Genome location of polyadenylated transcripts of herpes simplex virus type 1 and type 2 DNA

VIROLOGY

75, 145-154 (1976)

Genome

Location

JOHN Department

of Polyadenylated Transcripts of Herpes Simplex Virus Type 1 and Type 2 DNA

E. OAKES,

of Microbiology,

RICHARD

W. HYMAN,

AND

FRED RAPPl

The Milton S. Hershey Medical Center, The Pennsylvania College of Medicine, Hershey, Pennsylvania 17033

State University,

Accepted July 9,1976 The genome location of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) polyadenylated RNA has been studied by hybridization of herpesvirus-specific nuclear and cytoplasmic polyadenylated RNA to the Eco R, restriction enzyme fragments of herpesvirus DNA, which have been separated by agarose gel electrophoresis and transferred to nitrocellulose paper. For both HSV-1 and HSV-2 DNA, the nuclear and the cytoplasmic polyadenylated RNA are distributed on all the Eco R, fragments of the homologous DNA. In a heterologous system, total polyadenylated RNA from HSV-linfected cells was annealed to the Eco R, fragments of HSV-2 DNA and vice versa. HSV-2 polyadenylated RNA hybridized to all fragments of HSV-1 DNA, and HSV-1 polyadenylated RNA hybridized to all but three of the fragments of HSV-2 DNA. These data indicate that the DNA base sequence homology between the two herpesvirus strains is dispersed throughout the genome and is not located in a single contiguous block. INTRODUCTION

cleavage of the DNA by an appropriate restriction endonuclease, separation of the The major pathway of mRNA synthesis resulting specific DNA fragments by elecin mammalian cells begins with transcriptrophoresis through an agarose gel, denation of the DNA to produce heterogeneous turation of the DNA within the gel, and nuclear RNA and proceeds to the addition quantitative transfer of the denatured of poly(A) to the 3’ end of selected tranDNA fragments to nitrocellulose paper scripts. This event is followed by specific cleavage of the heterogeneous nuclear po- where the DNA is ready for RNA-DNA measurements. Using this lyadenylated RNA and transport of the hybridization technique, transcription of the individual, polyadenylated mRNA to the cytoplasm (Darnell, 1973). Evidence has been pre- specific HSV DNA fragments during productive infection can be detected by hysented which suggests that herpes simplex virus (HSV)-specific RNA is also processed bridization of radiolabeled RNA extracted in the nucleus (Kozak and Roizman, 1974). from HSV-infected cells to a nitrocellulose strip containing the denatured HSV DNA. During processing in the nucleus, approxiIn this communication, we report the use mately 50% of the primary HSV transcripts become polyadenylated. A large of the system described by Southern (1975) to examine the genome location of polyfraction of these polyadenylated transcripts is then transported into the cyto- adenylated transcripts appearing in both the nucleus and cytoplasm of HSV-inplasm (Silverstein et al., 1973). Recently, a new technique has been de- fected cells. In addition, taking advantage of the fact that the DNA of the oral (herpes veloped for the study of transcription (Southern, 1975). This technique uses simplex virus type 1; HSV-1) and venereal (HSV-2) strains of HSV are approximately 1 Author to whom requests for reprints should be 50% homologous (Ludwig et al., 1972; Kieff addressed. et al ., 1972), we tested whether the homol145 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

