Cell, Vol. 7, 557-566,

April

1976,

Copyright0

1976

by MIT

Low Molecular Weight Viral RNAs Transcribed by RNA Polymerase Ill during Adenovirus 2 Infection R. WeinmanrF, T. G. Brendler”, H. J. Raskas*, and R. G. RoedeP Department of Biological Chemistry* and Departments of Pathology and of Microbiology and Immunology* Division of Biology and Biomedical Sciences Washington University St. Louis, Missouri 63110

Summary Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5s or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated VIs6. The other three species are novel and have been designated VZoO, VlhO, and V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the VZoO, V156, and VIJO RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the VZoO, VIs6, V14,,, and VIsO RNAs are all synthesized by DNA-dependent RNA polymerase Ill, since synthesis is sensitive to high but not to low concentrations of a-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between 8.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5s rRNA species which was monitored by its hybridization to purified 5s DNA from Xenopus laevis. Introduction Low molecular weight RNA species have been identified both in the nucleus and cytoplasm of eucaryotic cells (for review see Ro-Choi and Busch, 1974). These RNAs include tRNAs, ribosomal5S RNA, and +Present 19104.

address:

The Wistar

Institute,

Philadelphia,

Pennsylvania

additional species whose cellular roles have not been established. An RNA of comparable size is transcribed from the adenovirus genome during productive infection (Reich et al., 1966; Ohe et al., 1969; Ohe, 1972). This viral RNA has been designated 5.5s or VA (virus-associated) RNA. The 5.5s RNA contains 156 nucleotides and has been sequenced (Ohe and Weissman, 1971), although its function remains unknown. Nuclei isolated from virus-infected cells have the capacity to synthesize the 5.5s RNA in vitro (Ohe et al., 1969; Price and Penman, 1972a; Weinmann, Raskas, and Roeder, 1974a, 1974b). The genes for 5.5s RNA are transcribed in vitro by a class III RNA polymerase, while the rest of the genome is transcribed by a class II RNA polymerase (Price and Penman, 1972b; Wallace and Kates, 1972; Weinmann et al., 1974a, 1974b). Quantitative studies of viral RNA synthesis during the course of infection have shown that the rate of 5.5s RNA synthesis increases during the period of viral DNA replication, with the maximum rate of synthesis observed in nuclei isolated at 14 hr after infection (Weinmann et al., 1976). In this paper, we report a further analysis of the synthesis of low molecular weight RNAs during late times in infection. In these studies, we show first, that additional low molecular weight viral RNAs are synthesized in isolated nuclei; second, that the DNA sequences which encode these RNAs are transcribed by RNA polymerase III; third, that low molecular RNAs of similar size are synthesized in intact cells; and fourth, that the DNA sequences encoding these RNAs are localized within a small region of the viral genome. Results Electrophoretic Resolution of Low Molecular Weight RNAs Synthesized in Isolated Nuclei Previous studies have demonstrated that the viral 5.5s RNA (V156) is the major low molecular weight RNA synthesized in nuclei isolated from infected cells (Ohe et al., 1969; Price and Penman, 1972a; Weinmann et al., 1974a, 1974b). High resolution electrophoresis and autoradiography have been used to analyze in more detail the RNAs synthesized in isolated nuclei (Figure 1). With this procedure, several radioactive components are detected. The lower component corresponds to cellular 5s RNA (see below), which contains 120 nucleotides (Forget and Weissman, 1969) and is designated CIZO. The other bands represent viral RNAs, since they are not synthesized in nuclei from uninfected cells (data not shown) and since they are transcribed from viral DNA (see below). The major band corresponds to the viral 5.5s RNA of 156 nucleotides

Cell 558

(Ohe and Weissman, 1971) and is designated VIS6. From the relative electrophoretic mobilities of the low molecular weight RNAs, two of the novel viral-

Figure 1, Polyacrylamide in Nuclei Isolated from

Gel Electrophoresis Adenovirus-Infected

of RNAs Cells

induced RNAs were calculated to be 205 and 143 nucleotides in length. For convenience, these RNA species are designated VzoO and VIdO, as indicated in Figure 1. Another component (designated VT3,,) migrates slightly faster than the VIA0 RNA and has an apparent length of about 130 nucleotides. Although its presence among the in vitro products is variable, it is apparent in most experiments.

Synthesized

Cells were harvested at 14 hr after infection, and detergent-washed nuclei were prepared as described in Experimental Procedures. Nuclei from 3 x lo6 cells were incubated for 30 min at 25°C in a final volume of 250 ~1, as described in Experimental Procedures. The radioactive precursor was a-32P-UTP (110 Ci/mmole) and was present at 0.05 mM. The RNA was extracted and subjected to electrophoresis on a 12% polyacrylamide gel. Autoradiography of the gel revealed discrete RNA species which are indicated by arrows and discussed in the text.

