RNA of low molecular weight in KB cells infected with adenovirus type 2

J. Mol. Biol. (1966) 17,428-439

RNA of Low Molecular Weight in KB Cells infected with Adenovirus Type 2 PAUL

R.

REICH, BERNARD

G.

FORGET, SHERMAN

M.

WEISSMAN

Metaholism Branch, National Cancer Institute AND

JAMES

A.

ROSE

Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014, U.S.A. (Received 27 December 1965) Chromatographic analysis on methylated albumin-kieselguhr columns of newly formed, radioactive RNA from KB cells infected with adenovirus type 2 revealed an unusual radioactive com pone nt which was eluted following 4 a RNA. This RNA was present pred ominantly in the 100,000 g ribosomal supernatant cell fraction, and was distinguished from 4 a, 16 sand 28 s RNA's on the basis of its behavior on m ethylated albumin columns, base com posit ion , sedimentation velocity and behavior on Sephadex GlOO columns. This RNA component has certain characteristics in common with a "5 a" RNA asso ciated with ribosomes from uninfected KB cells and a variety of other organisms. The effect of deoxyfluorouridine and a ct inomycin D on the m etabolism of this unusual RNA suggests that DNA synt hesis and transcription of DNA into RNA are required for its formation.

1. Introduction The biochemical events following adenovirus infection of human cells have been the subject of several investigations (cf. review by Levintow, 1965). Cell division is inhibited after infection, but RNA synthesis continues at an essentially unchanged rate (Green & Daesoh, 1961), and is necessary for the formation of adenovirus DNA and protein (Flanagan & Ginsberg, 1964). The RNA form ed in infected cells includes sRNA,t ribosomal RNA and RNA complementary to both host and virus DNA (Rose, Reich & Weissman, 1965). Chromatographic analysis of radioactive RNA from KB cells infected with adenovirus type 2 reveals an unusual component (VA. RNA) which is eluted from MAK columns after sRNA (Kohler & Odaka, 1964; Rose et al., 1965). Recently several workers (Rosset, Monier & Julien, 1964; Comb, Sarkar, DeVallet & Pinzino, 1965; Galibert, Larsen , Lelong & Boiron, 1965) have detected a small amount of an RNA ("5 s" RNA, "t -like" RNA) associated with ribosomes from uninfected KB cells and several other organisms. This RNA is also elut ed from MAK columns directly after sRNA. It lacks pseudo-uridine and methylated bases, and has a uridine nucleotide at the 3' -hydroxyl end of the molecule. Its function or relationship to other cellular RNA species is not known. t Abbreviations used: sRNA , soluble or 4 s RNA; MAK, methylated albumin kieselguhr; SDS, sodium dodecyl sulfate. 428

RNA IN ADE N OVIRUS-INFECTED KB CE L L S

429

In the present study, we have examined certain chemical and metabolic properties of VA-RNA. It resembles " 5 s" RNA in several respects. How ever , it is readily detectable in RNA prepared from 100,000 g supernat ant fr actions of disrupted KB cells, whereas "5 s" R NA appear s only in RNA from t he ribosomal fracti on. The effect of metabolic inhi bit ors on the metabolism of VA-RNA is also described.

