RNA Synthesis in cells infected with herpes simplex virus

J. Mol. Biol. (1970) 52, 247-263

RNA Synthesis in Cells Infected with Herpes Simplex Virus III.?

Absence of Virus-specified Arginyl- and Seryl-tRNA HEp-2 Cells VINCENT L. MORRIS, EDWARD K. WAQNER mu

in Infected

BERNARD ROIZMAN

University of Chicago Department of Microbiology, Chicago, Ill. 60637, U.S.A. (Received 6 October 1969, and in revised form 12 May 1970) Experiments designed to test whether herpes simplex virus specifies its own orginine- or serine-specific transfer RNA in productively infected HEp-2$ cells revealed the following. (i) Co-chromatography on reverse-phase Freon columns failed to show differences between the tRNA extracted from cytoplasm or polyribosomes of infected cells and charged with labeled arginine or serine by means of enzymes prepared from infected cells, and the corresponding aminoacyl-tRNA synthetase prepared from tRNA and enzymes extracted from uninfected cells. (ii) Purified 4 s RNA prepared from extracts of infected cells hybridized with herpes simplex virus DNA. However, the RNA extracted from nuclei of infected cells and excluded from Sephadex GlOO (i.e. >50,000 daltons in molecular weight) precluded the hybridization of 4 s RNA with viral DNA in hybridization competition tests. (iii) Arginine-specific tRNA and serine-specific tRNA extracted from infected cells and purified by the method of Gillam, Blew, Warrington, von Tigerstrom & Tener (1968) failed to hybridize with viral DNA. The experiments indicate that herpes simplex virus does not specify, in HEp-2 cells, amounts of these RNA species detectable by the procedures used in these studies. We have not excluded the possibility that the virus specifies tRNA specific for derivatives of arginine or serine.

1. Introduction We have previously reported that wild (dk-) strains of herpes simplex multiply in permissive human cells but not in non-permissive canine cells (Aurelian & Roizman, 1964,1965; Roizman & Aurelian, 1965). A number of observations suggested that proteins specified by dk- virus in DK cells are either not made or do not perform their function (Spring & Roizman, 1967 ; Spring, Roizman $ Schwartz, 1968 ; Sydiskis t Roizman, 1967,1968) and we were led to consider that translation of viral messenger RNA in DK cells lacks completeness or fidelity. This phenomenon might therefore be related to suppression in bacteria, in which it has been shown that the permissive state is distinguished by altered tRNA’s capable of reading different codons. It has been reported that certain phages either alter the pre-existing tRNA populations or direct the synthesis of their own tRNA (Sueoka & Kano-Sueoka, 1964; Brenner, Kaplan & Stretton, 1966; Kano-Sueoka & Sueoka, 1966; Neidhardt & Earhart, 1966; Smith, t Part II in this series is Wagner & R&man, 1969b. $ Abbreviations used: HEp-2 cells, human epidermoid carcinoma no. 2 cells; NENH, 2-naphthoxyacetyl ester of N-hydroxy succinimide; B-D cellulose, benzoylated DEAE-cellulose. 241

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Abelson, Clark, Goodman 85 Brenner, 1966; Sueoka, Kano-Sueoka $ Gartland, 1966; Wainfan, Srinivason & Borek, 1966; Waters & Novclli, 1967; Chrispeels, Boyd, Williams & Neidhardt, 1968; Daniel, Sarid & Littauer, 1968aJ; Hung & Overby, 1968; Kan, Kano-Sueoka & Sueoka, 1968; Wainfan, 1968; Weiss, Hsu, Foft & Scherberg, 1968; Daniel, Sarid & Littauer, 1969). Subak-Sharpe and co-workers reported that herpes virus specifies a serine and an arginine tRNA in infected cells (Subak-Sharpo Shepherd & Hay, 1966; & Hay, 1965; Subak-Sharpe, Bark et al., 1966; Subak-Sharpe, Hay, Koteles & Keir, 1966) and one possibility was that the virus was unable to produce these tRNA’s in non-permissive cells. We were, however, unable to confirm their findings for infected permissive cells, and in this paper we report) these experiments.

2. Materials and Methods (a) Chemicals DEAE (Cellex D), from Bio-Rad Laboratories, Richmond, Calif., was washed with 0.5 M-sodium hydroxide and rinsed with deionized distilled water. It was then washed in 0.5 M-hydrochloric acid and rinsed with water twice. The final water rinse was continued until the column effluent had a pH above 4.0. NENH was prepared by the procedure of Gillam, Blew, Warrington, von Tigerstrom & Tener (1968); it had a melting point of 146 to 148°C. Gillam et aE (1968) reported a melting point of 146.5 to 147°C. Columns of benzoylated DEAE-cellulose (50.to 100 mesh, Schwarz BioResearch, Orangeburg, N.Y.), were washed with a buffer consisting of 2.0 M-sodium chloride, 0.01 M-magnesium chloride and 0.01 M-sodium acetate adjusted to pH 4.5. The washing wa8 continued until the effluent had an absorbance of less than 0.02 at 260 nm. Yeast RNA (Sigma Chemical Co) was purified first by repeated phenol extraction of solutions containing 5 to 10 mg/ml. in 1 x SSC (0.15 M-sodium chloride, 0.015 M-sodium citrate) until the solution was colorless and then by several extractions with chloroform containing 2% isoamyl alcohol. The purified RNA W&B precipitated with ethanol, collected by centrifugation and redissolved in SSC at a concentration of 4 to 5 mg/ml. (b) Cell culture

media

HEp-2 cells were grown in the minimal essential medium of Eagle (1959) supplemented with 10% calf serum. The medium and calf serum were obtained from Microbiological Associates, Inc., Bethesda, Md. Complete Mixture 199 (Grand Island Biological Co., Grand Island, N.Y.) was supplemented with 1% calf serum (199-1) and was used to maintain virus-infected cells. (c) Cells The HEp-2 cell Bethesda, Md.

line

was

originally

obtained

(d) Virus and infection

from

Microbiological

Associates,

Inc.,

of cells

The MPGX- strain of herpes simplex virus (Roizman & Aurelian, 1965) was used in all experiments. Rapidly growing and dividing cells in uniform monolayers in 10 cm x 40 cm roller bottles (approx. 5 x 1Oe cells) were infected as described by Roizman & Spear (1968) at a multiplicity of 10 to 20 plaque-forming units per cell for 90 min at 37°C in 25 ml. of phosphate-buffered saline (Dulbecco & Vogt, 1954) containing 10% glucose and 1% bovine serum albumin. The residual inoculum was then removed and the cells were incubated in maintenance medium containing 1% dialyzed calf serum at 37%. Elapsed time after infection was calculated from the time of addition of the virus. For preparation of tRNA and aminoacyl-tRNA synthetase, the cells were harvested 6 to 8 hr after infection. At this time viral protein synthesis is proceeding at maximum rate (Sydiskis & Roizman, 1966,1967) and thus a viral-induced tRNA would be expected to be present at its highest ooncentration. The infected cells were rinsed with ice-cold phosphate-buffered saline, and then scraped, pelleted by centrifugation at 1000 g for 3 to 5 min, resuspended in the saline with 8% dimethylsulfoxide and frozen at -70°C until use.