Figure 2. Electrophoretic Analysis of Purified RNAs following Denaturation in Formamide

Low Molecular

Weight

Low molecular weight RNAs synthesized in nuclei isolated from infected cells were separated electrophoretically as in the experiment shown in Figure 1. The individual RNA species were recovered from the gels as described in Experimental Procedures, precipitated with ethanol, and dissolved in 95% formamide. Aliquots of these samples containing 1000-2000 cpm were individually run on 25 cm 12% polyacrylamide slab gels. Lanes 1, 2, 3, 4, and 5 contained, respectively, the VZOO, VIs6, V,.K,, VIaa, and CIX species.

Low Molecular 559

Weight

Adenovirus

RNAs

That these low molecular weight RNAs represent discrete size classes is indicated by the following experiment. After electrophoretic separation, the RNAs were eluted from the polyacrylamide gels, denatured in formamide, and subjected to electrophoresis for a second time. As shown in Figure 2, each component retained its distinct electrophoretic mobility when subjected to this procedure. Viral Origin of Low Molecular Weight RNAs Synthesized in Nuclei from infected Cells The VZOO, V156, and V14O RNAs synthesized in nuclei hybridize very efficiently to viral DNA (Table I), indicating that these RNA species are encoded by the viral genome. The V13O species also hybridizes efficiently to viral DNA, as shown in other experiments and in the studies described below. The putative 5s RNA species (C120) synthesized in nuclei from infected cells hybridizes to purified X. laevis 5s DNA with approximately the same efficiency as does bona fide 5S RNA from uninfected cells (Table 2). Furthermore, hybridization of both the in vitro and in vivo RNA species was prevented by the presence of excess unlabeled 55 RNA from uninfected cells. These results demonstrate that this species is comprised predominately (>92%) of cellular 5s RNA. Properties of the Low Molecular Weight Viral RNA Species There are several features common to the low molecular weight viral RNAs synthesized in isolated nuclei. First, most of the newly synthesized RNA species exit rapidly from the incubated nuclei (data not shown) as shown previously for other small RNAs synthesized in nuclei from uninfected cells (Price and Penman, 1972a; Weinmann and Roeder, 1974). Second, these RNA species as well as their putative in vivo counterparts (see below) do not bind to oligo(-dT)-cellulose columns under conditions where viral mRNA species are bound, sug-

Table 1. Hybridization of Low Molecular in Nuclei Isolated from infected Cells cpm RNA Species

Input (cpm) 2,420

211

VI56

16,630

904

VI40

3,085

345

RNAs

Synthesized

Hybridized

1.2 pg DNA

V200

Weight

5 pg DNA 537 3,145 943

% of Input Hybridized (Total)

cpm

31 24 42

Electrophoretically purified 32P-RNA species were isolated from the polyacrylamide gel shown in Figure 1 and hybridized with the indicated amounts of adenovirus DNA, as described in Experimental Procedures, Background values of IO-12 cpm, obtained with filters containing 1 pg of E. coli DNA, were subtracted from thwxa

rlnta

gesting that poly(A) tails are either absent or very short (data not shown). Third, the synthesis of at least two of these RNA species (VZOO and V1s6) continues linearly in isolated nuclei for prolonged periods of time (Figure 3). These latter data confirm that the VZOO and Vlsb species are synthesized at markedly different rates as suggested by visual inspection of electropherograms (Figure 1). Fourth, as shown below, these RNA species are transcribed by a single class of RNA polymerases. Synthesis of Low Molecular Weight Viral RNAs by RNA Polymerase III Previous studies demonstrated that the viral 5.5.S RNA (VIs6) and the cellular 5s RNA and tRNA species were transcribed by an RNA polymerase III activity (Weinmann et al., 1974a, 1974b), whereas precursors to viral mRNA were transcribed predominately, perhaps entirely, by an RNA polymerase II activity (Wallace and Kates, 1972; Price and Penman, 1974b; Weinmann et al., l974a, 1974b). It was therefore of interest to determine the RNA polymerase activity responsible for the synthesis of the new viral RNAs (VZOO, V14,,, and VI&. The toxin ai-amanitin can be used to answer this question unambiguously, since RNA polymerases I, II, and III show distinct sensitivities to this inhibitor (Weinmann and Roeder, 1974). At 1 pg of cu-amanitin per ml, RNA polymerase II is completely and se-

Table 2. Hybridization of CjZO RNA Synthesized from Infected Cells to 55 DNA from X. laevis cpm

in Nuclei

Isolated

Hybridized

Input (cwm)