2. Materials and Methods (a) Cells, medium and viruse8 Two KB cells lin es, one ob t ained from Microbiological Assoc ia tes and one from Dr M. Green, were used in t hese studies. The cells were grown in suspe nsi on culture in Eagle's medium (Eagle, 1959) supp leme nted wit h 5 % unheated hor se serum and 2 m x -glu t arnine . Under these conditions, t he cells have a generation time of 24 hr. Ad enovirus type 2 (adenoid 6) was obtained fro m Dr W . Rowe. Virus ino cula , gr own in KB cells, contained no adenovirus-associated particles (Melnick, Mayor, Smith & Rapp, 1965) as determined by elect r on microscopic examinat ion . There was no complem ent-fixing reactivity between the virus inoculum and a preparation of anti-serum against a de no virus-a ssociat ed particles (Hoggan, 1965; Atchison, Casto & Hammond, 1965). The procedure used to titrate adenovirus has been described elsewhere (Rose et al., 1965). KB cells at concentrations of approximately 2 X 105 cells per ml. were inoculated with a m u lt iplicit y of 100 plaqueforming units per cell. In order to be sure of t he completeness of infect ion under these con d it ions , cloning efficiencies of infect ed and contro l KB cells were determined . E x cep t for enrich me n t of medium with O· 2 m M non-essential amino acids , t he p rocedure described by Green & Daesch (1961) was used . Infected cell centers were plated 4 hr a fte r v irus inoculation. Duplicate as says of 200 and 400 infected cell centers revealed no clones . There was 100% cloning efficiency of 150 cont rol centers. Mixing infect ed cell cente rs with control cell centers resulted in re covery of a number of clone s approximating t hat of the number of plated cont rol cell cen ters . The possib ilit y of secondary cell cent er infect ion after plating was therefore unlikely. Thus , essentiall y a ll KB cells were infected. (b) Radioactive labeling of cellula r R N A Tritiat ed uridine (20 elm-mole, N uclear-Chicago ), uniformly labeled [14C]uridine (60 mojm -mole, Nuclear-Chicago), [2_14C]uridine (36' 9 m cjrn -mole, Nuclear-Chicago); L.[methyp4C]methionine (29'5 mc/m -mo le, New England Nuclear Corp.), [S-14C]azaguanine (1'6 mc/m-rnole, I sotope Specialties Corp .), and [32P]ph osphor ic acid (carrier free, Oak Ridge, neutralized with NaOH) were adde d to ce ll cu ltures t o la bel R NA. Labeling with sodium [32P ]phosphate was carried out after t he cells were washed twice with phosphate -free medium and re suspended in m ed ium cont a in in g 1O- 5M-phosphate and 5 % dialyzed h orse serum . In general, radioactive p recursor(s) was added 16 hr after v irus in oculation of exponen. tially dividing cells. Incubation was continued for p er iods varying in duration from 20 min to 6 hr. Cells were harvest ed by centrifugation (800 g for 10 min); RNA was isolated and chromatographed on MAK columns. (c) KB cell f ractionation Ribosomal and 100,000 g supe rnatant frac tions were obtaine d as described by Galibert et al. (1965). Int a ct cells, nuc lei an d mi t och ondria were removed by low-sp eed centr ifuga tion prior to ultrac entrifugation.

(d) R NA preparati on R NA was prepared b y eit he r a hot phenol-SDS (Scherrer & Darnell, 1962) or cold phenol-SDS (Salzman, Shatkin & Sebring, 1964) procedure. Similar result s were obtained with bo th m ethods. In certain exper im ent s, RNA wa s in cubat ed with D Na se (10 fLg/ml., elect rophoretically purified , Worthington Bioch emical Corp.) for 15 min at 37°C. The RNA wa s precipitated 3 t imes fr om a lcoho l prior to in cubation wit h DN as e.

