Hybridization between a repeated region of herpes simplex virus type 1 DNA containing the sequence [GGC]n and heterodisperse cellular DNA and RNA

69

Virus Research, 7 (1987) 69-82 Elsevier VRR 00317

~yb~dization between a repeated region of herpes simplex virus type 1 DNA containing the sequence [GGC] n and heterodisperse cellular DNA and RNA

David J. Spector *, Thomas R, Jones **, C~istop~er L. Parks, Alison M. Deckhut and Richard W. Hyman Departmeni of Microbiology and Cancer Research Center, The Pennsylvania State University, College ofMedicine, Hershey, PA i ?033, U.S.A.

(Accepted for publication 28 October 1986)

A small DNA fragment containing the simple sequence [GGC],, from the long repeat of herpes simplex virus type 1 (HEW-l) DNA hybridized to cellular DNA and polyadenylated RNA from different mammalian species. The number and intensity of blot hybri~zation signals were increased in human compared with rodent and simian nucleic acids. The hybridization was blocked specifically by human 2813 ribosomal DNA, which shares only the GGC repeats with the herpes simplex virus DNA. These data indicate that GGC repeats were common components of cellular DNA and were expressed in mRNA. BIot hyb~di~tion analysis of viral KNA from the HSV-2 gene regions encompassing the GGC repeats revealed abundant stable mRNAs from portions of the virus genome not previously analyzed in detail and indicated that the viral GGC sequence was not expressed in stable cytoplasmic mRNA.

Herpes simplex virus; GGC repeats; Cellular DNA and mRNA

* To whom carr~~~dence should be addressed. ** Presenr address: Departnxent of Mokcuiar Biology. Princeton Wniversity, Princetonn, NJ 08544, U.S.A. ~16~-~7~2/~?/~~.5~

@ 1987 Elsevier Science Publishers B.V. (Biomedical Division)

70

Introduction Simple repetitive sequences are common components of the genomes of members of the herpesvirus family (Heller et al., 1982, 1985; Gomez-Marquez et al., 1985; Parks et al., 1986) and higher eukaryotic cells (see Tautz and Rentz, 1984). These repeated DNA sequences probably are responsible for much of the cross-hybridization observed between herpesvirus and cellular nucleic acids (Peden et al., 1982; Puga et al., 1982; Arrand et al., 1983; Jones and Hyman, 1983; Umene et al., 1984; Gomez-Marquez et al., 1985; Heller et al., 1985; Jones et al., 1985; Shaw et al., 1985; Parks et al., 1986). We reported previously that the tandemly repeated triplet sequence [GGC]. is part of a much longer repeated region of the human herpes simplex virus type 1 (HSV-1) DNA (Parks et al., 1986). GGC repeats occur as well in the genes for human 28s ribosomal RNA (rRNA) (Gonzalez et al., 1985). A ten-copy tandem GGC repeat in the viral DNA and seven- to twelve-copy tandem repeats in three separate regions of the human 28s rRNA gene are sufficient to yield hybridization between the two nucleic acids under stringent blot hybridization conditions (Jones et al., 1985; Parks et al., 1986). This hybridization must be accounted for in studies of the detection of viral nucleic acids in cell or tissue samples. In the course of these studies, we discovered that the same small region of HSV-1 DNA hybridized to heterodisperse DNA and RNA from human and other mammalian cells. In this communication, we present these data and provide evidence that the hybridization resulted from the GGC repeats. We also show that, whereas cellular mRNA sequences contained GGC or the complementary CCG repeats, the repeated region of the HSV-1 genome that contains the [GGC],, sequence was not expressed as detectable stable cytoplasmic mRNA.

Materials and Methods Cells and viruses

KB cells were grown in Dulbecco’s minimal essential medium (DMEM) containing 5% fetal bovine serum (FBS). TC7 monkeys cells and LT-Amouse cells were a generous gift of M.J. Tevethia. Human embryo lung (HEL) cells were generously provided by B. Wigdahl. HSV-l-transformed hamster embryo fibroblast (HEF) cell line 14012-8-l (Duff and Rapp, 1973) subclones 3 and 140 were propagated as described (Eizuru et al., 1983). HSV-1 strain KOS was a generous gift of S.S. Tevethia. For infections, virus was adsorbed to cells for 1 hr in DMEM without serum and culture was continued in DMEM containing 5% FBS. Antibiotics, when present, were added at the time of addition of the virus. Recombinant

DNAs

Recombinant plasmids containing HSV-1 DNA (strain Patton) BamHI and EcoRI fragments are designated by “p” preceding the fragment name and have been described previously (Russell et al., 1982; Jones and Hyman, 1983). Subclones