Without Comoetitor

2,520

548

5

3H-CIZ0 RNA Synthesized in Nuclei from Infected Cells

2,494

494

26

3*P-V15, RNA Synthesized Nuclei from Infected Cells

2.550

RNA Saecies 3zP-5S RNA Synthesized Uninfected Cells

With Comwetitor

in

in 3

1

Unlabeled and 3*P-labeled 5S RNA were electrophoretically purified, respectively, from the cytoplasm of uninfected KB cells and from 32P-labeled uninfected KB cells, as described in Experimental Procedures. The 3ZP-labeled VIs6 and the IH-labeled CZO RNAs were similarly purified following synthesis in separate reaction mixtures in which nuclei from infected cells (14 hr) were incubated with 3ZP-UTP or 3H-UTP under conditions described in Figure 1 or in Weinmann et al. (1974a). The 32P-5S RNA and the 3H-C12,, RNA were hybridized simultaneously with two filters, one containing 0.2 pg purified 55 DNA from X. laevis (a gift of D. D. Brown) and a second containing 1 pg of E. coii DNA. In a separate experiment, 3ZP-V15a RNA was incubated with filters containing these same DNAs. Parallel hybridizations were also performed in the presence of a vast excess of unlabeled 5s RNA competitor (2 pg). Background values of 15 and 20 cpm for the 32P and 3H channels, respectively, obtained with the filters containing E. coli DNA, were subtracted, but no correction for spillover of the 32P into the 3H channel has been made (~3%).

Cell 560

lectively inhibited. At 200 pg a-amanitin per ml, RNA polymerase II is inhibited completely, RNA polymerase III is inhibited up to 90%, and RNA polymerase I is completely resistant (Schwartz et al., 1974; Weinmann et al., 1974a, 1974b). In our experiments, nuclei from infected cells were incubated in the presence of varying concentrations of oi-amanitin, and the synthesis of the small RNAs was monitored by electrophoresis and subsequent autoradiography (Figure 4). The synthesis of the VZoO, V15&, VIED, V1sO, CITY, and the presumptive tRNA precursor species (Weinmann and Roeder, 1974) are inhibited only by the high (Yamanitin concentrations which inhibit the activity of RNA polymerase III (Schwartz et al., 1974; Weinmann and Roeder, 1974; Weinmann et al., 1974a, 1974b). The data in Table 3 summarize the results obtained when the RNA species shown in Figure 4 were eluted from the gel and the radioactivity quantitated by scintillation counting. A significant fraction of the high molecular weight RNA which does not enter the gel is synthesized by RNA polymerase II, in agreement with previous findings (Weinmann et al., 1974a, 1974b). However, there appears to be no significant synthesis of any distinct low molecular weight RNA species by an RNA polymerase II activity, since no inhibition is ob-

served at 1 pg oc-amanitin per ml. The low molecular weight species are predominantly, if not exclusively, transcribed by RNA polymerase III, since synthesis is inhibited by 70-90% at an a-amanitin concentration which inhibits RNA polymerase III approximately 90%. Previous experiments showed that 5.5s RNA (VIs6) synthesis could in fact be completely inhibited at higher concentrations of a-amanitin (400 pg/ml), which completely inhibit purified RNA polymerase III, but which have no effect on RNA polymerase I (Weinmann et al., 1974a, 1974b). The levels of radioactivity in the high molecular weight RNA species remaining at the top of the gel (Figure 4) are the same whether nuclei were incubated at 1 pg cu-amanitin per ml or at 200 pg per ml, suggesting that all RNA polymerase III transcripts in this system are low molecular weight RNA species.

Analysis of Low Molecular Weight RNAs Present in infected Cells The low molecular weight RNA fraction in infected cells was analyzed to determine if species similar to those synthesized in isolated nuclei are also found in vivo. Infected cells were cultured in the presence of SzP-phosphate from 6 to 15 hr after infection. Cytoplasmic RNA was extracted, and a low molecular weight RNA fraction (4S-12s) was prepared by sucrose gradient centrifugation (see Experimental Procedures). This fraction was subjected to high resolution polyacrylamide gel electrophoresis. As shown in Figure 5A (lanes 1 and 2), autoradiography reveals several distinct radioactive species. Of the five cytoplasmic RNA species denoted by arrows, the lower two are also synthesized in uninfected cells (data not shown) and correspond to the cellular 5s RNA (C12,, band) and tRNA species. The identity of the 5s RNA band has been established by hybridization to purified 5s DNA

Table thesis

3. ol-Amanitin Sensitivity in Nuclei Isolated from

of Low Molecular Infected Cells cpm

Weight

Incorporated

RNA Syn-

(“2P-UMP)

ol-Amanitin Concentration

0

30

90 Min

6o

RNA Species

Control

1 pg/ml

200 &g/ml

78,180

29,851

30,005

VZOO

1,506

1,489

477

V156

10,001

9,275

1,106

V140

1,698

1,590

223

Vl30

902

908

26

Cl20

2,oi 0

2,110

519

High Figue 3. Kinetics Isolated Nuclei

of Synthesis

of Low

Molecular

Weight

RNAs

in

Nuclei isolated from infected cells were incubated in 25 pl reaction volumes with n-x*P-UTP as the radioactive precursor. At the indicated times, individual reactions were terminated, and the total RNA extracted and subjected to electrophoresis on 12% polyacrylamide slab gels as described in Experimental Procedures. After localization of individual RNA species by autoradiography, the appropriate regions of the gels were cut out and the radioactivity measured. The insert shows the kinetics of synthesis of VIM& RNA during very short incubation periods, as determined in a separate experiment using “H-UTP as the labeled nucleotide.