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8.0). Following centrifugation at 25,000 rpm for 2.25 hr in a Beckman SW 27 rotor, the virus band was identified by its characteristic light scattering and was removed and dialyzed against 0.01 M Tris and 0.001 M EDTA (pH 7.5). After dialysis, the virus preparation was treated MATERIALS AND METHODS with 50 pg/ml of RNase A (Sigma ChemiCells and virus. Vero cells, obtained cal Co.) and 80 pg/ml RNase T, (Worfrom Flow Laboratories, were maintained thington Biochemical Corp.) for 1 hr at 37”. Sodium dodecyl sulfate (SDS) (Bio-Rad as roller cultures in Bellco disposable Labs, electrophoretic grade) was added to roller bottles in medium 199 supplemented a final concentration of 0.5%. Previously with antibiotics, 10% fetal calf serum pronase (Calbiochem) was (FCS), and NaHC03. HSV-1 (strain Pat- autodigested ton) and HSV-2 (strain 333) available in added to 500 pg/ml, and the solution was incubated at 37” for an additional hour. this laboratory were plaque-purified three times in Vero cells. Virus stocks were pre- The HSV DNA was extracted by gentle rocking with an equal volume of freshly pared from plaque-purified virus by infectredistilled phenol (buffer saturated in 0.1 ing Vero cell monolayers at a multiplicity of 0.1 plaque forming unit (PFU) per cell. M Tris, pH 7.6) for 15 min. Following centrifugation, the aqueous phase was reVirus was harvested from cells 28 hr later moved and extracted a second time with by three cycles of freezing and thawing. The supernatant was clarified and assayed an equal volume of phenol. The aqueous for PFU in primary rabbit kidney cells as phase was then extracted with chloroform-isoamyl alcohol (24:1, v/v). The DNA previously described (Rapp, 1963). Purification of virus and extraction of was precipitated with 2.5 volumes of NaClDNA. The following procedures have been saturated 95% ethanol, redissolved in 0.01 derived, in part, from the work of Spear M Tris (pH 7.9), and measured for concen(uv) aband Roizman (1972) and of Gibson and tration by reading the ultraviolet sorbance of the solution. Roizman (1972). Endonuclease digestion and gel electroVero cell monolayers were infected at a phoresis. Eco Ri restriction endonuclease multiplicity of 1 PFU per cell. Eighteen hours later, infected cells were scraped off was purchased from Miles Laboratories, Inc. The digestion mixtures contained apthe surface, collected by centrifugation, proximately 10 pg of HSV DNA and 100 and washed twice in phosphate buffered saline (PBS). The infected cells were re- units Eco R, in 100 ~1 of 0.10 M Tris (pH 7.5), 0.005 M MgC12, and 0.050 M NaCl. suspended in lysis buffer (0.16 M KCl, 0.1% Triton X-100, 0.01 M Tris, and 0.02 M The digestion was allowed to proceed for 12 MgC12, pH 8.3) and allowed to stand for 5 min at 37”, and was then terminated by addition of 5 ,ul of 0.1 M EDTA (pH 8.0). min at 4”. This procedure lyses cytoplasElectrophoretic separation of the HSV mic membranes, thus releasing the cytoDNA fragments generated by Eco R, was plasm into the buffer, but leaves nuclear performed by the procedure of Sharp et al. membranes intact. Intact nuclei (containStock againg the host cell DNA) were removed by (1973) with slight modification. centrifugation at 2000 rpm for 10 min in a rose was prepared by dissolving agarose (0.5% w/v) (Bio-Rad Labs, electrophoretic Damon/IEC HN-S centrifuge. The supernatant was clarified by centrifugation at grade) in 0.04 M Tris (pH 7.9), 0.005 M 10,000 rpm for 10 min in a Sorvall SS34 sodium acetate, and 0.001 M EDTA (E buffer) (Sharp et al., 1973). The solution rotor. The clarified crude virus preparation was layered onto a preformed 28-ml was autoclaved and cooled to room temperlinear CsCl gradient (density 1.14-1.34 g/ ature. Gels were prepared by melting ml) containing 10% sucrose and 0.15 M stock agarose solutions and pouring the KCl, 0.10 M Tris, and 0.02 M MgCl, (pH melted agarose into acid-cleaned slab gel

ogous DNA sequences are grouped or dispersed by challenging the specific DNA fragments of HSV-1 with the polyadenylated RNA transcripts of HSV-2, and whether this process also occurs in reverse.

HSV POLYADENYLATED

molds (6 x 15 x 0.2 cm) containing a Xi-ml polyacrylamide plug (7.5% acrylamide and 0.3% bisacrylamide) at the bottom. A wellformer was inserted into the warm agarose and removed 1 hr after the gel had solidified. Eco R,-treated HSV DNA samples were adjusted to 8% sucrose and 0.02% bromophenol blue, and were loaded into the preformed wells. Sample size was 20 ,ul containing 1-2 pg of HSV DNA. Both reservoirs contained E buffer. Electrophoresis was carried out for 24 hr at 1.6 V/cm. The gel was then removed from the glass plates and soaked in E buffer containing 2 pglml of ethidium bromide for 45 min. The DNA bands were visualized by illumination of the gel with a short-wave uv light and photographed using a Wratten G (orange) filter and type 107 Polaroid film. Transfer of DNA fragments from agarose gels to nitrocellulose paper. Eco RI fragments of HSV DNA contained in agarose gels were denatured by soaking for 40 min in 0.2 N NaOH plus 1.0 M NaCl. The gel was then transferred to a solution of 1.0 M Tris (pH 7.5) plus 1.0 M NaCl, and allowed to stand for 45 min. The blotting technique described by Southern (1975) was used to transfer the denatured, neutralized DNA fragments from the gel to a nitrocellulose membrane (B6; Schleicher and Shuell, Inc.). The nitrocellulose paper containing the HSV DNA fragments was air-dried and baked in uacuo for 4 hr at 80”. Extraction of RNA. Vero cell monolayers (5 x lo8 cells) in Bellco disposable roller bottles were infected with 40 PFU/ cell of HSV-1 or 25 PFU/cell of HSV-2. After adsorption for 1 hr at 37”, the cells were washed with medium 199 without phosphate; this medium, supplemented with 75 &i/ml of 32P (Orthophosphate, AmershamlSearle Co.) and 2.5% dialyzed FCS, was added to the infected cells. The cells were harvested and collected by centrifugation 6 hr after addition of the radioactive label to HSV-l-infected cells (7 hr p.i.) or 7 hr after addition of the radioactive label to HSV-2-infected cells (8 hr p.i.). The infected cells were washed twice with PBS, resuspended in lysis buffer, and allowed to stand for 5 min at 4”. The nuclei