71 containing parts of the BumHI-E DNA fragment, designated pEB, pEC, pED, and pEBB, cover the regions depicted in Fig. 1 and their constructions have been described previously (Jones et al., 1985). A subclone of the BumHI-K (BarnHI-S/P) DNA fragment designated pKB also has been described previously (Jones and Hyman, 1986). The KB region also is shown in Fig. 1. The recombinant plasmid pE-28s contains a 7.3~kilobase pair (kbp) EcoRI DNA fragment that includes the human 28s rDNA gene, internal transcribed spacer sequences and a small portion of the 18s rDNA gene (Jones et al., 1985). Blot hybridiz~iion

Cellular DNA was extracted and blots of agarose gels were prepared and hybridized as described previously (Jones et al., 1985). Using 67% as the average percent G + C for HSV-1 DNA (Roizman, 1979) the hybridization and washing conditions were such that hybrids containing 18% or less mismatch would be stable (Howley et al., 1979). Cytoplasmic RNA was prepared from uninfected and HSV-linfected cells by phenol extraction in an isotonic buffer; poly(A)-containing RNA was selected by oligo(dT)-cellulose chromatography, and RNA blots were prepared after electrophoresis of samples in formaldehyde-containing gels and hybridized to nick-translated probes, all as described previously (Tevethia and Spector, 1984). The blots were hybridized under conditions similar to those used for DNA blots; however, with the ass~ption that RNA : DNA hybrids would be more stable than DNA : DNA hybrids, the washing temperature was 5°C higher.

Results Hybridization of HSV-1 DNA to heterodisperse mammalian DNA

The genome of HSV-1 contains three separate regions of repeated DNA sequences (Fig. 1). We previously identified a l.l-kbp region within the long repeated sequence that hybridizes to human 28s rDNA and rRNA by virtue of shared GGC repeats (Parks et al., 1986). This region, defined by a SmaI fragment of HSV-1 DNA (Fig. l), is designated EBB. EBB DNA was used to probe blots of EcoRI-digested HEL cell DNA, and the blots were exposed for longer times than that necessary to visualize the signal produced by hyb~d~ation to the 7.3-kbp fragment containing the moderately repetitive 28s rRNA genes. Under these conditions, hybridization was detected to many different size classes of EcoRI fragments of human DNA (Fig. 2A). The hybridization signals were diminished by the presence of unlabeled calf thymus DNA compared with unlabeled salmon sperm DNA. These results suggest that sequences within the EBB region hybridized to many different regions of human and bovine DNA but not to DNA of lower vertebrates. The specificity of the hybridization of EBB DNA to heterodisperse human DNA was tested by repeating the hybridization reaction in the presence of unlabeled guanine-rich nucleic acids (Fig. 2B-G). The inclusion of such a competitor DNA eliminates artifactual hyb~dization signals due to association of guanine-rich nucleic acids with py~~dine tracts in the absence of true Watson-Crick duplexes (Opara-

72 IRL,IRs

“L

USTRS P

J+

lR,_ ,lRS U,

“L

TRL

m’+j &QFlI-B

TRS ‘L

B

B

I I

B

Fig. 1. Physical map of HSV-1 DNA. The top line shows a schematic diagram of the complete HSV-1 genome in the prototype (“P”) orientation (see Roizman, 1979) and the location of the EcoRI-B DNA fragment. The hatched areas represent inverted (IR) and terminal repeat (TR) sequences that flank the unique (U) sequences from the long (L) and short (S) segments of the DNA. The junction between the long and short segments is indicated by the vertical dotted line. The second line shows the genome with Roizman, 1979) with the BumHI-E and BainHI-K (S/P) the long segment inverted (“IL” orientation; DNA fragments in expanded form below. The regions corresponding to various restriction enzyme fragments used in these studies (EB, EC, ED, KB, and EBB) also are shown. In the expanded map, individual capital letters represent restriction enzyme cleavage sites: B, BarnHI; H, HpuI; S, SucI. The fragment EBB (1.2 kbp) is shown as a solid bar and is defined by two SmaI cleavage sites within the EB region. All sizes of restriction enzyme fragments are in kbp.