tRNA

IN

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VIRUS-INPECTEI)

(e) Rudioactive

labeling

HEp-2

CELLS

249

oj RX8

Cultures of 5 x 10s HEp-2 cells were incubated, beginning with 1.5 hr post infection in 200 ml. of minimal essential medium supplemented with 1 yc dialyzed calf serum and twice the concentration of essential amino acids specified by Eagle (1959). At 4.5 hr post infection the medium was replaced with fresh supplemented minimal essential medium containing 500 PC of [3H]uridine (spec. act. 20 c/m-mole, Schwarz BioResearch). After 5 more hours, the radioactive medium was removed and the cells were harvested as described above. (f) Nuclear RNA Nuclear RNA was prepared by the method of Pemnan (1966) as described by Wagner & Roizman (19690). Nuclei from infected or from uninfected cells were obtained by suspending cells in ice-cold phosphate-buffered saline containing 0.5% Nonidet P-40 (Borun, Scharff t Robbins, 1967). After incubation for 10 min, the nuclei were collected by low-speed centrifugation. The pellet was disrupted by incubation in high-salt buffer (0.5 m-sodium chloride, 0.05 M-magnesium chloride, 0.01 rvr-Tris-HCl), pH 7.4. Approximately 50 pg of electrophoretically purified DNase (Schwarz BioResearch)/ml. was then added and the mixture stirred vigorously at room temperature until chromatin was no longer visible (usually about 2 min). The solution was adjusted to 0.5% sodium dodecyl sulfate and 0.01 M-ETDA, pH 7.4, and the nuclear RNA isolated by several extractions at 55°C with equal volumes of redistilled phenol. The aqueous phase from the last phenol extraction was extracted twice with chloroform containing l”/b isoamyl alcohol and the nuclear RNA was precipitated by addition of 2 vol. of 95% ethanol. After 1 hr at was collected by centrifugation for 20 min at 27,000 g and 4”C, -20°C the precipitate resuspended in reticulocyte standard buffer containing 50 pg/ml. of electrophoretically purified DNase and incubated at 37°C for 30 min. DNase was removed by chloroform oxtraction, and the RNA was further purified by exclusion from Sephadex GlOO (Pharmacia, Uppsala) equilibrated in 1 x SSC. (g) Preparation

of viral

DNA

and DNB-RNA

hybridization

The isolation of DNA from the MPdlcstrain of herpes simplex virus has been described by Wagner & Roizman (1969a). Cultures of 5 x lo* cells in 10 cm x 40 cm roller bottles were infected and after 24 hr of incubation at 34°C in mixture 199-l%, the cells were shaken off the glass and collected by centrifugation. The cells were then swollen in 10 vol. of deionized water for 1 hr, lysed with 20 strokes of a tight-fitting Dounce homogenizer, and after 5 to 10 see of sonication, centrifuged at 20,000 g for 1 hr. The pellet contained 90 to 95% of the infectious virus. DNA was extracted from the pellet by digestion for 2 to 3 hr at 37°C in 25 to 50 vol. of a solution adjusted to pH 8 and containing 1% sodium dodecyl sulfate, 0.15 M-sodium chloride, 0.05 M-EDTA and lsb heat-treated pronase (Calbiochem, Los Angeles, Calif.). The digestion was followed by repeated extraction with chloroform containing 2% isoamyl alcohol. When no denatured protein could be seen at the chloroform-water interface, the DNA was precipitated with 2 vol. of 95% ethanol and collected by winding on a glass rod. The DNA was redissolved in half the original volume of 0.1 x SSC and digested for 1 hr at 37°C with 50 pg of heat-treated pancreatic RNase/ml. The solution was then adjusted to 1 x SSC and extracted first with redistilled phenol at 55°C and then several times with chloroform containing 2% isoamyl alcohol. The final material was usually precipitated with ethanol, collected by centrifugation and redissolved in 0.1 x SSC at a final concentration of about 100 rg/ml. The DNA obtained in this manner contained 40 to 0% viral DNA as determined by isopycnic centrifugation in C&l or by fractionation on methylated albumin-kieselguhr columns (Sueoka & Cheng, 1962) using a 0.2 M-sodium chloride gradient. Viral DNA was eluted from methylated albumin-kieselguhr columns at 0.37 to 0.4 M-sodium chloride -a concentration similar to that required to elute equine abortion (herpes) virus DNA (O’Callaghan, Cheevers, Gentry & Randall, 1968). Cellular DNA was eluted with 0.58 Msodium chloride. The DNA used for RNA hybridization was finally purified either from the first isopycnic centrifugation in CsCl solutions or from methylated albumin-kieselguhr chromatography followed by another isopycnic centrifugation in CsCl solution. Viral

250

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E. K.

WAGNER

ANI)