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were pelleted by centrifugation and the cytoplasmic supernatant was decanted and held at 0”. The nuclei were washed twice with PBS and resuspended in high salt buffer (HSB) (0.5 M NaCl, 0.05 M MgC12, and 0.01 M Tris, pH 7.4). DNase I (100 pg/ml) (electrophoretically purified, RNase-free; Sigma Chemical Co.) was added and the mixture was digested for 40 min at 37”. The DNase digestion is essential to reduce viscosity of the preparation. The cytoplasmic fraction and nuclear fraction were individually extracted with phenol at room temperature in the presence of 0.5% SDS and 0.01 M EDTA. The phenol phase was reextracted with distilled water; the aqueous phases were combined and extracted with phenol-chloroform-isoamyl alcohol (24:24:1). After centrifugation, the aqueous phase was removed and extracted with chloroformisoamyl alcohol (24:l). The RNA was precipitated overnight by addition of 2.5 volumes of NaCl-saturated 95% ethanol at -20”. To eliminate any contaminating DNA from the RNA preparations, the precipitated RNA was redissolved in HSB containing 100 pg/ml of DNase I and incubated for 40 min at 37”. The solution was then extracted with phenol and, subsequently, with chloroform-isoamyl alcohol (24:l). The RNA was again precipitated with 2.5 volumes of ethanol at -20”. The precipitated RNA isolated separately from both nuclei and cytoplasm were individually washed in 70% ethanol followed by a second wash in 100% ethanol. Following drying, the RNA was dissolved in concentrated salt buffer (CSB) plus 0.2% SDS and 25% formamide, and applied to the appropriate column. of polyadenylated RNA. Isolation Poly(U)-Sepharose B (Pharmacia Fine Chemicals) was prepared and the columns used according to the manufacturer’s specifications. One milliliter of swelled gel was packed in glass wool-stoppered pipets. The column was washed with five bed volumes of CSB (0.7 M NaCl, 0.050 M Tris, pH 7.5, and 0.010 M EDTA) in 25% formamide. The flow rate was adjusted to 0.4 ml/min. The RNA sample was applied to the column, which was then washed with four

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bed volumes of CSB in 25% formamide. The RNA which did not bind to the poly(U)-Sepharose column, i.e., the flowthrough RNA, was diluted into the appropriate hybridization buffer. Approximately 1% of the 3’P-radiolabeled RNA bound to the column. The bound RNA was eluted with four bed volumes of elution buffer (0.010 M Tris, 0.010 M EDTA, pH 7.5, and 0.2% SDS) in 90% formamide. This polyadenylated RNA was precipitated with 2.5 volumes of NaCl-saturated 95% ethanol at -20”. RNA-DNA hybridization. The nitrocellulose strip containing the HSV DNA fragments was presoaked in 2 x SSC (0.015 M Na citrate and 0.15 M NaCl), 500 pg/ml of yeast transfer RNA (Sigma Chemical Co.), and 50% formamide for 2 hr at 37”. The filters were then transferred to 25- x 200mm Kimax tubes containing the isolated polyadenylated RNA (20 pg/ml), 2 x SSC, 500 pg/ml of tRNA, and 50% formamide in a total volume of 4 ml. The tubes were incubated at 39” with gentle rocking for 48 hr. Following hybridization, the strips were washed twice in 0.1 x SSC and incubated in 8 ml of 0.1 x SSC containing 50 pg/ml of pancreatic RNase (Sigma Chemical Co.) (previously held at 80” for 10 min) at 37” for 1 hr, rinsed twice in 0.1 x SSC, and air-dried. After drying, the strips were placed on Kodak Medical X-ray film. The film was exposed for an appropriate time, usually 4 days, and then developed. Photoscans of photographs of gels or autoradiographs were made on an Optronics P-100 Photoscanner connected to a Digital Equipment Corp. POP-11/40 computer. Following analysis, composites of several scans were plotted to determine relative positions and densities of the bands. Analysis of poly(A)-containing RNA. Polyadenylation of RNA was tested by the method of Darnell et al. (1971). One microgram of RNA in lo-p1 aliquots was added to 200 ~1 each of 0.1 x SSC and 2 x SSC. Twenty microliters of a 2 mg/ml solution of RNase A plus 80 units/ml RNase T, (Worthington Biochemical Corp.) were added to each, and the solutions were incubated for 1 hr at 37”. Carrier tRNA was added to 500 pug/ml, and the total acid-precipitable radioactivity was measured.