430

REICH, ROSE, FORGET AND WEISSMAN

The nucleic acid content of solutions was estimated from the absorption of ultraviolet light at 260 uu». An optical density of 0·025 was assumed equivalent to an RNA concentration of 1 p,gjml. (e) RNA fractionation and analysis Methylated albumin columns were prepared by a modification (Karon, Henry, Weissman & Meyer, 1965) of the method described by Mandell & Hershey (1960). The columns were eluted with a linear sodium phosphate and NaCl gradient. The mixing chamber contained 180 to 300 ml. of 0·2 M-NaCl, 0·02 M-sodium phosphate (pH 6'7), and the reservoir held an equal volume of 1·0 M-NaCl, 0·04 M-sodium phosphate (pH 6'7). Three- to 5-ml. fractions were collected every 10 to 15 min. Radioactive material corresponding to the sRNA region of the chromatogram and the radioactive material eluted after sRNA (VA-RNA) was subjected to the following analyses. Sedimentation analyses were performed by centrifugation in 5 to 20% linear sucrose gradients as previously described (Rose et al., 1965). RNA fractions were examined by chromatography on Sephadex 0100 (Pharmacia) in 1'0 M-NaCl, 0·05 M-sodium phosphate (pH 6,7) (Schleich & Goldstein, 1964). In one experiment, 8·0 M-urea was substituted for 1·0 M-NaCl, and the column was equilibrated and eluted with 8·0 M-urea, 0·05 M-sodium phosphate (pH 7,0). Base analyses of alkaline hydrolysates of 32P-labeled KB cell RNA were performed by paper electrophoresis at pH 3·5 in a pyridine-acetate buffer (Salzman et al., 1964). The relative quantities of radioactive pseudouridylic and uridylic acids in RNA from cells labeled with [14C]uridine were determined by hydrolysis of the RNA with 0·3 N-KOH at 37°0 for 18 hr. The neutralized digest was chromatographed on Dowex-l-formate columns (Cohn, 1960). Ribonuclease sensitivity of fractions eluted from MAK columns was determined by incubation of the radioactive material with pancreatic RNase (5 times crystallized, Sigma Chemical Co., heated to 80°C for 10 min to inactivate DNase). Two concentrations of RNase and NaCI (in 2 ml, 0·05 M-tris-chloride, pH 7-4), and two different incubation periods were used. The fraction of acid-insoluble material which remained after incubation was precipitated with 0·5 vol. of 15% trichloroacetic acid. The precipitates were collected on Schleicher & Schuell B6 nitrocellulose membrane filters, and assayed for radioactivity. The same procedure was used to assay for acid-insoluble radioactive material before exposure to RNase. (f) Assay oj radioactivity The 32p of aqueous samples was determined by plating portions at infinite thinness on aluminum planchets. These were assayed in a low background (2 ctsjmin) gas-flow counter (efficiency, 40%). Membrane filters and aqueous samples containing 3H and 14C were assayed in a threechannel liquid-scintillation spectrometer (Nuclear-Chicago, model 725). A scintillation fluid containing dioxane (Davidson, 1958) was used with aqueous samples containing 3H (efficiency, 10%). Liquifluor (Nuclear-Chicago) was added to the membrane filters after they were washed with 95% ethanol and toluene (SH efficiency, 20%; 32p efficiency, 80%). Corrections for quenching with the membrane filters were made by the channel ratios method. Dual isotope counting of aqueous samples was carried out in the dioxane-based scintillation fluid. 3H and 14C efficiencies were: 3H, 0·2% in 14C channel, 6,0% in 3H channel and 14C, 35'0% in 14C channel, 8'5% in 3H channel. 3H and 32p efficiencies were: 3H, 0% in 32p channel, 5% in 3H channel, and 32p, 70% in 32p channel, 7% in 3H channel. The statistical counting error was ±4'10 or less.

3. Results (a) Occurrence of VA-RNA

Methylated albumin column chromatography of RNA from KB cells infected with adenovirus type 2, and exposed to [3H]uridine for periods varying in duration from 20 minutes to six hours, revealed a prominent radioactive component (VA-RNA)

431

R N A IN ADE NOVIRU S -INF E CT ED K B CELL S

which was elute d with 0·45 }I·NaCI afte r t he bulk of sR NA (Fig. l(a)). Compared to sRNA, there was little mat erial which absorbed light at 260 m/-, associated with VA-RNA. No l4C-Iab eled VA-RNA was detectable in RNA from uninfected KB cells (labeled with [l4CJuridine) mixed with infected KB cells (lab eled with [3HJuridine) prior to preparation and chromatography of cellular R NA (Fig. l(a)). VA-RNA was prominent in RNA from KB cells exposed to radioacti ve RNA precursor for periods (of varying duration, from) 14 t o 24 hours after adenovirus infection. E arly in the course of infection (0 t o 10 hours), little or no VA-R NA was det ected. Sodium [32PJphosphate and [l4CJuridine could be substit uted for [3HJuridin e wit hout affecting the chroma t ographic characterist ics of VA-RNA. This RN.A component was demonstrated equally well in both KB cell lines. (c)

500

Whole cell RNA

400 300 0'6

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200 100

40 20

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60 40

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35

45

55

Tube no.

FIG. 1. MAK column chrom ato gr aphy of RNA fro m uninfected and adenovirus infected whole KB cells and cell fractions. Norm al KB cells (l 08 cells in 700 ml.) were ex p osed for 2 hr to [H C]uridine (0·04 /Lc/ml .). KB cells (l 08 cells in 700 m l. ) inoculated 16 hr previously with aden ovir us type 2 were exposed for 2 hr to [3R]uridine (1 /Lc/ml. ). (a) The cells from 200·ml. portion s fro m each culture were mixed, R NA extracted and chromatographe d as descr ib ed in t he text . The seco nd rad ioa ct ive peak is VA- R NA . The remai n ing cells were mixed and cell fr ac t ion s p re pare d . R NA from 100,000 g r ibo somal supe rnatant (b) a nd r ibosom al (c) fract ions was pre pare d and chromatographed . No n -radioactive E . coli B sRNA (1 mg) was added t o the RNA from t he ribo somal fr ac t ion . The arrow shows t he position of " 5 s " RNA in t he uninfected KB cell r ibosomal fraction . T hroe ml. of elu at e from the MAK columns wa s collect ed in each t u be and O·l-ml. p ortion assay ed for radioactiv ity. HC·l abeled RNA fr om normal K B cells ; - 0 -0-, 3R-label ed RNA from infected KB cells ; - /::;- 6.-, absorbanc e at 260 mJL.