13. ROIZMAN

DNA collected after centrifugation had a buoyant density of 1.72 g/orn, wharoas cell&l DNA bands at 1.68 g/cm. The viral DNA was dialyzed 48 to 60 hr against 4 to 5 changes of 1000 vol. of 0.1 X SSC at 4”C, diluted to 10 to 20 pg/ml., and stored at -2O’C. Hybridization of tRNA to herpay virus DNA was carried out by two methods : (1) using denatured DNA in solution and (2) using denatured DNA immobilized on membrane filters. Experiments were carried out using radioactive whole cell RNA isolated from herpes virus-infected cells in a manner similar to that described for nuclear RNA (section (f)) to determine optimal conditions for hybridization. It was found that by using either of the two methods described below, the amount of RNasu-resistant RNA bound to the viral DNA reached a plateau love1 after 18 to 20 hr of incubation at the tomporaturo indicated. This level remained constant for approximately 12 hr and than began to decline. For hybridization in solution, 72°C was chosen bcrcauso (i) Giacomoni Cy: Spiegelman (1962) using hybridization in solution, found that DN&tRNA hybridization lovolcd off between 70 and 75°C and thus adopted 72°C for t,hc:ir hybridization experiments; (ii) Sub&k-Sharpo, Shepherd & Hay (1966) using the proccduro of Ciucomoni & Spicgolman (1962) took 73°C for their hybridization experiments bctwcon infcctcd ccl1 4 s RNA and viral DNA. On the other hand, for hybridization on discs, 67 to 69°C wo,s chosen because (i) Martin (1969) reported that significant t,hermal denaturation between DNA -RNA hybrids (10%) began at 7O”C, (ii) Weiss et al. (1968) using T4 DNA and 35S-labc1(~tl 4 s RNA and Ma,rtin (1969) using polyoma-transformed hamstor cell RNA and Syrian hamster polyoma tumor DNA, both obtained good hybridization at 68”C, and (iii) exporiments in our laboratory indicated that 67 to 69°C was tho optimum tomporuture for hybridization between infected cell 4 s RNA and viral DNA. Method 1. Hybridization of tRNA to denatured DNA in solution was carried out using a modification of the method of Bolle, Epstein, Salser & Ceiduschek (1968). Stoppered test tubes containing 50 pg DNA denatured by boiling for 10 min in 0.1 x SSC, 250 pg of purified yeast RNA and radioactive 4 s RNA from infected cells in 0.5 ml. of 2 x SSC were incubated for 22 hr at 72°C. The reaction mixtures wcro then trcatod with 25 pg through 25-mm of heat-inactivated pancreatic RNase, adjusted to 4 x SSC and filtered type HA Millipore filters (Millipore Corporation, Bedford, Mass.) which had been soaked in 4 x SSC. Experiments with radioactive DNA showed that 95 to lOOo/o of the DNA was retained on the filter disc by this method. The discs were rinsod with 50 ml. of 2 x SSC, IO ml. of 75% ethanol and then dried in an oven at 60°C. Tritium disintegrations were measured in a Packard scintillation spectrophotometer. Background was determined by use of 50 pg of E. coli DNA with the largest amount of radioactive RNA used and was typically less than 0.02% of input radioactivity. Method 2. A modification of the procedure of Gillespie & Spiegelman (1965) described previously (Wagner & Roizman, 1969a,b) was the second method of hybridization. Heat-denatured viral DNA in 4 x SSC was slow-filtered onto 23 mm Millipore HA membrane filters (Millipore Corporation) which had been soaked for 1 to 2 hr in 4 x SSC. The filter discs were dried for 2 to 3 hr in air and then 6 hr at 80°C. The discs were then soaked in 4 x SSC and incubated with the DNA side up for 20 to 22 hr at 67°C in the bottom of glass scintillation vials containing 1 ml. of 4 x SSC with the radioactive RNA being tested for hybridization. The discs were then washed at least four times with lo-ml. amounts of 2xSSC, incubated for 1 hr at room temperature in 5 ml. of 2 x SSC containing heat-treated pancreatic RN&se (50 pg/ml.), rinsed twice with lo-ml. portions of 2 x SSC and then soaked for 2 to 3 hr in 25 to 50 ml. of 2 x SSC. After 15 min in 75% ethanol, the discs were dried in an oven at GO’C. Blank discs or discs loaded in the same fashion with Bacillus subtilis DNA retained less than 0.01% of the input radioactivity after such treatment. Saturation experiments using 3H-labeled unfractionated tRNA prepared from infected cells gave the same maximum level of RNA bound to a given amount of DNA with either method 1 or 2, but saturation was reached with about 20% less input radioactive material with method 2. Hybridization competition was carried out by pre-incubating the DNA-containing discs with the unlabeled competing RNA for 12 hr at 68°C before addition of radioactive RNA (Wagner & Roizman, 1969b).

tRNR

IN

HERPES

VIRUS-INFECTED

HEp-2

CELLS

251

In an experiment designed to estimate the over-all efficiency of hybridization in the presence of excess DNA, RNA complementary to herpes virus DNA was synthesized with E. coEi polymerase according to the procedure of Milanesi, Brody & Geiduschek (1969). It was found that, over a wide range of DNA excess ranging from a ratio of DNA to RNA of 25 to 2500, about 10% of the input RNA hybridized using method 2. This experiment thus indicates that using our hybridization techniques we might be able to detect 10% viral-specific counts out of the radioactive material used in hybridization. It should be pointed out, however, that the efficiency of hybridization for the complementary RNA will be higher than the efficiency for a species of viral arginine-specific tRNA, because the complementary RNA will bind to many sites on the viral DNA, whereas an amino acid-specific tRNA can only bind to a restricted region on the viral DNA genome. Therefore, the efficiency of hybridization of complementary RNA is an upper limit to the efficiency of hybridization of any arginineor serine-specific tRNA. (11) preparation

of 4 s RNA

4 s RNA was prepared from infected or uninfected cells stored m described in section (d), by a method adapted from Rosenbaum & Brown (1961). Each gram of wet packed cells (approximately 5 x lOa cells) in 1 ml. of phosphate-buffered saline, 8% dimethyl sulfoxide was extracted with an equal volume of redistilled phenol at 4°C. The phases were separated by centrifugation for 10 min at 1000 g and the upper, aqueous phase was removed and extracted 3 more times with phenol. The aqueous phase was then extracted with peroxidefree ether at 4°C. The ether extraction was repeated (usually 3 to 5 times) until the aqueous phase ceased to be cloudy. Residual ether was removed from the aqueous phase by bubbling nitrogen through the solution. The aqueous phase wae then made 1 molar with sodium chloride and the solution was allowed to sit overnight at 0°C. The solution was then centrifuged at 7710 g for 15 min, the pellet containing high molecular weight RNA was discarded, and the RNA preoipitated with 2 vol. of 95% ethanol. The RNA was collectod by centrifugation as described and resuspended in 0.2 m-Tris buffer, pH 8.0. Residual amino acids bound to the tRNA were discharged by incubating for 30 min at 37°C. with The solution was then made 0.4 M in sodium chloride and the RNA was precipitated ethanol and redissolved in the desired buffer. For hybridization experiments the RNA preparation was digested for 30 min at 37°C with 25 to 50 pg of electrophoretically purified DNaae (Schwarz BioResearoh)/ml. of 10 x reticulocyte standard buffer to remove residual low molecular weight DNA fragments ; the DNase was removed by chloroform extraction. It was shown by chromatography on Sephadex GlOO columns (Fig. 5, Results section) that RNA prepared in the above described manner contained mostly 4 s RNA as well as some low molecular weight material ; large RNA molecules were not generally found. 4 s RNA was also prepared from polyribosomes which were isolated by a modification of the procedure of Soeiro & Amos (1966) as described by Wagner & Roizman (19695). Fresh-packed cells were suspended in reticulocyte standard buffer (Warner, Knopf & Rich, 1963) containing 0.5% Nonidet P-40 and stored for 15 min at 4°C. The debris and nuclei were spun out at 1000 g. The supernatant fluid was then adjusted to contain 5% w/w of sucrose and layered over a 50”/0 sucrose pad in a centrifuge tube. The tube was centrifuged at 195,000 g for 3 hr. The 4 s RNA was then extracted from the pelleted polyribosomes as described. The yield of 4 s RNA from polyribosomes was 5% of that obtained from whole cells. (i) Isolation

of aminoacyl synth,etase and charging of transfer RNA with amino a&d8

Aminoacyl-tRNA synthetase was extracted by a procedure adapted from Stephenson & Zamecnik (1961) and Hoskinson & Khorana (1965). Frozen cells were thawed and centrifuged at 6780 g for 20 mm and the supernatant fraction saved. The pelleted cells were resuspended in the enzyme buffer, O+OOl M-EDTA, 0.002 M-magnesium chloride, 0.001 M-dithiothreitol, 0.05 M-Tris-HCl, pH 7.2. The cells were then incubated at 4°C for 10 min, lysed by 20 strokes with a tight-fitting Dounce homogenizer and centrifuged at 6780 g for 20 min. This supernatant fraction was pooled with the supernatant fraction from the frozen cells and centrifuged at 160,000 g for 2 hr. A saturated ammonium sulfate

252

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Ii.