AND

RAPP RESULTS

Cleavage of HSV DNA by Eco RI Restriction Endonuclease Following separate treatments with Eco RI, HSV-1 and HSV-2 DNA were subjected to electrophoresis through 0.5% agarose gels. Figure 1 shows the gels photographed under uv light following staining with ethidium bromide. Figure 2 shows photoscans of photographs of the gels. The patterns obtained for the Eco R, cleavage of HSV-1 and HSV-2 DNA are virtually identical to those previously reported by Skare

FIG. 1. Products of digestion of HSV DNAs with Eco R,. HSV-1 or HSV-2 DNA was digested with Eco R, endonuclease as described in the text. Twentymicroliter aliquots of the reaction mixture, containing 2 pg of DNA adjusted to 8% sucrose and 0.02% bromophenol blue, were applied to a 0.5% agarose gel. Electrophoresis was carried out for 24 hr at 1.6 V/cm. Following electrophoresis, the gels were stained with ethidium bromide. The DNA bands were then visualized by illumination of the gel with short-wave uv light and photographed using a Wratten G (orange) filter and type 107 Polaroid film. Major fragments are designated with letters and minor fragments are designated with numbers. (A) HSV-1 DNA; (B) HSV-2 DNA.

HSV

POLYADENYLATED

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TRANSCRIPTS

FIG. 2. Photoscans of photographs of the ethidium bromide-stained agarose gels of Fig. 2. The original photographs of the DNA bands illuminated under uv light following staining in ethidium bromide (Fig. 1) were made on 107 Polaroid film. The Polaroid photograph was rephotographed and reduced on Kodak panotomic X film. The negative of the second photograph was analyzed by an Optronics P-100 Photoscanner.

et al. (1975) and by Hayward et al. (1975a). Following the nomenclature of the latter group, the fragments present in equimolar amounts are called “major” fragments and are given letter designations. The fragments present in submolar amounts are called “minor” fragments and are given number designations. It should be noted that the HSV-1 DNA fragments B, C, and D comigrate and appear as one band, as do HSV-2 DNA fragments B and C (Hayward et al., 1975a). It should also be noted that a major fragment may obscure the presence of a minor fragment. It has been postulated that the presence of fragments in submolar amounts is caused by permutations in the arrangement of base sequences within the HSV genome (Hayward et al., 197513;Clements, et al., 1976). If this model is correct, the minor fragments share DNA base sequences with each other and, in certain cases, with some major fragments. Characterization Bound RNA

of Poly(U)-Sepharose-

RNA which bound to the poly(U)-Sepharose column and later eluted with buffer plus 90% formamide was presumed to contain sequences of poly(A). An additional test for polyadenylation was applied based on the report that poly(A)-containing regions of RNA isolated from herpesvirusinfected cells are resistant to RNase treatment in 2 x SSC but are sensitive in 0.1 x SSC (Bachenheimer and Roizman, 1972; Harris and Wildy, 1975). Therefore, the cytoplasmic and the nuclear poly(A)-containing RNA from both HSV-l- and HSV2-infected cells were tested for sensitivity to RNase in the presence of high and low salt. Table 1 presents the data. Under low

TABLE RNase SENSITIVITY POLYADENYLATED

Poly-

Treatment

adenylated RNA samule

HSV-1

HSV-2

2 x ssc RNase RNase ssc RNase RNase x ssc 2 x ssc RNase RNase ssc RNase RNase x ssc

1

OF THE 32P-L~~~~~~, RNA SAMPLE@

A plus T, in 2 x A plus T, in 0.1

A plus T, in 2 x A plus T, in 0.1

Percentage of 3ZP-labe1 sensitive to RNase treatment cvtopl&mic RNA (%I

Nuclear RNA (%)

0 82

0 65

96

94

0 86

0 62

96

93

n Nuclear and cytoplasmic 32P-labeled RNA isolated from HSV-infected cells were individually eluted from poly(U)-Sepharose columns with 90% formamide. One-microgram aliquots of each RNA preparation were diluted into 2 x SSC or into 0.1 x SSC. The samples were treated with 20 ~1 of 2 mg/ ml solution of RNase A and 80 units/ml RNase T,. mateAfter each treatment, the TCA-precipitable rial was counted on Millipore filters. The counts per minute (cpm) which precipitated following incubation of the RNA samples in 2 x SSC without RNase represented 100% resistance or 0% sensitivity. The percentage of the total cpm which remained acidprecipitable for each sample was subtracted from 100% to yield the percentage sensitive to nuclease treatment.

salt conditions, all four RNA samples are essentially completely sensitive (96% sensitive for the cytoplasmic RNA) to RNase treatment. Under high salt conditions, only 82-86% of the cytoplasmic RNA and