-e-e-,

REICH, ROSE, FORGET AND WEISSMAN

432

KB cells infected with adenovirus type 2, and labeled in the usual manner with [3H]uridine, were fractionated into ribosomal and 100,000 g supernatant components to determine with which fraction VA-RNA was primarily associated. RNA from both fractions was chromatographed and showed a radioactive component in the VA-RNA region (Fig. I(b) and (c}). The quantity of radioactive VA-RNA was tenfold greater in RNA from the ribosomal supernatant fraction than in the ribosomal fraction. Chromatography of ribosomal RNA from unifected KB cells (Fig. I (c» showed small amounts of radioactivity in the "5 s" RNA region, as described by Galibert et al. (1965). The ribosomal supernatant fraction was devoid of significant radioactivity in this region (Fig. I(b}). Chromatographic analysis of ribosomal and 100,000 g supernatant RNA from three other uninfected KB cell cultures labeled with large amounts of tritiated uridine or sodium [32P]phosphate demonstrated that radioactivity in the "5 s" region was detectable only in ribosomal RNA. (b) Susceptibility of VA-RNA to RNase and DNase

Radioactive material from the sRNA and from the VA-RNA regions of a MAK column chromatogram was used to investigate their susceptibility to RNase and DNase. Radioactive VA-RNA was insoluble in 5% trichloroacetic acid. It was made completely soluble by prior incubation with pancreatic RNase (20 /Lgjml., incubated 20 minutes in 0·01 M-NaCI) and remained completely insoluble after incubation with DNase (100 /Lgjml., incubated 20 minutes at 37°C in 0·002 M-MgCI 2). DNase treatment of RNA from KB cells infected with adenovirus type 2 did not change the elution pattern of VA-RNA from MAK columns. The susceptibility to RNase of VA-RNA compared to sRNA is shown in Table 1. VA-RNA and sRNA differed slightly in their resistance to RNase digestion. VARNA appeared to be more resistant to RNase, especially in the presence of low TABLE

I

Ribonuclease susceptibility of sRNA and VA-RNA

Molar concentration of NaCI

0·01 0·01 0·01 0·01 0·5 0·5 0·5 0·5

Time of incubation (min)

0·5 20 0·5 20 0·5 20 0·5 20

Concentration of ribonuclease (f£g/ml.)

0·1 0·1 20 20 0·1 0·1 20 20

Acid-insoluble fraction remaining (%) Type of RNA sRNA

VA-RNA

9

35 18 7 1 96 93 77 43

2

1 1 95 83 62 18

Approximately 2000 cts/min of each 32P_Iabeled RNA component, obtained by methylated albumin column chromatography, was digested with RNase in 2 ml, 0·05 M-tris-ehloride (pH 7,4) at 37°C. The acid-insoluble fraction remaining was precipitated with 0·5 vol. of 15% triohloroacetic acid. Precipitates were collected on membrane filters and radioactivity determined 8S described in Materials and Methods.

RNA IN ADENOVIRUS -INFECTED KB CELLS


concentrations of NaO!. This increased resistance was observed despite the larger chemical amount of sRNA tested. (c) Base composition ofsRNA and VA-RNA

The r esult s of base analyses of 32P-labeled material from the sRNA, "5 a" and VA-R NA regions of MAK column chromatograms, and 4 a, 16 sand 28 a KB cell ribosomal RNA, fractionated by centrifugation in sucrose density-gradients, are presented in Table 2. Base analysis of 4 s, 16 sand 28 s ribosomal RNA resembled that previously reported from HeLa cells (Salzman et al., 1964). There were differences between the base composition of VA-RNA and that of sRNA and "5 s" RNA. VA· RNA contained more guanylic acid and less adenylic acid than the latter two RNA species, which had similar base compositions. Ribosomal RNA also had base composition different from VA-RNA. TABLE

2

Base composition of newly synthesized RNA components Moles/I 00 moles nucleotides Experiment no.