WAGNER

AND

U. HOlZMAN

solution was added at 4°C to the pooled supernatant fraction to yiold an 80(x, saturatctl solution. After 10 min at 4’C, the solution was centrifuged at 6780 g for 20 min. The pollct was resuspended in the enzyme buffer. The preparation was then put through a 1.5 cm >: 30 cm Sephadex G150 column equilibrated with the enzyme buffer, and the clucnt fractions with high absorbance at 280 nm were pooled. The procedures used for charging tRNA with amino acids were adapted from Holly et ~1. (1961). 4 s RNA (section (h)) or purified amino acid-specific tRNA (section (k)) were charged with arginine or serine by one of three charging procedures. Procedure (1). RNA was incubated at 37°C in 1 ml. of solution, containing, per mg of tRNA, O-064 m-mole magnesium chloride, 2 ,XI~O~CSEDTA, 12 pmoles ATP, 1.2 pmolcs CTP, 0.1 pmole amino acids, 0.08 m-mole Tris, and a predetermined optimum amount of enzyme in 1 ml. total volume with a final pH of 7.2. After 20 min of incubation at 37”C, the charging mixture was diluted so as to contain 0.01 M-sodium acetate, 0.01 M-Inagnesium chloride, 0.003 M-p-mercaptoethanol, 0.001 M-EDTA and 0.25 M-sodium chloride. This mixture was then applied to a DEAE-cellulose column (0.9 cm x 30 cm) and the protein was rinsed off with this buffer. The aminoacyl-tRNA was eluted by rinsing tho column with the same buffer but containing 0.8, M-sodium chloride. The recovery of aminoacyltRNA from the DEAE-cellulose column was lOOo/,. The optimum love1 of activating enzyme was determined by incubating different concentrations of cnzymo with constant amounts of RNA. Procedure 2 was identical with that described above, except that the charging mixturo was adjusted to pH 7.5 and contained, per mg RNA: 0.36 m-mole magnesium chloride (final concentration), 6.0 pnoles EDTA, 0.025 m-mole ATP, 2*5prnoles CTP, 0.1 pmole amino acid, 0.34 m-mole Tris and an optimum amount of enzyme in 1 ml. (final volume). Procedure 3 was also identical with charging procedure 1, except that the charging mixture contained, per mg tRNA: 0.36 m-mole magnesium chloride (final concentration), 2 pmoles EDTA, 0.12 m-mole ATP, 1.2 pmoles amino acid, 0.08 m-mole Tris, and an optimum amount of enzyme in 1 ml. (final volume). (j) Reverse-phase

Freon chromatography of arginylfrom infected and uninfected cells

and seryl-tRKA

Reverse-phase chromatography was done by the method of Weiss Bc Kelmcrs (1967). Tricaprylylmethylammonium chloride, 12.5 ml. (Aliquat 336, General Mills, Inc., Kankakee, Ill.) was dissolved in 250 ml. t~ctrachlorotetrafluoropropane (Freon 214, E. I. du Pont de Nemours & Company, Wilmington, Del.). The solution was extracted once with 2 vol. of 1 M-sodium hydroxide, once with 2 vol. of 1 M-hydrochloric acid and twice with 2 vol. of 0.5 M-sodium chloride. The organic phase was thon filtered through no. 1 Whatman filter paper. The organic phase (56 ml.) was added dropwise to 100 g of acid-washed dimethyldichlorosilane-treated Chromosorb W, 100-120 mesh (Johns-Manville Products Corporation, New York, N.Y.). The coated Chromosorb W was then agitated for 1 hr and allowed to sit overnight to ensure even distribution of the organic phase over tho Chromosorb W. A 0.9 cm x 150 cm column was then packed under 10 lb./sq. in. with the coated Chromosorb W suspended in a buffer containing 0.2 M-sodium chloride, 0.01 M-magnesium chloride, 0.003 M-fl-mercaptoethanol, 0.001 M-EDNA, 0.01 M-sodium acetate, pH 4.0. The column was loaded and run under the same pressure (10 lb&q. in.) at 6°C with 0.1 to 0.5 mg radioactive arginylor scryl-tRNA at a concentration of 4 to 9 pg/ml. in the same buffer. Aminoacyl-tRNA was olutcd with a linoar gradient of 0.2 to 0.8 M-sodium chloride with a total volume of 1200 ml. Tho amounts and specific radioactivity of the arginylor seryl-tRNA are shown in the legends to the appropriate Figures. The recovery of input radioactivity as trichloroacetic acid-prccipitable matorial was not less than 75% and usually better than 85%. (k) Preparation

of 3H-labeled

m&r&mund se&e-specijic of high specific radioactivity

transfer

RNA

Tritiated uridine-labeled arginine and serine-specific tRNA of high specific activity were prepared by the method of Gillam et al. (1968) from RNA extracted from cells incubated with [3H]uridine from 4.5 to 9.5 hr after infection. The procedures for argininespecific tRNA are outlined below.

tRNA

IN

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253

The buffers used for the elution of RNA from the B-D cellulose columns contained 0.01 Msodium acetate and 0.01 M-magnesium chloride adjusted to pH 4.5 with acetic acid, and the following: buffer 1, O-3 M-sodium chloride; buffer 2, 1.0 M-sodium chloride; buffer 3, 1.0 M-sodium chloride and 4.7 (v/v) ethanol; buffer 4, 1.0 M-sodium chloride and 19% (V/V) ethanol. The acylation mixture consisted of 2.5 mg NENH in 0.1 ml. tetrahydrofuran. For serine-specific tRNA isolation, the buffers also contained 0.02 M-merCaptoethanol. A sample (0.05 ml.) of acylation mixture was added to 4 mg of 4 s RNA in 1 ml. of 0.1 M-triethanolamine and 0.01 M-magnesium sulfate (pH 4.0) at 0°C. The pH was raised to 8 with 1 M-sodium hydroxide and the mixture was stirred for 5 min at 0°C. At this time the remaining 0.05 ml. of acylation mixture was added and the solution incubated for 5 more minutes at 0°C. The pH was adjusted to 4.0; then the RNA was precipitated with 2 vol. of ethanol, and collected by centrifugation. After washing the precipitate twice with 95% ethanol, the RNA was resuspended in buffer 1. This procedure (sham acylation) was carried out to remove any radioactive material which would stick to the B-D cellulose columns and be co-eluted with naphthoxacetyl-aminoacyl-tRNA. The sham-acylated RNA was then applied to a B-D cellulose (0.9 cm X 28 cm) column, the column was washed with buffer 1, and the tRNA was eluted with buffer 2. The fractions containing RNA were pooled and the sodium chloride concentration was adjusted to 0.3 M by diluting the material with O-01 M-sodium acetate and 0.01 M-magnesium chloride (pH 4.0). This solution was then applied to a 0.9 cm x 15 cm DEAE-cellulose The tRNA was eluted with buffer 2, precipitated with 2 vol. ethanol, and column. collected by centrifugation. It was then charged with [14C]arginine (procedure (1)) or [14C]serine (procedure (3)) and precipitated with ethanol. The W-labeled aminoacyltRNA was then redissolved in the triethanolamine buffer, pH 4.0, and the entire procedure of acylation with NENH as described above was repeated. The naphthoxyacetyl14C-labeled aminoacyl-tRNA in buffer 1 was applied to a second B-D cellulose column (0.9 cm x 28 cm). The column was washed with buffers 1, 2, 3 and naphthoxyacetyl14C-labeled aminoacyl-tRNA was eluted with buffer 4. For the serine-specific tRNA isolation, the wash with buffer 3 was not used. The fractions containing naphthoxyacetyl14C-labeled aminoacyl-tRNA were pooled and diluted fivefold with 0.01 M-sodium acetate, O-01 M-magnesium chloride (pH 4*0), and re-applied to a third B-D cellulose column (0.9 cm x 10 cm). The column was washed with only buffers 1 and 2, and naphthoxyacetyl-14C-labeled aminoacyl-tRNA was eluted with buffer 4. This process was repeated once more and the naphthoxyacetyl-14C-labeled aminoacyl-tRNA was concentrated by use of a DEAE-cellulose column aa described. The naphthoxyacetyl-14C-labeled aminoacyl[3H]uridine tRNA W&S eluted from the DEAE-cellulose column by 1 M-sodium chloride, 0.01 M-magnesium chloride, and 0.01 ivr-Tris, pH 3.8. The pH of this buffer was adjusted to 9.0 with 0.5 M-sodium hydroxide and the naphthoxyacetyl-14C-labeled amino acid was then removed from the tRNA by incubation for 1.5 hr at room temperature. The tRNA specific for either arginine or serine was then recovered by precipitation with 2 vol. of et’hanol as described before.