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62-65% of the nuclear RNA from HSV-land HSV-2-infected cells is sensitive to RNase treatment. Preincubation of the polyadenylated RNA in 2 x SSC plus 50% formamide at 37” for 2 days did not increase the fraction resistant to RNase treatment. We conclude from these measurements that the nuclear and cytoplasmic RNA eluted from poly(U)-Sepharose columns are polyadenylated and do not contain detectable amounts of duplex RNA or sequences capable of self-annealing. In a very recent paper, Silverstein et al. (1976) have extensively examined the adenylic acid sequences of HSV-specific polyadenylated RNA. Hybridization of 32P-Labeled Nuclear Polyadenylated RNA from HSV-Infected Cells to Specific HSV DNA Fragments The Eco R, fragments of HSV-1 and HSV-2 DNA were separated by agarose gel electrophoresis. The DNA was denatured within the gel and transferred to nitrocellulose paper (Southern, 1975). Radiolabeled nuclear polyadenylated RNA from HSV-l-infected cells was hybridized to a nitrocellulose strip containing HSV-1 DNA and, analogously, HSV-2 nuclear polyadenylated RNA was hybridized to a nitrocellulose strip containing HSV-2 DNA. Under identical conditions, two control RNA were also hybridized to the DNA: radiolabeled nuclear polyadenylated RNA from mock-infected cells and, for the RNA from HSV-2-infected cells, the runthrough of the poly(U)-Sepharose column. This run-through RNA represents 32P-labeled nuclear RNA from infected cells which did not bind to poly(U)-Sepharose. Following hybridization, the filters were washed, treated with RNase, and placed on Kodak Medical X-ray film. Figure 3 shows representative autoradiographs of nuclear polyadenylated RNA from infected cells hybridized to its homologous DNA. Figure 4 shows photoscans of the autoradiographs. We could not detect hybridization to either HSV-1 DNA or HSV-2 DNA by radiolabeled nuclear polyadenylated RNA from mock-infected cells or by radiolabeled nonpolyadenylated RNA

i, J ,T;BF FIG. 3. Autoradiographs

of nuclear polyadenylated 32P-labeled RNA hybridized to electrophoretitally separated Eco RI fragments of HSV DNA immobilized on nitrocellulose paper. Nitrocellulose strips containing HSV DNA fragments were placed in Kimax tubes containing 20 pg/ml of polyadenylated 32P-labeled RNA (1.5 x 10’ cpm), 2 x SSC, 500 Kg/ml of tRNA, and 50% formamide in a final volume of 4 ml, as described in the text. Following hybridization, the strips were washed, treated with RNase, rinsed, and dried. The strips were placed on Kodak X-ray film and exposed for 4 days. (A) Polyadenylated 32P-labeled RNA, isolated from nuclei of HSV-l-infected cells 7 hr p.i. and hybridized to the Eco RI fragments of HSV-1 DNA; (B) polyadenylated 32P-labeled RNA, isolated from nuclei of HSV-2-infected cells 8 hr p.i., and hybridized to the Eco RI fragments of HSV-2 DNA.

from the nuclei of HSV-2-infected cells to HSV-2 DNA. While HSV-specific sequences may be contained within the runthrough RNA from infected cells, presum-

HSV POLYADENYLATED

TRANSCRIPTS

32P-labeled RNA annealed to the FIG. 4. Photoscans of autoradiographs of nuclear polyadenylated separated Eco RI fragments of HSV DNA immobilized on nitrocellulose strips. Autoradiographs from Fig. 3 were analyzed by the use of an Optronics P-100 Photoscanner. (A) Scan of an autoradiograph of polyadenylated 32P-labeled RNA, isolated from the nuclei of HSV-l-infected cells 7 hr p.i. and hybridized to Eco R,treated HSV-1 DNA; (B) scan of an autoradiograph of polyadenylated 32P-labeled RNA, isolated from the nuclei of HSV-2-infected cells 8 hr pi. and hybridized to Eco R,-treated HSV-2 DNA.

DISTANCE

IN

CENTIMETERS

FIG. 5. Photoscans of autoradiographs of cytoplasmic polyadenylated 32P-labeled RNA hybridized to the separated Eco Ri fragments of HSV DNA immobilized on nitrocellulose strips. Nitrocellulose strips containing HSV DNA fragments were placed in Kimax tubes containing 20 pgiml of polyadenylated 32P-labeled RNA (1.1 x 10’ cpm), 2 x SSC, 500 pglml of tRNA, and 50% formamide in a final volume of 4 ml. Following hybridization, strips were processed as described and placed on Kodak X-ray film and exposed for 4 days. (A) Polyadenylated 3ZP-labeled RNA, isolated from the cytoplasm of HSV-l-infected cells 7 hr p.i. and hybridized to Eco RI-fragmented HSV-1 DNA; (B) polyadenylated 32P-labeled RNA isolated from the cytoplasm of HSV-Z-infected cells 8 hr p.i. and hybridized to Eco RI-fragmented HSV-2 DNA.