Molecular

Weight

RNA

These data are from the experiment of the polyacrylamide gel containing cut out and radioactivity determined Weinmann and Roeder (1974).

detailed in Figure 4. Regions specific RNA species were as described previously by

Low 561

Molecular

Weight

Adenovirus

RNAs

(data not shown). The upper three bands (labeled V 2001 VrS6, and VIdo) represent viral-induced RNA species. In similar experiments with uninfected KB cells, no radioactive RNA species corresponding to these bands could be detected in the cytoplasmic fraction (data not shown). These data, however, do not exclude the possible synthesis and accumula-

-1”

2

pre-

t RNA A, Figure Weight Figure 4. a-Amanitin ular Weight RNAs

Sensitivity

of Synthesis

of Specific

Low Molec-

Nuclei isolated from 6 x 105 infected cells were incubated for 20 minat25oCin50plreactionvolumeswith~-~~P-UTP(10Ci/mmole) as the radioactive precursor and in the presence of the indicated concentrations of a-amanitin. The reactions were terminated, and the total RNA extracted and subjected to electrophoresis on 10% polyacrylamide slab gels. Lane 1 shows the reaction products synthesized in the absence of a-amanitin, while lanes 2 and 3 show, respectively, the reaction products synthesized in the presence of 1 pg and 200 pg of a-amanitin per ml. Specific RNA species discussed in the text are indicated by the arrows.

5. Polyacrylamide Gel Electrophoresis RNAs Isolated from Infected Cells

of Low

Molecular

Virus-infected cells were harvested at 15 hr after infection, and the low molecular weight RNAs in both nuclear and cytoplasmic fractions analyzed by electrophoresis in 12% polyacrylamide gels. (A) shows an autoradiogram of RNA species labeled with 3*P04 from 6-15 hr post infection. The samples analyzed were derived from 1.2 X lo7 infected cells. The cytoplasmic sample was divided between lanes 1 and 2 of the gel, while the total nuclear sample was run in lane 3. (5) shows the results of a different experiment in which unlabeled RNA species were stained with methylene blue (Peacock and Dingman, 1967). In this experiment, the cytoplasmic sample (lane 1) and the nuclear sample (lane 2) were each derived from 3 X lo* cells. The discontinuities in the photograph are a result of cutting gels into sections prior to staining. In both (A) and (8) the nuclear RNAs were isolated from detergent-washed nuclei. The arrows indicate discrete bands of low molecular weight RNAs which are discussed in the text. The unlabeled arrow in (8) indicates the expected position of the VzoO species,

Cell 562

tion in the cytoplasm of low levels of undetected cellular RNA species similar in size to the RNAs observed here (for example, see Walker et al., 1974). The major band in the upper three species (labeled VIs6) is the viral 5.5.S RNA. The additional viralinduced species have mobilities similar to those of the VZoo and VIdo species synthesized in isolated nuclei (see above). In experiments performed to date, we have not detected a cytoplasmic RNA species with a mobility similar to that of the V,so RNA synthesized in nuclei. Relative to the radioactivity accumulated in the Vrs6 species, the radioactivity accumulated in other cytoplasmic RNA species varied in several experiments from 5-20% for the putative VZoo RNA, from lo-40% for the putative VIJo RNA, and from 20-40% for the host 5s RNA. To determine if the low molecular weight cytoplasmic RNAs synthesized in intact cells are viral in origin, these species were eluted from polyacrylamide gels and hybridized to viral DNA immobilized on nitrocellulose filters. As shown in Table 4, the VIs6 and the putative VZoo and VIeo species synthesized in infected cells hybridize to viral DNA, but not to a heterologous DNA. Thus the VZoo, VIs6, and VIho RNA species appear to contain predominantly viral RNAs. Although these hybridization data do not rule out the possibility that these RNA species contain some cellular encoded RNAs, this seems improbable since no significant levels of RNA species of similar size were detected in the cytoplasm of uninfected cells (data not shown; see above). To characterize further these RNA species, they have been eluted from the gel fractions, denatured in formamide, and individually subjected to electrophoresis, under conditions identical to those used for analysis of the RNAs synthesized in isolated nuclei (see Figure 2). The C1zo, VIho, and Vrsb species from intact cells maintained their unique electrophoretic properties. Although the major fraction of the putative Vzoo species exhibited the migration rate expected for an RNA of 200 nucleotides, approximately 20-30% of the radioactive component eluted from the first gel showed a migration rate similar to that of the Vlsb RNA (data not shown). The basis for this observation is currently being investigated. The labeled RNA species present in nuclear fractions were also analyzed by electrophoresis as shown in Figure 5A (lane 3). Comparison of the data indicates that most of the labeled V200, VIs6, and VIdo species are found in the cytoplasmic fraction. Although a significant fraction of the VIao component appears to be localized in the nucleus, other experiments (not shown) suggest that the nuclear RNA migrating close to the cytoplasmic V,ao species has a slightly greater mobility and may therefore represent a distinct RNA species. Most of the labeled nuclear RNA species shown in Figure 3 may