3. Results (a) Chromatography

of argkyl-

and seryl-tRNA

from infected a,nd uninfected

cells

(i) Argkyl-tRNA The purpose of these experiments was to determine whether arginyl-t,RNA’s extracted from infected and uninfected cells and charged with arginine differ with respect to the pattern of elution from reverse-phase Freon columns. Three series of experiments were done. In the first series, conditions for charging of 4 s RNA with arginyl-tRNA synthetase were studied. With charging procedure 2 (see Materials and Methods), arginyl-tRNA res.ched a maximum level after 15 minutes at 37°C and remained unaltered for at least 15 additional minutes, showing that little or no RNase was present. The hydrogen ion concentration was varied because enzyme activity is often very dependent on pH,

264

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60 50

’

’ 20

’

’ 40

’

’ 60

’

’ 80

Ratio Mg/ATP I

I

7060So

6.2

I

I 6.6

I

I 7.0

I

1 7.4

1

pH of charging buffer

FIQ. 1. Effect of pH and ratio of magnesium ion to ATP on the amount of [3H]arginine bound to infected cell tRNA. This was measured by charging infected cell tRNA as described in Materials and Methods (charging procedure (1)) and altering the pH or magnesium ion to ATP ratio as indicated.

and the ratio magnesium ion : ATP was varied because it has been reported that the activity of some tRNA synthetsses from mammalian cells is very sensitive to changes in this ratio (Yang & Novelli, 1968). Figure 1 shows that the arginyl-tRNA synthetase extracted from infected HEp-2 cells has broad optima for pH and for the ratio magnesium ion : ATP, and the experiments discussed below indicate that changes in the pH and in the ratio magnesium ion : ATP do not alter the populations of tRNA charged with arginyl-tRNA synthetase. The second series of experiments dealt with chromatography on reverse-phase Freon columns of arginyl-tRNA extracted from whole cells. 4 s RNA extracted from infected and uninfected cells was charged with [3H]- and [14C]arginine, respectively, mixed and co-chromatographed. The elution profile obtained in one experiment in which the 4 s RNA’s were charged with the enzyme extracted from infected cells is shown in Figure 2. In other experiments, we either reversed labels or charged the 4 s RNA with enzymes extracted from uninfected cells. The chromatogram of arginyltRNA extracted from HEp-2 cells shows two peaks and a shoulder preceding the first peak. In none of the experiments performed in this study have we found differences in the elution profiles of the arginyl-tRNA extracted from infected and uninfected cells as determined from an inspection of 3H/14C ratios and from the relative amounts of arginyl-tRNA eluted in the first and second peaks (Table 1). Arginyl-tRNA extracted from polyribosomes of infected cells was studied in the third series of experiments. Previous work from this laboratory (Wagner & Roizman, 1969a) established that the half-life of tRNA is longer than the reproductive cycle of herpes simplex virus. It could therefore be argued that if the virus specifies only a small fraction of the total arginyl-tRNA and the acceptor function of host

tRNA

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CELLS

265

500

200

O

Fraction no.

FIG. 2. Reverse-phase Freon chromatography of whole-cell infected and uninfected arginyltRNA. 4 8 RNA was extracted from infected and uninfected cells as described in Materials and Methods (section (h)). Infected 4 s RNA (0.1 mg) was charged with [“Hlarginine (spec. act. 1.3 c/m-mole) and uninfected 4 s RNA (O-25 mg) with [“Clarginine (spec. act. 220 me/m-mole) (charging procedure (2)). The infected and uninfected arginyl-tRNA’s were then co-chromatographed on reverse-phase Freon columns aa described (Materials and Methods, section (j)). [“Cjarginyl-tRNA from infected cells. (- - -) [3H]Arginyl-tRNA from infected cells; (-)

arginyl-tRNA is not destroyed after infection, any viral tRNA might be obscured. However, polyribosomes extracted from infected cells should contain enriched amounts of a virus-specific arginyl-tRNA. In one experiment, tRNA was extracted from polyribosomes of six-hour infected cells, charged with rH]arginine with infected cell enzyme, and co-chromatographed with [14C]arginyl-tRNA from uninfected cells charged in the same way (Fig. 3). Again, as shown in Figure 3 and Table 1, no differences were found in the elution profiles of arginyl-tRNA extracted from polyribosomes of infected cells and that of uninfected cells. We repeated the chromatography of 4 s RNA extracted from polyribosomes but charged in two different ways, using procedure 1 (pH 7.2 and Mg2+/ATP= 5.3) and TABLE

1

Effect of charging enzyme on the ratio of area peak 1 :peak 2 joor arginyl-tRNA Uninfected cell tRNA tRNA charged with uninfected cell enzyme

0.66 0.44 0.63

tRNA charged with infected cell enzyme

0.56

Average

0.66 0.63

Infected cell tRNA

Average

0.51

0.51

043t 0.65t 0.86

0.69

0.61 0.56

tRNA was extracted, charged with arginine and run on reverse-phase described in Materials and Methods, section (h), (i), (j) and the text. t tRNA isolated from polyribosomes. 17

Freon

columns

as

256

V. L.

MORRIS,

E. K.

WAGNER

AND

B. ROIZMAN

Fraction no.