RNA species

Cytidylic acid

Adenylic acid

Guanylic acid

Uridylic acid

32-1

25·6 28·8

15·2 21·9 18·6

35·6 29·0 31·2

17·0 23'4 21-6

sRNA "5s" RNA

27·8 27·4

18·0 18·8

32·6 31· 3

21·6 22'3

sRNA VA ·RNA

29·9 29·1

19·2 16·1

30·8 34·4

20'1 20'6

Ribosomal 28 s 16 s sRNA

3§

The results are representative of duplicate analyses performed on each of two preparations 3~P .labeled RNA. t Prepared fr om uninfected KB cells exposed to sodium [32]phosphate for 3 hr. RNA fractionated by ultracentrifugation in sucrose density-gradients. t Prepared from uninfected KB cells exposed to sodium [32P]phosphate for 48 hr. RNA was prepared from ribosomal cell fractions, and fractionated by MAK column chromatography. § Prepared fro m cells inoculated with adenovirus 16·5 hr prior to exposure to sodium [3 ~P]phosphate for 3 hr. RNA fractionated by MAK column chromatography. of

The relative amounts of uridylic acid and pseudouridylic acid in sRNA and VARNA were determined by labeling infected KB cells with [2. HO]uridine for four hour s and fractionating the RNA from these cells on MAK columns. The HO-labeled sRNA and VA-RNA were hydrolyzed and their base composition analyzed (see Materials and Methods). The ratio of radioactive pseudouridylic acid to uridylic acidinsRNA was 14%, whereas in VA-RNA it was 1%, indicating a ma rked difference in the amount of pseudouridylic acid present in these RNA species. The small amount of pseudouridylic acid in VA-RNA could represent contamination with sRNA. Dowex-L column chromatography revealed radioactivity corresponding to one

REICH, ROSE, FORGET AND WEISSMAN

434

uridine nucleoside for each 17 to 22 uridylic acid residues in digests of VA-RNA. No uridine nucleosides were found in sRNA digests. To determine whether any of the methylated ba ses found in sRNA were present in VA-RNA, infected KB cells were incubated for four hours with L-[methyP40 ]. methionine and [3H]uridine, and the RNA chromatographed on a MAK. column. Although a prominent tritium-labeled RNA component was seen in the VA-RNA region of the chromatogram (Fig. 2), essentially no radioactivity due to 14 0 label was present in this region. There was radioactivity du e to tritium and 140 labels in the sRNA region of the chromatogram. 30 . --

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FIG. 2. Incorporation ofL·[methyp4C]methionine into RNA from adenovirus-infected KB cells. KB cells in oculated 16 hr previously with virus were exposed to L-[methyl·14C]methionine (0 ,1 Jic/m1.) and [3H]uridine (0,4 Jic/mI.) for 4 hr. RNA was prepared and chromatographed on a MAK column. A O·I-mI. portion from each fraction (5 ml.) of eluate was assayed for radioactivity. - 0 -0-, L-[methYP'C]methionine; - .A.- .A.-, [3H]uridine; absorbance at 260 ttu».

- e-e-,

(d) Incorporation of [8- 140 ]azaguanine into sENA and VA -EN A

There is an eight- to t enfold greater incorporation of 8-azaguanine into sRNA t han into ribosomal or high er molecular weight RNA from uninfecte d KB cells exposed to low concentrations of this purine analog (Karon etal., 1965). The mechanism of selective incorporation into sR NA is unknown. The incorporation of 8-azaguanine and uri dine into sRNA was compared with their incorporation into VA-RNA. Sixteen hours after adenovirus inoculation, KB cells were incubated for two hours with [140]azaguanine and [3H]uridine. The MAK. column chromatogram of RNA from these cells (Fig. 3) revealed relatively large quantities of 14 0 label in the sRNA region compared to the VA·RNA region. Radioactivity du e to tritium label was great er in the VA-RNA region than in the sRNA region. The dissimilar distribution of 14 0 label compared to 3H label throughout the sRNA region was also observed with uninfected KB cells.

435

RNA IN ADENOVIRUS-INFECTED KB CELLS

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Tube no.