Kubinski and Szybalski, 1964; Szybalski et al., 1971). In this experiment, the relative hybridization efficiency of the probe and blots was controlled by including a lane containing digested pEBB DNA in each blot. Three different guanine-rich competitor DNAs (Fig. 2; panels C-E) did not reduce the hybridization of EBB DNA to heterodisperse human DNA. This result suggests that the hybridization between the EBB and cellular DNA sequences was due to authentic Watson-Crick base pairing. When human 28s rDNA was used as unlabeled competitor, all of the hybridization signals were reduced (Fig. 2; panels F and G). The only known homology between EBB DNA and human 28s rDNA is the GGC repeats (Parks et al., 1986). Therefore, the blocking of the hybridization of EBB DNA to heterodisperse cell DNA by 28s rDNA indicated that GGC repeats were responsible for the hybridization signals.

73

Fig. 2. Blot hybridization of HSV-1 EBB DNA to human cellular DNA. Panel A: HEL cell DNA was digested with EcoRI and mixed with 1.0 copy per haploid genome equivalent of HSV-1 EcoRI-B fragment and vector DNA. The probe was purified EBB DNA. The sonicated and denatured carrier DNA (100 pg/ml) used in the prehybridization and hybridization solutions was either salmon sperm DNA (lane 1) or calf thymus DNA (lane 2). Panels B-G: Hybridization performed in the presence of excess unlabeled guanine-rich nucleic acids. Lanes 1: HSV-1 EBB DNA fragment and vector DNAs present at 10 copies per haploid genome equivalent. Lanes 2: EcoRI-digested HEL cell DNA. The probe was the purified viral DNA insert from pEBB. The total concentration of unlabeled nucleic acid was 200 ug/ml. The added unlabeled guanine-rich nucleic acids were: panel B, none; panel C, 50 pg/ml each of poly(G) and poly(U,G) (U : G = 1: 1); panel D, 100 pg/ml of MicrococcusluteusDNA [71% guanosine + cytosine (G+C)]; panel E, 25 pg/ml of the cloned HSV-1 BarnHI-K (S/P) DNA fragment; panel F, 10 pg/ml of the cloned human 28s rDNA gene-containing fragment; and panel G, 50 pg/ml of the same DNA as in panel F. The letter “B” to the left of Panel A, lane 1 indicates the position of the HSV-1 EcoRI-B DNA fragment. The position of the HSV-1 EBB DNA fragment in the reconstruction samples in Panels B-G is indicated as “EBB”. The closed circle indicates the position of the human 28s rDNA gene fragments. The lines to the right of Panels A and G indicate the migration of Hind111 fragments of lambda DNA (approximate sizes, from top to bottom, 23.7-, 9.5-, 6.7-, 4.3-, 2.3-, and 2.0-kbp).

To investigate directly whether the EBB DNA sequences would hybridize to heterodisperse DNA from other mammals besides human, EBB DNA was used to probe blots of EcoRI-digested DNA from HEF cells and HEF cells transformed by

74

HSV-1 (Duff and Rapp, 1973) (Fig. 3). EBB DNA hybridized to many size classes of hamster DNA but did not produce a distinct hybridization signal of a size corresponding to hamster 28s rDNA (Fig. 3; panel A, lanes 1 and 3 and panel B), whereas human 28s rDNA probe hybridized to hamster 28s rDNA (Fig. 3; panel A, lane 2). The efficiency of hybridization was monitored by including one copy per haploid genome equivalent of the EcoRI-B fragment of HSV-1 DNA in the samples. EcoRI-B contains the EBB sequence (Fig. 1); however, the complexity of EcoRI-B is too large to permit detection of the hybridizing cell DNA under the conditions

C

Fig. 3. Blot hybridization of the HSV-1 EBB DNA fragment to hamster cellular DNA. Panel A: HEF DNA (lanes 1 and 2) and HEL DNA (lane 3). Panels B and C: HEF DNA plus HSV-1 EcoRI-B DNA fragment and vector DNAs present at 1.0 copy per haploid genome equivalent (lane 1); HSV-l-transformed HEF cell line 14012-8-l (clone 3) DNA (lane 2); and 14012-8-l (clone 140) DNA (lane 3). All cellular DNAs were digested with EcoRI. The probes were the purified EBB DNA insert from pEBB (panel A, lanes 1 and 3; panel B, all lanes); the purified 7.3-kbp EcoRI DNA fragment insert from the human 28s rDNA clone, pE-28s (panel A, lane 2); or a plasmid (pEcoRI-B) that consists of the cloned ~coRI-B DNA fragment (panel C, all lanes). For panels B and C, the hybridization conditions were adjusted such that hybrids containing 24% or less mismatch would be stable. The positions of the HSV-1 EcoRI-B and vector DNA fragments are indicated as “B” and “v”, respectively. The migration of 28s rDNA is indicated as in Fig. 2.