FIG. 3. Reverse-phase Freon chromatography of arginyl-tRNA extracted from polyribosomes of infected cells. Infected cell of 4 s RNA was extracted from polyribosomes as described; uninfected cell 4 s RNA was extracted from whole cells (Materials and Methods, section (h)). Infected cell 4 s RNA from polyribosomes (0.2 mg) was charged with [3H]arginine (spec. act. 19 c/m-mole) using infected cell arginyl-tRNA synthetase (charging procedure (1)). Uninfected cell 4 s RNA (0.1 mg) was charged with [‘%]arginine (spec. act. 220 me/m-mole) by uninfected cell arginyl-tRNA synthetase (procedure (2)). The infected and uninfected arginyl-tRNA’s were then co-chromatographed on reverse-phase Freon columns as described (Materials and Methods, section (j)). (- - - -) r3H]Arginyl-tRNA from polyribosomes of infected cells; (-----) [r%]arginyl-tRNA from uninfected cells.

also procedure 2 (pH 7.5 and Mg2+/ATP = 14.4). The two elution profiles of the arginyl-tRNA were indistinguishable from the patterns shown in Figure 3. (ii) Seryl-tRNA Experiments similar to those described above for arginyl-tRNA were carried out on seryl-tRNA from infected and uninfected cells. It was found that the magnesium ion, pH and ATP concentration for optimal charging of tRNA with serine was independent of whether infected or uninfected cells were used as sources of tRNA and aminoacyl-tRNA synthetase. Chromatography of seryl-tRNA on reverse-phase Freon columns showed one peak of seryl-tRNA in both infected and uninfected cells (Fig. 4). Charging of tRNA from infected cells with serine with aminoacyl-tRNA synthetase from uninfected cells produced no changes in chromatographic properties. We conclude from this series of experiments that in HEp-2 cells productively infected with herpes simplex virus : (i) new species of arginyl-tRNA or seryl-tRNA with different chromatographic behavior on reverse-phase columns do not arise ; and (ii) the population of pre-existing species does not become substantially altered. (b) Hybridization of 4 S RNA from infected cells with viral DNA The purpose of these experiments was to determine whether purified 4 s RNA contains RNA complementary to viral DNA and to describe the nature of this RNA. Two series of experiments were done.

tRNA

IN

HERPES

VIRUS-INFECTED

HEp-2

2000 .c F 2-2 .I .c

267

1000 p-5 : : : I ,--ir- ’ : J

I

loo:

CELLS

, ,

,-

: : -...,-.

so0

,

,

.f E ::” 9-6(I) .z

o

‘. 50

150

200

Fraction no. FIG. 4. Roversc-phase Freon chromatography of wholo-cell infcctcd and uninfected seryltRNA. 4 s RNA was extracted from infected and uninfected cells (Materials and Methods, section (h)). Infected 4 s RNA (0.07 mg) was charged with [3H]serine (speo. act. 3.7 c/m-mole) and uninfected 4 s RNA (0.7 mg) was charged with [r4C]sorine (spot. act. 112 me/m-mole). In each case the corresponding aminoacyl-tRNA synthetase was used (charging procedure (2)). The infected and uninfected seryl-tRNA’s were then co-chromatographed on reverse-phase Freon columns as described (Materials and Methods, section (j)). (- - -) [“HJArginyl-tRNA from infected cells; (-) [14C]seryl-tRNA from uninfected cells.

In the first series of experiment,s 4 x 10s HEp-2 cells were labeled with [3H]uridine from three to six hours after infection. The RNA was extracted, digested with DNase, precipitated with ethanol and dissolved in 1 x SSC. This RNA bands on acrylamide gel electrophoresis in a position characteristic of 4 s RNA. 4 s RNA was loaded on a 1 cm x 80 cm Sephadex GIOO column and eluted in l-ml. fractions (Fig. 5). The fractions indicated in Figure 5 as containing 4 s RNA were pooled, precipitated with ethanol and dissolved in 2 ml. of 2 x SSC. The final yield was 220 pg of purified 4 s RNA with a specific activity of 9000 cts/min/pg. Various amounts of this purified 4 s RNA were then hybridized with 50 yg of viral DNA by method 1 (see Materials and Methods, section (g)). In Figure G the amount of hybridized RNase-resistant radioactive material bound to the DNA is plotted as a function of the RNA added. At high concentrations of 4 s RNA, the level approaches saturation, and the saturation level, as estimated from a double reciprocal plot (Fig. 7), was 220 f 50 cts/min of [3H]uridine in purified 4 s RNA per 50 tug of viral DNA. On the basis of a specific radioactivity of 9000 cts/min/pg for the purified 4 s RNA, it can be oaIculated that approximately 2 mobcules, 25,000 daltons each, can anneal t,o a viral DNA molecule of approximately IO8 daltons (Becker, Dym & Sarov, 1968)t. This should, however, be regarded as an upper estimate, since the purified 4 s RNA is not uniformly labeled; very likely it contains unlabeled cellular 4 s RNA made prior to infection. The purpose of the second series of experiments was to determine whether RNA excluded from a 1 cm x 80 cm Sephadcx GlOO column (i.e. >50,000 daltons in molecular weight) competes with purified 4 s RNA for hybridization with viral DNA. t 1 pg of viral DNA is equivalent to 10-s pmole; 10-s pmole of 4 s RNA of 25,000 daltons is approximately 2.5 x 10s4 pg. 220 cts/min of RNA of specific radioactivity of 9000 cta/min/pg bound to 50 rg of viral DNA is equivalent to 5 x 10e4 fig of 4 s RNA annealing to 1 pg of viral DNA or 2 pmoles of RNA of 25,000 daltons annealing to one mole of viral DNA.

258

V. L.

MORRIS,

E.

K.

WAGNER

AND

B. ROIZMAN

Fraction no. FIG. 5. Fractionation of radioactive 4 s RNA from infected HEp-2 cells on Sephadex GIOO. 4 s RNA extracted (summary, section (i)) from infected HEp-2 cells was labeled from 4.5 to 9.5 hr after infection. This RNA was then digested with DNase and loaded onto a 1 cm x 80 cm Sephadex GlOO column in 1 x SSC. Fractions (1 ml.) were taken and assayed for sH radioactivity (- - 0 - - 0 - -) and absorbance at 260 nm (-a-@-). The fractions indicated were pooled and precipitated as purified 4 s RNA. A solution of 1 y0 Blue Dextran 2000 (Pharmacia) and 1 oh potassium ferricyanide was used to mark the exclusion volume (I’,) and total inolusion volume (V,) in a separate experiment with the same column.

The competing unlabeled RNA was extracted from the nuclei of five-hour infected cells. The 4 s RNA was prepared from HEp-2 cells labeled with [3H]uridine from 45 to 9.5 hours after infection. This RNA had an estimated minimum specific activity of 10,000 cts/min/pg based on a.mino acid acceptor activity. The results, summarized in Table 2, show that RNA extracted from infected cell nuclei a.nd excluded from a

pg RNA added FIG. 6. Hybridization of purified 4 s RNA (speo. act. 9000 cts/min/pg) extracted from infected HEp-2 cells with herpes simplex virus DNA. Various amounts of the purified 4 s RNA were hybridized with 50 ag of herpes DNA by method 1 (Materials and Methods, section (g)). The range and average amount of RN&se-resistant radioactivity bound in two separate tubes was plotted as a function of the RNA added to the DNA.

tRNA

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CELLS

259

0 (pg RNA added)-’ FIG. 7. Estimation of maximum amount of C3H]RNA bound at saturation. The data shown in Fig. 6 wore replotted in a double reciprocal plot. The Figure shows that 220 f 50 ots/min (0.047,t 0.008) would be bound at saturation.