FIG. 3. Incorporation of [8- 14C]azaguanine into RNA from adenovirus-infected KB cells. KB cells inoculated 16 hr previously with virus were exposed to [8- 14C]azaguanine (0,001 p.c/ml.) and (3H]uridine (0'1 p.c/ml.) for 2 hr. RNA was prepared and chromatographed on a MAK column. Assays for radioactivity were carried out as described in Fig. 2. -0-0-, [8- 14C]azaguanine; -A-A-, [3H]uridine; absorbance at 260mI"

-e-e-,

(e) Molecular volume and sedimentation velocity of

VA-RNA compared to sRNA

311P-Iabeled sRNA and VA-RNA, obtained from the same infected KB cell culture, were each mixed with unlabeled Escherichia coli B sRNA (1 mg, General Biochemicals) and placed on separate Sephadex GlOO columns (125 em X 0·9 em). These were eluted with phosphate buffer containing 1·0 M-NaCl. The 32P-Iabeled VA-RNA was eluted ahead of the bulk of E. coli B sRNA (Fig. 4). The 32P-Iabeled sRNA was

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FIG. 4. Sephadex GI00 column chromatogram of non-radioactive E. coli B sRNA and 32P·labeled VA-RNA. The RNA fractions were obtained as described in the text. -0-0-, 32P-Iabeled VA-RNA; absorbance at 260 mI" The ultraviolet-absorbing material in tubes no. 10 to 13 represents a contaminant found in E. coli B sRNA preparations (Brown, 1963).

-e-e-,

436

REICH, ROSE, FORGET AND WEISSMAN

eluted with the main peak of E. coli B sRNA (Fig. 5). An experiment performed with 32P-Iabeled VA-RNA and a Sephadex G100 column pre-equilibrated with phosphate buffer containing 8·0 M-urea yielded results similar to those shown in Fig. 4. This suggested that aggregation of RNA macromolecules was not responsible for the be. havior of VA-RNA on Sephadex and MAK columns. These experiments indicated that the molecular volume, and presumably the molecular weight, of VA-RNA is greater than that of sRNA.

20 c:

~ ...,'" U

0-

10 :::

~o 40

20 Tube no.

FIG. 5. Sephadex GIOO column chromatogram of non-radioactive E. coli B sRNA and 82P-labeled sRNA from an adenovirus-infected KB cell culture. -0-0-, 82P-Iabeled sRNA; absorbance at 260 mJL.

-e-e-,

The sedimentation velocities of 3H-Iabeled VA-RNA relative to 32P-Iabeled sRNA were determined by centrifugation through linear sucrose density-gradients. Labeled sRNA and VA-RNA were prepared separately under standard conditions and mixed prior to layering on the sucrose gradients. Analysis of sucrose gradients from four separate experiments showed the VA-RNA to have a sedimentation velocity significantly greater than sRNA, with an estimated value of 5 to 6 s (Fig. 6). (f) Effect of metabolic inhibitors on the occurrence of VA-RNA

Incubation of KB cells infected with adenovirus with actinomycin D was performed to determine whether production of VA-RNA was dependent on DNA transcription into RNA (Reich, Franklin, Shatkin & Tatum, 1962). Sixteen hours after adenovirus inoculation, KB cells were exposed to actinomycin D (5 /Lg!ml., Merck, Sharpe & Dohme) for two hours and then to sodium [32P]phosphate for three hours. This concentration of actinomycin D was shown to reduce the incorporation of radioactive precursors into RNA by more than 95%. After MAK column chromatography of the cellular RNA, no concentration of radioactivity in the VA-RNA region was seen. Base analysis of the 32P-Iabeled sRNA showed radioactivity predominantly associated with cytidylic and adenylic acids. To determine whether VA-RNA is rapidly degraded or metabolized, actinomycin D was employed to depress VA-RNA synthesis after infected KB cells had been exposed to labeled RNA precursors. Eighteen hours after adenovirus inoculation, KB cells

RNA IN ADENOVIRUS-INFECTED KB CELLS

437

100

150

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50

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Tube no.