ably they are present in such low concentration as to hybridize to an extent below our level of detection. It can be seen in Figs. 3 and 4 that all the resolvable major and minor bands of both HSV-1 and HSV-2 DNA contain sequences complementary to nuclear polyadenylated RNA from homologously infected cells. Hybridization of Cytoplasmic lated RNA to Specific Fragments

PolyadenyHSV DNA

The distribution of HSV DNA sequences complementary to cytoplasmic polyadenylated RNA was investigated in HSV-infected cells by first separately isolating poly(A)-containing RNA from the cytoplasm of cells infected with HSV-1 or HSV2. The 32P-labeled cytoplasmic polyadenylated RNA was then hybridized to the homologous HSV DNA Eco RI restriction enzyme fragments. Two control RNA were

also hybridized to HSV DNA: radiolabeled cytoplasmic polyadenylated RNA from mock-infected cells and, for HSV-2 DNA, the 32P-labeled cytoplasmic nonpolyadenylated RNA from HSV-2-infected cells. Photoscans of representative autoradiographs (Fig. 5) demonstrate that all the resolvable Eco RI DNA fragments from HSV-1 and HSV-2 DNA contain sequences to which the homologous cytoplasmic polyadenylated RNA hybridizes. In addition, the photoscans of these autoradiographs are qualitatively indistinguishable from the scans of the annealed nuclear polyadenylated RNA (Fig. 4) and from the scans of the DNA patterns (Fig. 2). Hybridization of the control RNAs to HSV-1 or HSV-2 DNA was not detected. These data indicate that the DNA sequences complementary to nuclear and cytoplasmic HSV-specific polyadenylated RNA are distributed over the entire HSV genome.

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Heterologous Hybridization of Polyadenylated RNA to Specific HSV DNA Fragments Previous studies have shown that HSV1 and HSV-2 DNA are approximately 50% homologous (Ludwig et al., 1972; Kieff et al., 1972). In addition, data have been presented which show that HSV-1 and HSV-2 DNA transcripts isolated during lytic infection are approximately 40-50% homologous (Frenkel et al., 1973; Murray et al., 1974). To test whether the homologous sequences between HSV-1 and HSV-2 DNA are tightly grouped or dispersed on the virus genome, the separated Eco Ri fragments of HSV-1 DNA were challenged with 32P-labeled total polyadenylated RNA isolated from HSV-2-infected cells; conversely, the Eco RI fragments of HSV-2 DNA were challenged with radiolabeled total polyadenylated RNA isolated from HSV-l-infected cells. If the common base sequences between the two virus DNA are located within particular segments of their genomes, then only a limited number of specific DNA fragments will hybridize with heterologous RNA. However, if the shared base sequences are dispersed throughout the genome, then all of the DNA fragments will hybridize heterologous RNA. Figure 6 shows photoscans of autoradiographs of such heterologaus RNA-DNA hybridization experiments. Figure 6A is a photoscan of the autoradiograph of HSV-1 32P-labeled total poly-

s c &

AND

RAPP

adenylated RNA hybridized to HSV-2 DNA. It can be seen by comparison of this figure to the DNA scan (Fig. 2B) that the polyadenylated HSV-1 RNA has hybridized to the major bands A, BC, and D of HSV-2 DNA and the minor bands 1,2,3,4, and 5 of HSV-2 DNA. This autoradiograph does not show hybridization between HSV1 RNA and major bands E and F and minor band 6 of HSV-2 DNA. In a second experiment, the total polyadenylated RNA from HSV-2-infected cells was hybridized to the Eco RI fragments of HSV-1 DNA (Fig. 6B). By comparison with the DNA pattern (Fig. 2A), it can be seen that the RNA has hybridized to all the major fragments A, BCD, E, F, G, and H and to the resolvable minor fragments 1 and 2. DISCUSSION

We have studied the organization within the HSV genome of sequences that are transcribed and the transcripts that are adenylated. By applying the procedure published by Southern (1975), it was possible to determine which specific Eco RI fragments of HSV DNA were complementary to nuclear and cytoplasmic polyadenylated RNA. Polyadenylated RNA isolated from the nuclei of HSV-l- or HSV-2-infected cells hybridized to all the Eco RI fragments of the homologous DNA (Fig. 4). The distribution of DNA sequences complementary to HSV-specific cytoplasmic polyaden-