TABLE 2 Hybridization

competition between injected cell 4 8 RNA and injected cell nuclear RNA

RNA pm-incubated for 12 hr with 20 pg viral DNA

3.6 mg yeast RNA 3.4 mg infected nuclear

4 s RNA cts/min incubated for 20 hr with DNA

233,000 233,000

Net RNese-resistant counts hybridized to 20 pg viral DNA 123 0

RNA 4 s RNA and nuclear RNA were prepared as described in Materials and Methods, sections (f) and (h); DNA extraction and hybridization competition were carried out as described in Materials and Methods, section (g).

Sephadcx GlOO column effectively competes with the crude 4 s RNA in hybridization tests carried out as described (see Materials and Methods, section (g), hybridization procedure 2). These experiments confirm and extend the finding of Subak-Sharpe, Shepherd & Hay (1966), that there is 4 s RNA which can hybridize with viral DNA, and show that viral DNA contains approximately two regions complementary to RNA molecules of 25,000 daltons molecular weight found in infected cells. However, we find that this 4 s RNA annealing to viral DNA has nucleotide sequences identical with those of RNA molecules greater than 50,000 daltons in molecular weight. (c) Attempts to hybridize arginine- and se&e-specific transfer RNA from injected cells with viral DNA (i) Arginine-speci$c tRNA Arginine-specific tRNA was purified from 4 s RNA extracted from 9.5-hour infected cells labeled with [3H]uridine for five hours prior to extraction. The specific activity of the purified arginine-specific tRNA was calculated to be 19,000 cts/min/pg; the

260

V. L.

MORRIS,

E.

K.

WAGNER

AND

B. ROIZMAN

calculations are based on the recovery of 22,000 cts/min of [14C]arginine bound to 49,000 cts/min of [3H]uridine-labeled tRNA which is assumed to have a molecular weight of 25,000 daltons. The hybridization test was done according to method 2 (see Materials and Methods, section (g)). Approximately 46,000 cts/min (2.3 pg) of discharged [3H]uridine-labeled tRNA was incubated in two vials, each vial containing one blank disc and one disc with 20 pg of heat-denatured viral DNA. As in previous hybridization tests, the blank discs contained only background amounts of ribonuclease-resistant counts. The two discs containing viral DNA bound only a total of 2 cts/min above the background. To determine that the viral DNA was capable of binding RNA, and moreover, the starting material had RNA complementary to viral DNA, the discs were removed from the toluene-based scintillation counting mixture, rinsed several times with 75O/, ethanol and soaked overnight in 4 x SSC at 4°C. Each disc was then incubated with 490,000 ct,s/min of the 4 s starting material from which the arginine-specific tRNA was cxtracted and purified. In this test the 40 pg of viral DNA bound a total of 700 cts/min of ribonuclease-resi&nt [3H]uridine-labeled RNA. Assuming the DNA was saturated and that the specific activity of 4 s RNA labeled between 4.5 and 9.5 hours after infection was the same as that of tRNA specific for arginine isolated from it, it could be calculated that the saturation level in the experiment was about 8 x 10m4 pg of 4 s RNA per pg of viral DNA-a result similar to that obtained in the preceding section. (ii) Se&e-speci$c tRNA Serine-specific tRNA was purified from 4 s RNA in a manner identical to that described for arginine-specific tRNA from cells harvested 9.5 hours after infection and labeled with [3H]uridine for five hours prior to harvesting (see Materials and Methods, section (k)). A final recovery of 29,000 3H cts/min of serine-specific tRNA with a specific radioactivity of 38,000 cts/min/pg was obtained. Hybridization was carried out incubating 29,000 cts/min (0.34 pg) of [3H]serinespecific tRNA in one vial with a blank disc and two discs each bearing 10 pg of denatured DNA (set Materials and Methods, section (g)). Less than 2 cts/min above background bound to the discs. In a second experiment identical to that described for the experiment with [3H]uridine-labeled arginine-specific tRNA, these discs were shown to be able to bind [3H]uridine-labeled 4 s RNA. The significance of the results obtained in these experiments stem from the following considerations. If each viral DNA molecule carries one gene for arginine-specific or serine-specific tRNA, 4Opg (4 x lo-l3 mole) of DNA should at saturation bind 0.01 pg, i.e., 4 x lo-l3 mole of arginine- or serine-specific tRNA of molecular weight 25,000 daltons. In this experiment, at saturation (190 cts/min of arginine-specific tRNA), 40 yg of DNA would be expected to bind 380 cts/min of serine-specific tRNA radioactivity. This amount of radioactivity would have been readily detected above background. In the preceding section, we showed that 50 pg of viral DNA was more than SOY/i, saturated when hybridized with 25 pg of purified 4 s RNA. If the purified 4 s RNA which hybridized was in fact tRNA, and if rarginine-specific tRNA represents 5% of the total tRNA, 1.2 tLg of arginine tRNA should saturate 50 pg of DNA to a level of approximately 800/,. However, no binding was detected when 2.3 pg of purifiecl arginine-specific tRNA was hybridized to 40 pg of DNA. Similarly, if serine-specific tRNA also represents 50/, of the total tRNA population, then approximately 0.5 pg

tRNA

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261

of serine tRNA would saturate 20 cogof viral DNA to about 80% of the maximum value. In the experiments with purified serine tRNA, O-3 pg should have halfsaturated the 20 tug of viral DNA used. We conclude that if there were genes for arginine-specific or serine-specific tRNA in the herpes virus DNA, and if these tRNA’s have the same molecular properties as the viral RNA contained in the purified 4 s RNA, then the transcription products of these genes are present in purified 4 s RNA at a level of only l%, or less than that of any other 4 s viral RNA species. Whereas viral DNA hybridized with 4 s RNA was more than 80% saturated and bound 2 to 3 moles of RNA per mole DNA, the DNA bound only I:,: or less of arginine- and serine-specific tRNA counts expected at saturation.

