scanned. The time after infection (in minutes) when the cells were harvested, is indicated at the top of each panel.
type of RNA synthesized in infected cells after removal of cycloheximide is analyzed. The results of this experiment are summarized in Fig. 10. As expected from the results described earlier, in the presence of cycloheximide, RNA complementary to KpnI fragments E and H only are synthesized; after removal of cycloheximide there is a dramatic switch from the transcription of KpnI DNA fragments E and H to the transcription of the early region of the DNA, predominantly I and J. It is interesting that the transcrip-
TIMI lmin.1 FIG. 9. Accumulation during the early stages of infection of viral transcripts complementary to different KpnI restriction fragments. The area under some of the peaks in the scans of autoradiograms obtained from an experiment similar to the one illustrated in Fig. 8 was determined and plotted. All strips were scanned with the same microdensitometer wedge so that the areas under the peaks in the different filter strips could be compared to one another.
326
FELDMAN
FIG. 10. Transition from the synthesis of IE RNA to that of early RNA after removal of cycloheximide kom infected cells treated with cycloheximide during the early stages of infection. All cultures were infected in the presence of eycloheximide and incubated with the drug up to 5 hr after infection. (C) IE RNA labeled in the presence of cycloheximide from O-5 hr; (B) RNA labeled in cyclaheximide treated cells from O-5 hr as well as labeled after removal of cycloheximide from 5-9 hr; (A) RNA labeled from 5-9 hr, aker removal of cycloheximide.
Because there is no evidence to show that herpes virions carry an RNA polymerase, the assumption has been made that the first transcripts from Pr viral DNA are synthesized by an unmodified host RNA polymerase. Indeed, studies using inhibitors have implicated RNA polymerase II in the transcription of the HSV genome (Alwine et at., 1974; Ben-Zeev and Becker, 1973). The question whether the unmodified RNA polymerase is capable of transcribing the entire incoming virion genome or whether the enzyme recognizes one or only a few promoters and is thereafter modified by a protein synthesized in the infected cells (or is replaced by a viral coded RNA polymerase) is controversial (see Introduction). The results in this paper show that IE RNA synthesized in Pr virus-infected RK cells is complementary to the DNA of approximately half the sequences of the inverted repeats. This DNA represents approximately 12% of the total viral genome
ET AL.
but has a sequence complexity of approximately 7% of that of the total complexity of the viral genome. By hybridization in solution (Fig. l), we have found that IE RNA is complementary to 10% of the total DNA sequences or to the equivalent of 20% of one complete strand of DNA. Thus, unless both strands of the fragments to which IE RNA is complementary are transcribed, the results obtained by hybridization in solution represent an overestimation by almost a factor of 2. It is unlikely that minor, undetected transcripts are synthesized in cycloheximide-treated, infected cells because we have not detected the presence of such transcripts even when the autoradiograms have been greatly overexposed. However, we cannot eliminate completely the possibility that other regions of the genome are transcribed at a very low rate. The possibility that transcripts to some regions of the genome are synthesized but are rapidly degraded thereafter has been eliminated, however, since identical transcripts are detected after a short (2 min) pulse and after longer labeling periods. Our data show the exclusive synthesis of IE RNA during very early stages (up to 30 min postinfection) during the normal course of infection (i.e., in the absence of inhibitors of protein synthesis). Thereafter, the rate of synthesis of this RNA decreases with a concomitant increase in the transcription of “early” sequences. A switch from the synthesis of IE RNA to early RNA occurs also after removal of cycloheximide from cells infected in the presence of the drug. The synthesis of RNA hybridizing to DNA fragments from which IE RNA is transcribed is reduced considerably after removal of cycloheximide, indicating that the cellular polymerase may have been modified and may have lost its affinity for the promoter of these regions of the genome. The data discussed in this paper show that stringent transcriptional controls operate in Pr virus-infected cells. However, processing of RNA in animal cells has by now been widely documented (Berget et al., 1977; Chow et al., 1977; Aloni et al., 1977). It seems likely that, in addition to these transcriptional controls, controls operate at the level of selection of tran-
TRANSCRIPTION
OF PSEUDORABIES
scribed sequencesthat are transported to the cytoplasm to act as messenger RNA. Our data, however, indicate that extensive degradation of IE RNA or RNA transcribed at late times of the infective process does not occur because the hybridization pattern and abundance classes of RNA complementary to different regions of the Pr genome (as determined by hybridization to the 30 djfferent restriction fragments which are generated from the Pr genome by KpnI and BamHI) synthesized during a 2- or 5-min pulse is the same as that found after a long 3- to 5-hr labeling period. Thus, the abundance classes of transcripts of various regions of the genome that we have observed are generated at the level of the rate of transcription of various regions of the genome rather than through selective degradation of some transcripts. After this manuscript had been completed, Watson and Clements (1978) reported that a temperature-sensitive mutant of HSV-1 makes only IE RNA at the nonpermissive temperature. This is the first direct evidence of a viral protein modifying the transcription of a herpesvirus genome. ACKNOWLEDGMENTS This work was supported by Grant AI-10947 from National Institutes of Health. Larry Feldman was supported by Training Grant 5-T32GM-07319. REFERENCES ALONI, Y., DHAR, R., LAMB, O., HOROWITZ, M., and KHOURY, G. (19’77).Novel mechanism for RNA maturation. The leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences.
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