.94 .47 0 0

32

6.4 DISTANCE

IN

96

CENTIMETERS

FIG. 6. Photoscan analysis of autoradiographs of total cell polyadenylated 32P-labeled RNA hybridized heterologously to Eco R,-digested HSV DNA immobilized on nitrocellulose strips. Nitrocellulose strips containing HSV DNA fragments were placed in Kimax tubes containing 20 pg/ml of polyadenylated heterologous 32P-labeled RNA (1.3 x IO1 cpm), 2 x SSC, 500 fig/ml of tRNA,and 50% formamide in a final volume of 4 ml. Following hybridization, strips were processed as described and placed on Kodak X-ray film and exposed for 6 days. (A) Total polyadenylated 32P-labeled RNA isolated from HSV-l-infected cells 7 hr p.i. and hybridized to Eco RI-fragmented HSV-2 DNA; (B) total polyadenylated 32P-labeled RNA isolated from HSV-2-infected cells 8 hr p.i. and hybridized to Eco R,-fragmented HSV-1 DNA.

HSV

POLYADENYLATED

ylated RNA was also studied (Fig. 5). As was the case for the nuclear RNA, ,the cytoplasmic polyadenylated RNA hybridized to all the Eco RI fragments of the homologous DNA. The problems of quantitation with the Southern (1975) technique, as noted by that author, make it difficult to compare the results reported here with other studies in the literature. In addition, we do not know what fraction of the polyadenylated RNA was HSV-specific, nor did we systematically vary the RNA concentration in any one experiment. Thus, whether the hybridization conditions were those of DNA excess or of RNA excess is unknown. Nevertheless, some comparison can be made. Roizman and his collaborators (Silverstein et al., 1973; Kozak and Roizman, 1974) have used the technique of RNADNA hybridization, under conditions in which RNA is in excess and DNA is radiolabeled, to examine total RNA and polyadenylated RNA in the nuclei and cytoplasm of HSV-l-infected HEp-2 cells at 8 hr p.i. Analysis of such data yields numbers for abundance classes of RNA expressed as the percentage of the genome in each abundance class. Kozak and Roizman (1974) examined the abundance classes of nuclear total RNA and cytoplasmic total RNA and concluded that the nuclear transcripts are complementary to 50% of the HSV genome, while the cytoplasmic RNA is complementary to 40% of the virus genome. Silver-stein et al. (1973) examined the abundance classes of nuclear polyadenylated RNA and cytoplasmic polyadenylated RNA and concluded that the nuclear polyadenylated RNA was complementary to 24% of the HSV genome and the cytoplasmic polyadenylated RNA was complementary to 22% of the virus genome. For these latter numbers and the data reported here to be in accord, it is necessary that the 22-24% of the HSV genome represented as polyadenylated RNA, as reported by Silverstein et al. (19731, be distributed over the total genome in such a way as to produce polyadenylated RNA complementary to each specific Eco R, DNA fragment (Figs. 4, 5). In previous studies on the base sequence

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153

homology between HSV-1 and HSV-2 DNA, Ludwig et al. (1972) used the technique of hybridization with the DNA immobilized on a filter. Ludwig reported that reciprocal and competition hybridization experiments demonstrated a maximum homology of 70%. Kieff et al. (1972) used the techniques of liquid and filter hybridization and thermal melting of the HSV-lHSV-2 DNA heteroduplex and concluded that 46% of the DNA had relatively good matching of base pairs. The data of these two groups are not in severe disagreement. These combined data provide the overall percentage of homology but not the segmental distribution. The reciprocal heterologous hybridization experiments (Fig. 6) provide information on the distribution of the base sequence homology between HSV-1 and HSV-2 DNA. When total polyadenylated RNA from HSV-2-infected cells was hybridized to the separated Eco RI fragments of HSV-1 DNA, all the DNA bands showed sequences complementary to the RNA (Fig. 6b). In the reciprocal experiment, total polyadenylated RNA from HSV-l-infected cells was hybridized to the separated Eco RI fragments of HSV-2 DNA (Fig. 6a). The major bands A, BC, and D, as well as the minor bands 1, 2, 3, 4, and 5, are complementary to the heterologous RNA. The three smallest bands in terms of molecular weight, i.e., E, F, and 6, are not seen in the photoscan trace (Fig.Ga). However, when the autoradiographs were heavily overexposed, these bands were faintly visible. Thus, it appears that all the HSV-2 DNA Eco Ri fragments contain sequences complementary to the polyadenylated HSV-1 RNA. We conclude, therefore, that the base sequence homology between HSV-1 and HSV-2 DNA is dispersed over the genome and is not confined to a single contiguous block. ACKNOWLEDGMENTS We wish to express our appreciation to Mr. Mike Katz, Mr. Doug Ednie, and Ms. Janet Chapman for their assistance in providing the large number of roller bottles of Vero cells required for our experiments. We also wish to thank Ms. Linda Kudler for her excellent assistance with the virus stocks. We are grateful to Biotechnology Research Re-