4. Discussion We may summarize our findings as follows. (i) The chromatographic properties of arginyl-tRNA and seryl-tRNA from infected HEp-2 cells cannot be differentiated from those of the corresponding tRNA’s of uninfected cells. (ii) Approximately two moles of purified 4 s RNA of molecular weight 25,000 daltons extracted from infected cells hybridize with one mole of viral DNA, but the nucleotide sequences annealing are homologous to those found in nuclear RNA of molecular weight 50,000 daltons or higher. (iii) No detectable amount of arginine-specific or serine-specific tRNA could be shown to anneal to viral DNA. We conclude from these experiments that viral arginine- and serine-specific tRNA are not specified in HEp-2 cells ; if they are specified, they are not made in amounts sufficient to be detected. Two questions, however, arise from the results. The first question concerns the significance of the hybridization of purified 4 s RNA from infected HEp-2 cells with viral DNA. Similar results have also been obtained with 4 s RNA extracted from infected BHK cells (Subak-Sharpe & Hay, 1965; Subak-Sharpe, Shepherd & Hay, 1966; Hay et al., 1966), but we have shown that this 4 s RNA shares common nucleotide sequences with larger RNA molecules. It is possible that the RNA which hybridizes is viral tRNA and that larger competing RNA is a tRNA precursor molecule similar to those described for cellular tRNA by Bernhardt & Darnell (1969). This interpretation cannot be rigorously excluded without further study, but the levels of this tRNA precursor would not be expected to be very high. The other, more likely, interpretation is that the RNA which hybridizes with the viral DNA consists of messenger RNA fragments. Weiss et al. (1968) have found viral mRNA fragments in 4 s RNA extracted from E. coli infected with T2 phage, and Wagner & Roizman (1969b) showed that some herpes simplex virus mRNA is derived from a high molecular weight precursor RNA made in the nucleus. It would be expected therefore that mRNA fragments would have nucleotide sequences in common with higher molecular weight RNA extracted from the nucleus. The second question arises from our failure to show virus-specific arginine- and serine-specific tRNA in infected HEp-2 cells, and whether anything could have gone amiss. The experimental design does in fact have a limitation. First, it seems clear that the results are valid for tRNA which can be charged with arginine or serine prior to purification; it could be argued that in spite of all the precautions taken, the virus specifies an activating enzyme which is inactivated during extraction and fails to charge the tRNA’s specified by the virus. The second limitation is based on the fact that we have chromatographed, purified and hybridized only the tRNA which accept’ed arginine and serine. We have not performed similar experiments with

262

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K.

WAGNER

AND

B.

ROIZMAN

arginine or se&e derivatives, and we cannot exclude the possibility specifies tRNA specific for derivatives of these amino acids.

that

t’he virus

These studies were aided by grants from the U.S. Public Health Service (CA 08494), the American Cancer Society (E 314E) and the National Science Foundation (GB 8242). One of us (V. L. M.) was a U.S. Public Health Service Pre-Doctoral Fellow (PHS AI 00238-06). Another author (E. K. W.) is a Helen Hay Whitney Foundation Post-Doctoral Fellow. We wish to thank Dr Oskar Grau, in the laboratory of Dr E. P. Geiduschek, for his aid in the synthesis of RNA complementary to herpes virus-specific DNA. REFERENCES Aurelian, L. & Roizman, B. (1964). F+oZogy, 22, 452. Aurelian, L. & Roizman, B. (1965). J. Mol. Biol. 11, 539. Becker, Y., Dym, H. & Sarov, I. (1968). Virology, 36, 184. Bernhardt, D. & Darnell, J. E. (1969). J. Mol. BioZ. 42, 43. Bolle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (1968). J. Mol. BioZ. 31, 325. Borun, T. W., Scharff, M. D. I% Robbins, E. (1967). Biochim. biophya. Acta, 149, 302. Brenner, S., Kaplan, S. & Stretton, A. 0. W. (1966). J. Mol. BioZ. 19, 574. Chrispeels, M. J., Boyd, R. F., Williams, L. S. & Neidhardt, F. C. (1968). J. Mol. BioZ. 31, 463.

Daniel, V., Sarid, S. & Littauer, U. Z. (1968a). Proc. Nut. Accd Sci., Wash. 60, 1403. Daniel, V., Sarid, S. & Littauer, U. Z. (19685). Israel J. Chem. 6, 94. Daniel, V., &rid, S. & Littauer, U. Z. (1969). Abstract, 6th Meeting, F.E.B.S., Madrid, p. 618. Dulbecco, R. & Vogt, M. (1954). J. Exp. Med. 99, 167. Eagle, H. (1969). Science, 130, 432. Giacomoni, D. & Spiegelman, S. (1962). Science, 138, 1328. Gillam, I., Blew, D., Warrington, R. C., von Tigerstrom, M. & Tener, G. M. (1968). Biochemidry,

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Gillespie, D. & Spiegelman, S. (1965). J. Mol. BioZ. 12, 829. Hay, J., Koteles, G. J. & Keir, H. M. (1966). Nature, 210, 387. Holley, R. W., Apgar, J., Doctor, B. P., Farrow, J., Marini, M. A. & Merrill,

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Hoskinson, R. M. & Khorana, H. G. (1965). J. BioZ. Chem. 240, 2129. Hung, P. P. & Overby, L. R. (1968). J. BioZ. Chem. 243, 5525. Kan, J., Kano-Sueoka, T. & Sueoka, N. (1968). J. BioZ. Chem. 243, 6684. Kano-Sueoka, T. & Sueoka, N. (1966). J. Mol. BioZ. 20, 183. Martin, M. A. (1969). J. Virology, 3, 119. Milanesi, G., Brody, E. N. & Geiduschek, E. P. (1969). Nature, 221, 1014. Neidhardt, F. C. & Earhart, C. F. (1966). Cold Spr. Hurb. Symp. Quunt. BioZ. 31, 657. O’Callaghan, D. J., Cheevers, W., Gentry, G. & Randall, C. C. (1968). Virology, 36, 104. Penman, S. (1966). J. Mol. BioZ. 17, 117. Roizman, B. & Aurelian, L. (1965). J. Mol. BioZ. 11, 528. Roizman, B. & Spear, P. G. (1968). J. Virology, 2, 83. Rosenbaum, M. & Brown, R. A. (1961). Anulyt. Biochem. 2, 15. Smith, J. D., Abelson, J. N., Clark, B. F., Goodman, H. M. & Brenner, S. (1966). Cold Spr. Hurb.

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Sueoka, N. & Cheng, T-Y. (1962). J. Mol. Biol. 4, 161. Sueoka, N. & Kano-Sueoka, T. (1964). Proc. Nut. Aced. Sci., B’ush. 52, 1535. Sueoka, N., Kane-Sueoka, T. & Gartland, W. J. (1966). Cold Spr. Hurb. Syrnp. Quant. Biol. 31, 571. Sydiskis, R. J. & Roizman, B. (1966). Science, 153, 76. Sydiskis, R. J. & Roizman, B. (1967). 17iroEogy, 32, 678. Sydiskis, R. J. & Roizman, B. (1968). Virology, 34, 562. Wagner, E. K. & Roizman, B. (1969a). Vi7iroZogy, 4, 36. Wagner, E. K. & Roizman, B. (1969b). Proc. Nat. Acrrd. Sci., Wash. 64, 626. Wainfan, E. (1968). Vi7irology, 35, 282. Wainfan, E., Srinivason, P. R. & Borek, E. (1966). Cold Spr. Harb. Syq. Quant. BioZ. 31, 525. Warner, J. R., Knopf, P. & Rich, A. (1963). Proc. Na,t. Acad. Sci., ?Va.sh. 49, 122. Waters, L. C. & Novelli, G. D. (1967). Proc. Nat. Acud. Sci., Wash. 57, 979. Weiss, S. B., Hsu, W., Foft, J. W. Bs Scherberg, N. H. (1968). Proc. Nat. Acad. Sci., W&t. 61, 114. Weiss, J. F. & Kelmers, A. D. (1967). Biochemistry, 6, 2507. Yang, W. & Novelli, G. D. (1968). Nucleic Acids in 1mmunoZogy, ad. by 0. J. Plescia & \V. Braun, p. 644. h’ew York: Springer-Verlag.