Table 4. Hybridization of Low Molecular the Cytoplasm of Infected Cells

RNA Species

Input (cpm)

V200 VI% VI40

cpm

Weight

Hybridized

RNAs

Isolated

2 pg DNA

4 kg DNA

% of Input Hybridized

1525

144

372

33.8

2013

112

223

16.6

2146

117

194

14.4

from

cpm (Total)

Electrophoretically purified 3*P RNA species from the cytoplasm of infected cells were hybridized with the indicated amounts of adenovirus 2 DNA, as described in Experimental Procedures. Background values of IO-12 cpm, obtained with filters containing 1 pg E. coli DNA, were subtracted from these data.

in fact represent predominantly cellular RNAs, since we have not attempted to distinguish them from the small heterodisperse nuclear RNAs present in uninfected cells (Weinberg and Penman, 1968; Ro-Choi and Busch, 1974; Marzluff et al., 1975). The accumulated mass of the various RNA species in infected cells was analyzed by staining the samples with methylene blue-after electrophoresis (Figure 5B). No species corresponding to VZoo could be detected in either the cytoplasm (lane 1) or the nucleus (lane 2) of infected cells. In contrast, species corresponding to Vrs6 and V14o were readily detected in the cytoplasmic fraction (lane l), suggesting that the specific activity and hence the turnover rate of the putative VZoo species is greater than that of the VIs6 or the VIdo species. In the nucleus (lane 2), a number of bands with mobilities greater than that of the VZoo species are apparent. Whether any of the RNA species detected in the nuclear fraction are synthesized as a result of viral infection is not yet clear. Many or all of these nuclear RNAs may correspond to the small stable nuclear RNAs present in uninfected cells (Weinberg and Penman, 1968; Ro-Choi and Busch, 1974; Marzluff et al., 1975). Organization of Genes Coding for Low Molecular Weight Viral RNAs Since there appear to be many features common to the expression of the low molecular weight viralcoded RNAs (above), the organization of the viral genes coding for these RNAs is of considerable interest. This problem has been approached by hybridization of the isolated RNA species synthesized in isolated nuclei to specific viral DNA fragments which were prepared by digestion with restriction endonucleases. Preliminary experiments were performed with the six DNA fragments produced by cleavage of adenovirus 2 DNA with endo R.Eco RI (Pettersson et al., 1973); all the small viral RNA species hybridized exclusively to the Eco RI-A fragment (data not shown). Since the Eco RI-A fragment comprises the left-hand 58% of the viral genome

Low 563

Molecular

Weight

Table 5. Hybridization to Restriction Enzvme

Adenovirus

RNAs

of Low Molecular Weight Fraaments of Adenovirus

corn

Discussion

Viral RNA Species 2 DNA

Hvbridized

RNA Species

Synthesized

Fragment

V200

VI56

Sma J

18

E

15

4

15

4

F

14

7

26

11

B

925

398

561

420

I

0

in Nuclei V140 9

v130 10

3

0

8

3

D

34

14

30

18

H

11

5

13

9

A-A

15

2

4

10

After cleavage of adenovirus 2 DNA with Eco RI, the largest fragment (A) was purified by sucrose gradient centrifugation. This fragment was subsequently subcleaved into 8 fragments with endonuclease Sma I. The genomic order of these fragments from the left-hand end is as indicated (H. Delius and C. Mulder, personal communication). The fragment A-A is that fragment of DNA extending from the seventh Sma I site to the right-hand terminus of the EGO RI A fragment. For each fragment, nitrocellulose filters were prepared which contained that amount of DNA derived from 1 ILg of whole genome DNA. Hybridization was performed as described in Experimental Procedures. The radioactive RNA species were synthesized in nuclei isolated from cells harvested at 14 hr after infection and were labeled with ol-32P-UTP. Input values of the Vzao, Visa, VI,~, and VIJO RNAs were, respectively, 4500, 5707, 8222, and 4787 cpm for the RNA species synthesized in isolated nuclei. Background values of 15 cpm, obtained with filters containing 1 pg E. coli DNA, were subtracted from these data.

(Pettersson et al., 1973), more detailed mapping was undertaken with a restriction endonuclease Sma I from Serratia marcescens (C. Mulder, personal communication). Table 5 shows the results obtained when the small RNAs synthesized in isolated nuclei were purified and hybridized with viral DNA fragments obtained by subcleavage of the Eco RI-A fragment with the Sma I enzyme. The Sma I fragments are listed in the order in which they appear within the viral genome (H. Delius and C. Mulder, personal communication). Fragment J comprises the left-most end of the viral genome, and fragment A.A represents the right-most segment of the Eco Rl-A fragment. As the data show, the V2,,0, VIs6, V14,,, and VIsO RNA species show significant hybridization only to Sma I fragment B. This fragment is located between 0.18 to 0.38 fractional lengths of the viral genome (H. Delius and C. Mulder, personal communication). Similar results have been obtained with the low molecular weight viral RNAs purified from infected cells (Jaehning et al., 1975). Thus the genes coding for the low molecular weight viral RNAs appear to be localized to a specific internal region of the viral DNA.