75 used in this experiment (Fig. 3; panel C). Similar results were obtained with hamster and mouse DNAs from other sources (data not shown). The sequences of rodent 2% rRNAs do not contain tandem GGC repeats (Goldman et al., 1983) so the finding that the EBB probe did not produce a strong hybridization signal corresponding to the 28s ribosomal genes was consistent. These results indicate that EBB DNA shared DNA sequences with many regions of the DNA of other mammals besides humans, as might be expected if the hybridizing element were tandem GGC repeats.

Hybridization of EBB DNA to stable cytoplasmic cellular RNA We next investigated whether the cell DNA sequences that hybridized to EBB DNA were expressed as stable cytoplasmic RNA. In this experiment, cytoplasmic RNA was prepared from human, monkey and mouse cells and separated into fractions enriched for and depleted of poly(A)-containing RNA, respectively. Blots of the RNA samples were probed with labeled EBB DNA under stringent hybridization conditions (see Materials and Methods). As expected, the poly(A)-containing RNA-depleted fraction [poly(A)-] from the human cells produced a strong 28s rRNA hybridization signal (Fig. 4, panel A, lane l), whereas the rodent RNA produced a barely detectable signal after a long exposure (Fig. 4, panel A, lane 3). Using a variety of probes from procaryotic and eucaryotic sources and different RNA preparations, we consistently observed signals of similar intensities to that obtained in lane 3 in the regions of RNA blots

Fig. 4. Hybridization of EBB DNA to cytoplasmic RNA from different mammalian species. Blots of cytoplasmic RNA samples depleted of [poly(A)-, panel A] and enriched for [poly(A)+, panel B] poly(A)-containing RNA were hybridized to pEBB DNA probe. For the poly(A)samples, 2 pg of RNA was run in each lane; for the poly(A)+ samples, the amount of oligo(dT)-cellulose purified RNA from 50 pg of total cytoplasmic RNA was used. Since the hybridization of 28s rRNA is equivalent in the two conditions, we estimate that about 4% of the initially present rRNA contaminated the poly(A)+ preparations. The positions of migration of 28s and 18s rRNA, as determined by ethidium bromide staining of the gel, are indicated. Lanes 1: human (KB) cell RNA; lanes 2: simian (TC7) cell RNA; lanes 3: mouse (LT-A) cell RNA.

76 corresponding to the mobility of the 28s and 18s rRNAs. Such signals are unlikely to have represented authentic base pairing but rather might have been due to nonspecific sticking of the probe to the abundant rRNAs in the blots. Thus the rodent RNA probably contained little if any true sequence match with EBB DNA. These results were consistent with the presence of GGC repeats in the human 28s rRNA and the lack of the same sequence in the rodent RNA. A hybridization signal, reproducibly stronger than that found with rodent RNA (Fig. 4, panel A, lane 3) was obtained with the monkey RNA sample (Fig. 4, panel, A, lane 2) suggesting that monkey 28s rRNA might have sequences capable of hybridizing weakly to EBB DNA. Besides the hybridization signals that corresponded to ribosomal species, there were a few other faint bands present in the human RNA sample and none detectable in the other RNA samples. We could not rule out the possibility that the faint bands in the human RNA sample were degradation products of rRNA. Thus, these data did not provide any clear evidence that heterodisperse cellular DNA sequences hybridizing to EBB DNA were expressed as stable, nonpolyadenylated cytoplasmic RNA. EBB DNA also was used to probe blots of poly(A)-containing RNA-enriched samples (Fig. 4, panel B). The RNA present in these lanes came from approximately 25 times as much cytoplasm as was used in the poly(A)RNA samples. The oligo(dT)-cellulose-selected fraction was not completely depleted of rRNA, as indicated by the hybridization signals obtained of the sizes and intensities expected for 28s rRNAs. In addition, other bands of various intensities and mobilities were obtained in samples from all three species. The hybridization signals were not strong, but this result would be expected if only a small portion of each RNA molecule shared sequence homology with the EBB probe. These data indicate that heterodisperse cellular DNA sequences that hybridize to EBB DNA and probably containing [GGC]. were expressed as mRNA in the different species and suggest that the occurrence of such sequences in mRNA was more common in human than in rodent or monkey cells. To obtain more evidence that the hybridizing RNA contained repeated GGC sequences, or the complementary sequence [CCG],,, a 68-base pair fragment of EBB DNA containing the ten copies of GGC was cloned into pBR322. This plasmid was used to probe blots of poly(A)-containing RNA similar to those shown in Fig. 4. The results obtained were identical to those using pEBB DNA as the probe (data not shown). Transcription of the region of the HSV-I genome containing EBB DNA Previous transcription maps of HSV-1 provide little information as to whether the region defined by EBB DNA is expressed as stable cytoplasmic RNA (Anderson et al. 1979, 1980; Holland et al., 1979). These studies were done with relatively high complexity probes and low resolution RNA separation methods. We therefore probed blots of RNA extracted from virus-infected cells with EBB DNA and DNA from neighboring regions of the HSV-1 genome. In the first experiment, cytoplasmic RNA was extracted from human cells early after infection in the presence of an