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sources of the Milton S. Hershey Medical Center (supported by NIH RR 00576-05) for use of the Optronics P-100 Photoscanner, and particularly to Dr. John Patterson, Director of the Research Computer Facility, who wrote the computer program for the photoscans of our autoradiographs and assisted us in the scanning procedure. This work was supported by Contract No. NO1 CP 53516 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS, by Grant No. CA 16498, awarded to R. W. H. by the National Cancer Institute, DHEW, and by Grant No. IN-109 from the American Cancer Society. REFERENCES BACHENHEIMER, S. L., and ROIZMAN, B. (1972). Ribonucleic acid synthesis in cells infected with herpes simplex virus. VI. Polyadenylic acid sequences in viral messenger ribonucleic acid. J. Viral. 10, 875-879. CLEMENTS, J. B., CORTINI, R., and WILKIE, N. M. (1976). Analysis of herpesvirus DNA substructure by means of restriction endonucleases. J. Gen. Viral. 30, 243-256. DARNELL, J. E. (1973). The origin of mRNA and the structure of the mammalian chromosome. Harv. Let. 69, l-45. DARNELL, J. E., WALL, R., and TUSHINSKI, R. J. (1971). An adenylic acid-rich sequence in messenger RNA of Hela cells and its possible relationship to reiterated sites in DNA. Proc. Nat. Acad. Sci. USA 68, 1321-1325. FRENKEL, N., SILVERSTEIN, S., CASSAI, E., and ROIZMAN, B. (1973). RNA synthesis in cells infected with herpes simplex virus. VII. Control of transcription and of transcript abundancies of unique and common sequences of herpes simplex 1 and 2. J. Virol. 11, 886-892. GIBSON, W., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. VIII. Characterization and composition of multiple capsid forms of subtypes 1 and 2. J. Viral. 10, 1044-1052. HARRIS, T. J. R., and WILDY, P. (1975). The synthesis of polyadenylated messenger RNA in herpes simplex type 1 virus infected BHK cells. J. Gen. Virol. 28, 299-312. HAYWARD, G. S., FRENKEL, N., and ROIZMAN, B. (1975a). Anatomy of herpes simplex virus DNA: Strain differences and heterogeneity in the locations of restriction endonuclease cleavage sites. Proc. Nat. Acad. Sci. USA 72, 1768-1772. HAYWARD, G. S., JACOB, R. J., WADSWORTH, S. C.,

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and ROIZMAN, B. (1975b). Anatomy of herpes simplex virus DNA: Evidence for four populations of molecules that differ in the relative orientations of their long and short components. Proc. Nat. Acad. Sci. USA 72, 4243-4247. KIEFF, E., HOYER, B., BACHENHEIMER, S. L., and ROIZMAN, B. (1972). Genetic relatedness of type 1 and type 2 herpes simplex viruses. J. Virol. 9, 738-745. KOZAK, M., and ROIZMAN, B. (1974). Regulation of herpesvirus macromolecular synthesis: nuclear retention of nontranslated viral RNA sequences. Proc. Nat. Acad. Sci. USA 71, 4322-4326. LUDWIG, H. O., BISWAL, N., and BENYESH-MELNICK, M. (1972). Studies on the relatedness of herpesviruses through DNA-DNA hybridization. Virology 49, 95-101. MURRAY, B. K., BENYESH-MELNICK, M., and BISWAL, N. (1974). Early and late viral-specific polyribosomal RNA in herpes virus-l and -2-infected rabbit kidney cells. Biochim. Biophys. Acta 361, 209-220. RAPP, F. (1963). Variants of herpes simplex virus: Isolation, characterization, and factors influencing plaque formation. J. Bacterial. 86, 985-991. SHARP, P. A., SUGDEN, B., and SAMBROOK, J. (19731. Detection of two restriction endonuclease activities in Haemophilus parainfkenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry 12, 30553063. SILVERSTEIN, S., BACHENHEIMER, S. L., FRENKEL, N., and ROIZMAN, B. (19731. The relationship between post-transcriptional adenylation of herpesvirus RNA and mRNA abundance. Proc. Nat. Acad. Sci. USA 70, 2101-2104. SILVERSTEIN, S., MILLETTE, R., JONES, P., and ROIZMAN, B. (1976). RNA synthesis in cells infected with herpes simplex virus. XII. Sequence complexity and properties of RNA differing in extent of adenylation. J. Virol. 18, 977-991. &ARE, J., SUMMERS, W. P., and SUMMERS, W. C. (19751. Structure and function of herpesvirus genomes. I. Comparison of five HSV-1 and two HSV2 strains by cleavage of their DNA with Eco R, restriction endonuclease. J. Viral. 15, 726-732. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517. SPEAR, P. G., and ROIZMAN, B. (1972). Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpesvirion. J. Virol. 9, 143-159.