We have shown that nuclei isolated from human cells productively infected with adenovirus 2 have the capacity to synthesize at least four discrete low molecular weight RNA species which are of viral origin. These RNA species include the VIs6 (or 5.5.S) RNA species, previously shown to be synthesized in vitro (see Introduction), and three novel RNA species which we have designated V200, VldO, and V130. That the Vlsb RNA is present in intact cells was originally shown by Reich et al. (1966). In addition to this RNA species, the present studies reveal the presence in infected cells of low molecular weight viral RNAs with electrophoretic mobilities similar to those of the VZoO and VIhO viral RNAs synthesized in nuclei. At present, we have not detected an in vivo counterpart to the V130 species synthesized in nuclei. The sequence identity of the VIs6 RNAs synthesized in vivo and in vitro has been established by hybridization-competition studies (Weinmann et al., 1974a; R. Weinmann, unpublished observations). The sequence relationships between the VZoO and VI40 RNAs synthesized in vitro and their in vivo counterparts have not yet been established. We tentatively assume, however, that the respective components are the same, since they have similar electrophoretic mobilities and since they hybridize to the same adenovirus DNA fragments (Jaehning et al., 1975). Function of Low Molecular Weight RNAs Our studies indicate that the low molecular weight viral RNAs are found primarily within the cytoplasmic fraction. However, because of the possibility of leakage from nuclei during isolation, we cannot exclude the possibility that any or all of these RNAs are localized predominantly within the nucleus in intact cells. Baum and Fox (1974) reported that at least a fraction of the 5.5s RNA is associated with ribosomes, suggesting a role in translation. Alternatively, some of these RNAs might function within the nucleus, possibly as primers for DNA replication, as reported for other low molecular weight RNAs (for example, see Hayes and Szybalski, 1975; Verma, Meuth, and Baltimore, 1972). A third possibility is that the low molecular weight RNAs represent structural or regulatory elements of adenovirus transcription complexes. As such, they may be important for effecting transcription of late viral genes; it has been suggested previously that low molecular weight cellular RNAs are associated with chromatin (Marzluff et al., 1975). Further analysis of the genome sites coding for these RNAs may provide insight into their function.

Cell 564

Organization of Viral Genes All the low molecular weight RNAs described here are transcribed from a single adenovirus 2 DNA fragment which comprises the region between 0.18 and 0.38 fractional lengths of the viral genome. Preliminary results in our laboratory, using fragments produced by endo R.Hpa I, indicate that the genes for these RNAs are localized between 0.27 and 0.38 on the genome, and that all three RNAs hybridize to the I strand (G. King, unpublished observations), which is transcribed from left to right (Sharp, Gallimore, and Flint, 1974). Although these results restrict the DNA sequences which encode these RNAs to 11% of the genome, a DNA segment coding for the VsoO, VIs6, and VIhO RNAs would represent only 1.79% of the genome if there are no common sequences. Recently, Pettersson and Phillipson (1975) have shown that the 5.5s RNA (V,U) is transcribed from the region of the I strand located between positions 0.27 and 0.32 on the unit map, in agreement with the present results. The precise sequence relationships between the V200, VIs6, and VIdO species are not known, although hybridization-competition and nearest-neighbor analyses of the RNAs synthesized in isolated nuclei indicate that the VZoO RNA contains sequences distinct from the VIs6 and VIJO RNAs (R. Weinmann, unpublished observations). While all three RNA species could be derived from one large precursor, two arguments against this possibility can be made. First, we have not yet detected an RNA polymerase III transcript equal to the combined lengths of the low molecular weight RNAs, suggesting that any processing would have to occur very rapidly. Second, the VZoO, VIs6, and VIJO RNAs are synthesized in greatly different molar amounts in isolated nuclei, suggesting that a certain fraction of the transcripts would have to be prematurely terminated at specific internal sites to account for the observed rates of synthesis. An alternative and more probable explanation is that the DNA sequences encoding the low molecular weight RNAs have independent initiation and termination sites, and are transcribed independently. The differences in transcription rates would then be determined by the relative frequencies of initiation by RNA polymerase III, Low Molecular Weight RNAs and RNA Polymerase III Previous studies have shown that the cellular 5S RNA and tRNAs are transcribed by RNA polymerase III in isolated nuclei (Weinmann and Roeder, 1974). Similarly, an RNA polymerase III was shown to synthesize the adenovirus VIs6 RNA (Weinmann et al., 1974a, 1974b). Evidence is presented here for a similar role for RNA polymerase III in the transcription of all the low molecular weight viral RNA spe-