FIG. 6. Sedimentation pattern of differentially labeled sRNA and VA-RNA through linear sucrose density-gradients. 32P-labeledsRNA (4000cts/min) and 3R-Iabeled VA-RNA (2900 otsjmin) were layered on 5 to 20% linear sucrose gradient. The preparation, centrifugation (45,000 g for 18 hr) and analysis of the sucrose gradients were carried out as described in the text. Approximately 0·4-mI. fractions were collected and 0·3-mI. portions assayed for radioactivity. -e-e-, alp-labeled sRNA; -0-0-, 3R-Iabeled VA-RNA. Tube no. 61 contains material from the top of the sucrose gradient.

were exposed to [l4C]uridine for two hours. Actinomycin D (5 p.gfml.) was then added and the incubation continued for an additional two hours. Radioactive RNA was separately prepared from infected cells incubated with [3H]uridine for two hours beginning 18 hours after virus inoculation, but not exposed to actinomycin D. The differentially labeled RNA's from each KB cell culture were mixed and chromatographed on one MAK column. Inspection of the chromatogram (Fig. 7) shows that VA-RNA did not undergo any significant change in chromatographic characteristics after exposure of the KB cells to actinomycin D. A potent inhibitor of DNA synthesis, deoxyfluorouridine (Salzman & Sebring, 1962), was used to study VA-RNA production after suppression of DNA replication. KB cells were exposed to 2 X 10- 6 M-deoxyfluorouridine (Hoffman-LaRoche) four hours after initiation of virus infection. Incubation was continued for 12 hours and [3H]uridine then added for 40 minutes. RNA was extracted and chromatographed in the usual manner. No significant tritium label was detected in the VA-RNA region of the chromatogram, although the sRNA region contained radioactive material.

4. Discussion The important characteristics of VA-RNA can be summarized as follows. It is an RNA component present in KB cells infected with adenovirus type 2. This component appears coincidentally with the exponential rise in infectious adenovirus titer and with the rapid production of RNA complementary to adenovirus DNA (Rose et al.,

438

REICH, ROSE, FORGET AND WEISSMAN

200r----r-------,-------,50

c

25

~

tl

u

:!

o

50

40 Tube no.

FIG. 7. MAK column chromatography of VA· RNA from adenovirus-infected KB cells exposed to actinomycin D 2 hr after labeling with radioactive RNA precursor. One of two KB cell cultures inoculated 18 hr previously with adenovirus type 2 was exposed to [14C]uridine (0,015 fJ-c/ml.), followed by actinomycin D (5 fJ-g/ml.) for an additional 2 hr. The other culture (control) was exposed for 2 hr to [3H]uridine (0·25 fJ-c/ml.). RNA was prepared from each culture; they were mixed and chromatographed on a MAK column. Assays for radioactivity were per· formed as described in Fig. 2. - 0 - 0 - , [14C]Uridine; -A-A-, [3H]uridine; absorbance at 260 mfJ-.

-e-e-,

1965). Its susceptibility to RNase indicates that it is RNA and may have a significant amount of secondary structure. Although VA-RNA can be detected in the ribosomal cell fraction, it appears predominantly in RNA from ribosomal supernatant fractions. Base analysis reveals significant differences from KB cell ribosomal and sRNA. On the basis of studies with actinomycin D, transcription of DNA into RNA is necessary for VA-RNA production. The inhibition by deoxyfluorouridine of VA-RNA formation indicates that its occurrence during infection requires DNA synthesis. VA-RNA can be distinguished from sRNA by its chromatographic properties on MAl( columns, base composition, sedimentation velocity and behavior on Sephadex G100 columns. Significant quantities of methylated bases and pseudouridine are not present in VA-RNA, and 8.azaguanine is not selectively incorporated into this RNA component. Since a uridine nucleoside was present in digests of VA-RNA, a uridine nucleotide is present at the 3' hydroxyl end of VA-RNA. No uridine nucleoside was present in sRNA digests. RNA with a sedimentation velocity of 4 to 6 s has been produced by degradation in vitro of high molecular weight RNA from mouse plasma cell tumors (Hymer & Kuff, 1964) and from other sources (Spirin, 1963). VA-RNA is not degraded RNA produced during isolation of high molecular weight RNA, since the preparation and chromatography of RNA from mixtures of differentially labeled, non-fractionated infected and uninfected cells revealed VA-RNA only in RNA from infected KB cells. Furthermore, high molecular weight RNA is absent from infected KB cell ribosomal supernatant fractions in which VA-RNA is predominantly found. The possibility that VA-RNA is derived from catabolism, in vivo, of a rapidly labeled RNA precursor is consistent with the present data. VA-RNA has many properties in common with a "5 s" RNA detected in preparations of ribosomes from E. coli (Rosset et al., 1964), the aquatic fungus Blastocladiella