ties detected thus far. In isolated nuclei, the viral genes encoding the small viral RNAs are transcribed for extended periods of time (Figure 3), suggesting the occurrence of specific site selection and reinitiation by RNA polymerase III in this system. Direct evidence for de novo synthesis of VIs6 (Price and Penman, 1972a) and the cellular 5s RNA (Marzluff, Murphy, and Huang, 1973; McReynolds and Penman, 1974) has previously been reported. In our studies, a minimum of IO-20 V1s6 RNA molecules are transcribed per nucleus per set, and probably an equivalent amount of all other low molecular weight RNA molecules (VZoO, V1aO, VIXO, CIZ,,, and pretRNA). Thus during the 60 min period of linear synthesis, at least 80,000-160,000 RNA molecules are synthesized per nucleus. At all times after adenovirus infection, there are about 2 x 104 RNA polymerase III molecules per cell (Weinmann et al., 1976) and approximately one third of the RNA polymerase III remains in nuclei following subcellular fractionation (J. Jaehning, unpublished observations). Assuming that all the remaining intranuclear RNA polymerase III molecules are actively engaged in transcription in vitro, a minimum of 12-24 reinitiation events must occur for each RNA polymerase III molecule. The reiterated genes for the 5s and tRNAs are clustered (Brown, Wensink, and Jordan, 1971; Clarkson, Birnsteil, and Purdom, 1973). Studies with isolated nuclei implicate RNA polymerase III in transcription of all these genes. It seems plausible that other cellular low molecular weight RNAs which have been isolated from various eucaryotic cells(Ro-Choi and Busch, 1974; Weinberg and Penman, 1968; Marzluff et al., 1975) may also be transcribed by RNA polymerase III. Like the low molecular weight viral RNAs described here, the functional significance of these species is not known. Continued analysis of the function and transcription of the viral RNAs may help to elucidate the function of the low molecular weight cellular RNA molecules and the mechanisms by which these RNAs are transcribed. Experimental

Procedures

Cell Culture and Virus Infection Exponentially growing cultures of KB cells were infected with adenovirus2 at a multiplicity of 40 PFU per cell as described previously (Craig and Raskas, 1974a). After a 1 hr adsorption period, the culture was diluted to 3 x 105 cells per ml. The virus used for infections and as a source of DNA was purified by two equilibrium centrifugations in cesium chloride (Craig, Zimmer, and Raskas, 1975). Labeling and Purification of RNA Synthesized in infected Cells To prepare radioactive RNAs, cultures were infected in phosphatefree Joklik MEM containing 2.5% horse serum (Craig and Raskas, 1974b). Carrier-free “2P04 (50-200 pCi/ml) was added 6 hr after infection, and the culture was harvested 9 hr later. The pelleted cells were washed with phosphate-buffered saline 2 times and re-

Low Molecular 565

Weight

Adenovirus

FiNAs

suspended in 5 vol (v/v) of hypotonic buffer [lo mM Tris (pH 7,9), 10 mM MgCb, 24 mM KCI, 0.5 mM dithioerythritol]. After swelling for IO-20 min at 0°C cells were disrupted with lo-20 strokes of the B pestle of a Kontes all-glass Dounce homogenizer. Nuclear and cytoplasmic fractions were separated by low speed centrifugation (800 x g, IO min). The nuclei were resuspended in the above buffer containing 500 mM NaCl and 50 mM MgQ, and treated with electrophoretically purified DNAase (10 pg/3 X 107 nuclei) at 0°C for 15 min. Sodium dodecyl sulfate (SDS, 0.5%) was added to both nuclear and cytoplasmic extracts. The preparations were then treated with phenol and CHCll consecutively, as described by Palmiter (1974). Following extraction, the material remaining in the aqueous phase was collected by ethanol precipitation and then redissolved in water. The concentrated RNA was then layered on IO-30% sucrose gradients containing IO mM Tris (pH 7.5), 1 mM EDTA, 0.5% SDS. Centrifugation at 40,000 rpm in the SW40 rotor in the Beckman ultracentrifuge (18°C 12 hr) yielded 3 peaks containing 28S, IBS, and low molecular weight RNA species, respectively. Fractions containing 12S-4s were pooled and the RNA concentrated by ethanol precipitation. Labeling and Purification of RNAs Synthesized in Nuclei Isolated from Infected Cells Nuclei were prepared from cultures 14 hr after infection (Weinmann et al., 1975a). After swelling and homogenization in hypotonic buffer (above), nuclei were separated from the cytoplasm by centrifugation. The nuclear pellet was resuspended at 3 x 107 nuclei per ml in hypotonic buffer containing 0.5% Triton X-100. This suspension was layered over 2 vol of a buffer solution containing 10 mM Tris (pH 7.9), 5 mM MgCl*, 0.1 mM EDTA, 25% glycerol, and 0.5 mM dithioerythreitol. The nuclei were collected as a pellet by centrifugation at 800 x g for IO min and resuspended in the same glycerol containing buffer at 3 x 107 nuclei per ml. The cytoplasmic supernatants were saved for purification of unlabeled RNA. The Triton-washed nuclei were as active in the synthesis of viral 5.5s RNA (VI& as were nuclei not treated with detergent, but since the cytoplasmic contamination was reduced in the former, the hybridization efficiency of the in vitro transcribed RNAs was increased (results not shown). RNA synthesis was performed at 25°C with 1.3 x 107 nuclei per ml in IO mM Tris (pH 7.9), 2 mM MnCIZ, 0.04 mM EDTA, 0.2 mM dithioerythritol, 0.5 mM ATP, CTP, and GTP, and 0.05 mM of labeled UTP. 3H-UTP was from Amersham Searle (spec. act. 49 Ci/mmole). QP-UTP (spec. act. loo-120 Ci/mmole) was prepared by the method of Symons (1974). Reactions were terminated by treatment with electrophoretically purified DNAase (1 pg/3 x lo5 nuclei) for IO min at 0°C. Following the addition of SDS, RNA was extracted with ohenol and chloroform as described above.

phoretically (Tal et al., 1975) into a dialysis bag at 4 mA per gel using Tris-acetate buffer [40 mM Tris (pH 7.5), IO mM Na acetate, 1 mM EDTA, 0.28% SDS]. The RNA was further concentrated by ethanol precipitation. Preparation of Viral DNA Fragments Restriction endonuclease Eco RI was purified from E. coli following the procedure of Yoshimori (1971). Restriction endonuclease Sma I was purified from Serratia marcescens according to the procedure outlined by C. Mulder (manuscript in preparation). Viral DNA was extracted from purified virions as described by Craig et al. (1975). The fragments obtained after digestion with the appropriate restriction endonuclease were separated in 0.7% or 1% cylindrical agarose gels. The bands stained with ethidium bromide were cut out under an ultraviolet lamp as described by Sharp et al. (1974). The fragments were eluted electrophoretically as described by Pettersson et al. (1973) and attached to nitrocellulosefilters after denaturation with base, as described by Craig and Raskas (1974a). The hybridization capacity of DNA immobilized in each set of filters was tested by hybridization of alkali-boiled radioactive adenovirus DNA. In all cases, the amounts of DNA bound were proportional to the molecular weight of the fragments. RNA-DNA Hybridization Conditions Nitrocellulose filters (7 mm diameter) were punched from larger filters containing either whole adenovirus DNA or specific viral DNA fragments or E. coli DNA. Incubations were in 100-200 ~1 of 0.6 M NaCI, 0.06 M Na citrate (4 x SSC), and 0.1% SDS at 66°C for 14 hr. RNA was heated briefly at 100°C before hybridization. Filters containing different DNA fragments, E. coli DNA, and a blank were hybridizedsimultaneously in thesame reaction mixture. After hybridization, the filters were washed with 2 x SSC and treated for 1 hr at room temperature with 20 kg/ml of boiled pancreatic RNAase in 2 x SSC. The filters were washed twice more in 2 x SSC, dried, and counted in a toluene fluor (4 g PPO, 0.05 g POPOP, toluene to 1 I). Acknowledgments We thank M. Telle for excellent technical assistance. This research was supported in part by grants from the National Cancer Institute and from the NSF to R. G. R., and by grants from the National Cancer Institute and from the American Cancer Society to H. J. R. R. G. R. is a research career development awardee of the NIH. Cell culture media were prepared in a facility funded by the NSF. Received

November

24, 1975;

revised

January

6, 1976

References Purification of Low Molecular Weight RNAs Following ethanol precipitation, RNAs were dissolved in formamide containing 10% (w/v) of sucrose. Gel slabs (40 x 15 x 0.1 cm) containing 10% polyacrylamide, 0.27% N, N’-methylenebisacrylamide, 0.075% ammonium persulfate, and 0.07% N, N’, N’-tetramethylethylenediamine in Tris-phosphate buffer (4.35 g Tris, 4.14 g NaH2P04Hz, and 0.29 g EDTA/I of HzO) were prerun for 3 hr at 150 volts in Tris-phosphate buffer containing 0.2% SDS. Electrophoresis was for 35 hr at 150 volts with one change of buffer (0.8 I). Analytical gels used ethylene diacrylate as the cross-linking agent, as previously described by Weinmann and Roeder (1974). The RNA species were localized by autoradiography of the wet slabs in the case of ‘2P-RNA or by the use of appropriate markers for 3H-RNA. Nonradioactive RNA species were localized by direct examination of the slabs under a short-wave ultraviolet lamp (Mineralight UVS 12). Gel regions containing specific RNA species were excised and placed over a 1% agarose plug in a 1 cm cylindrical gel tube; after the addition of 50-100 pg of E. coli tRNA in 20 ~1 of electrophoresis buffer, 1 ml of a 1% agarose solution was added and allowed to solidify. The RNA was then eluted electro-

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