RNA IN ADENOVIRUS -INFE CTED KB CELL S

43 9

emersonii , sea ur chin embryo (Comb et al ., 1965), rat liver, a nd uninfect ed mon olayer cult ures ofKB cells (Galibert et al., 1965). It b ehaves similarly on MAK and Sephadex Gl00 columns; does not cont ain pseudouridine or methylated bases; sediments with a velocity gr eater than sR NA ; and has a uridine nu cleotide at the 3' hydroxyl end. However , VA-RNA is readily detect abl e in non-fracti onated , KB cells infected with adenovirus type 2, and it s production is as sociated with the appearance of newly synt hesized infectious adenovir us 14 hours after virus ino culation. Unlike " 5 s" RNA, it appears predominantly in RNA from ribosomal supe rnat ant fractions. Analysis of its base composition also reveals significant differenc es fr om "5 s " RNA. Although t he relationship of VA -RNA to "5 s " RNA found in uninfected cells is not certain, it is po ssible that VA-RNA represents an in creased production or decreased degradation of an RNA component normally present in KB cells. The time of appearance of VA-RNA and the inhibition of its formation by deoxyfluorouridine suggest that its occurrence is related to processes associated with virus synthesis. Preliminary experiments indicate that an R.NA component similar to VA-RNA has been found in RNA from KB cells infected with adenovirus type 1. Other adenovirus infections of mammalian cells are currently being investigated . The assistance of Dr Patrick Henr y, Miss Carol Meyer and Miss Iris J effries is gratefully acknowledged. The work of one of us (J .A.R.) was carried out in pa rt during the t enure of a Nati onal Cancer Institute special post- doctoral fellowship. Th e elect ron microscope examinat ion and compl ement fixati on tes t ing for ad enovirus associate d particles was performed by Dr M. David Hoggan. REFERENCES At chison, R. W., Cast o, B. C. & Hammond, W. McD. (1965) . Science, 149 , 754. Brown , G. L. (1963). In P roqress in N u cleic Acid Research, ed. by J. N. David son & W. E. Cohn, vol. 2, p. 278. New York: Academi c Press. Cohn, W. E. (1960). J. B ioI. Ohem, 235, 1488. Comb, D. G., Sarkar, N., De Vallet , J. & Pinzino, C. J. (1965) . J. M ol. B ioI. 12, 509. Davidson, J . D. (1958). In L iquid Scintillation Oounting, ed . by C. G. Bell & F. M. Hayes, vol. I , p. 211. New York: Acad emic Press. Eagl e, H. (1959). S cience, 130, 432. Flanagan, J. F. & Ginsberg, H . S. (1964). J. Bact. 87, 977. Galibert, F., Larsen, C. J., Lelong, J . C. & Boiron, M. (1965) . Nature, 207, 1039. Green, M. & Daeseh , G. E. (1961). Vir ology, 13, 169. Hoggan, M. D. (1965). F ed. Proc, 24, 248. Hymer, W. C. & Kuff, E. L. (1964). Biochem, Biophq«. R e8. Oomm., 15, 507. Karon, M., Henry, P., Weissman, S. M. & Meyer, C. (1965). Oancer Re8. 25, 185. Kohler, K. & Odaka, T. (1964). Z. Naturf. 19b, 331. Levintow, L. (1965). Ann. R ev. B iochem. 34, 499. Mandell, J. C. & Hershey, A. O. (1960). Analyt. B iochem, I, 66. Melnick, J. L., Mayor, H. D., Smith, K. O. & Rapp, F. (1965) . J . Bact. 90, 271. Reich, E ., Franklin, R. M., Shatkin, A. J. & Tatum, E. L. (1962) . Pr oc. N at. Acad. Sci., Wa8h. 48, 1238. Rose, J . A., Reich, P. R. & Weissman, S. M. (1965) . Virology, 27, 571. ROBBet, R., Monier, R. & Julien, J . (1964) . B ull. Soc. Ohim. Bioi. 46, 87. Salzman, N. P. & Sebring, E . D. (1962). B iochim. biophy8. A cta, 61, 406. Salzman, N. P., Shatkin, A. J . & Sebring, E. D. (1964). J. M ol. B ioI. 8, 405. Scherrer, R. & Darnell, J. E. (1962). Biochem, Biophq«, R e8. Oomm . 7, 46. Schleich, T. & Goldst ein, J. (1964). Proc. Nat. Acad. Sci., Wash . 52, 774. Spirin, A. S. (1963). In P roqress in Nucleic A cid Research, ed. by J. W. Davidson & W. E. Cohn, vol. I, p. 311. New York: Academic Pre ss. 29