77 i~bitor of viral DNA replication and late after infection, as well as from uninfected human cells. The blots of poly(A)+ RNA were probed with four different recombinant DNAs containing viral DNA fragments covering most of the long repeated region of the genome and a small portion of the long unique sequences as shown in Fig. 1. The EBB region is contained in fragment EB. The data (Fig. 5) indicate that, as expected, pEB DNA was the only probe to hybridize to poly(A)+ RNA from uninfected human cells (Fig. 5, panel A, lane 2). As expected, pEB DNA also hybridized strongly to the 28s rRNA contaminating the poly(A)” RNA preparations. Two additional prominent bands of about 2.4 and 1.7 kilobases (kb) were obtained in late RNA samples with pEB DNA (Fig. 5, panel C, lane 2), suggesting that virus-specific mRNA was transcribed from this region at late times after infection. The unique sequence DNA present in pED hyb~d~ed to prominent 1.9- and 1.2-kb bands in early time samples (Fig. 5, panel B, lane l), and at least six abundant mRNAs in late samples ranging from 3.3 to 0.7 kb (Fig. 5, panel C, lane l), two of which had the same mobilities as RNA hybridizing to the EB region. pKB and pEC DNA hybridized to a prominent 2.5-kb species in both the early (Fig. 5, panel B, lanes 3 and 4) and late (Fig. 5, panel C, lanes 3 and 4)

Fig. 5. Stable mRNA species transcribed from the long repeat region of HSV-1. Blots were prepared with cytoplasmic p~ly(A)-cont~~ng RNA isolated from uninfected KB cells (panel A); KB cells infected with 15 plaque-forming units (PFU) per cell of HSV-1 for 6 hr in the presence of 20 fig/ml of cytosine arabinoside (early or beta RNA, panel B), or KB cells infected with 6 PFU per cell of HSV-1 for 12 hr (late or gamma RNA, panel C). The amounts of RNA run on the gel were purified from 100 ylg (panel A) or 50 pg (panels B and C) of total cytoplasmic RNA. Blots were hybridized to recombinant DNAs containing the HSV-1 DNA fragments indicated in Fig. 1. Lanes 1: pED; lanes 2: pEB; lanes 3: pEC; lanes 4: pKB. Panel C, lane 1’ is a shorter exposure of lane 1. The migration of 28s and 18s rRNA markers was determined as in Fig. 4. The approximate sizes of virus-specific transcripts were estimated by comparison using the published length of 5025 bases for the 28s RNA (Gonzalez et al., 1985) and an approximate length of 1850 bases for the 18s RNA by analogy to other mammalian small subunit rRNAs (Nelles et al., 1984).

78 samples and other less abundant RNAs. The prominent species was of the mobility expected for the immediate early 1 (IE-1) RNA of HSV-1, which is transcribed from this region and encodes a IlO-kilodalton polypeptide (ICPO) (Watson et al., 1979; Anderson et al., 1980). In the next experiment, cytoplasmic RNA was prepared from human cells infected in the presence of the protein synthesis inhibitor cycloheximide. This condition defines immediate early (or alpha) HSV-1 gene expression (Honess and Roizman, 1974). In addition, early (or beta; Honess and Roizman, 1974), late (or gamma; Honess and Roizman, 1974), and uninfected cytoplasmic RNA samples were prepared as before. Identical blots of poly(A)+ RNA were probed with a recombinant DNA containing a fragment (BarnHI-E) covering the region spanned by fragments EC, EB, and ED, or with pEBB DNA. The rest&s (Fig. 6) show, as expected, that hybridization to both probes was detected in the uninfected cell samples as seen before with pEB DNA as the probe. However, whereas additional RNAs were detected in the infected samples by the BamHI-E probe (Fig. 6, panel A), no bands clearly identifiable as viral RNAs could be detected with the EBB probe (Fig. 6, panel B), although there were differences in intensities of some of the RNA bands in the different samples. These results suggest that the region of HSV-1 DNA that includes the tandemly repeated GGC sequences was not expressed as detectable stable mRNA in the cytoplasm of infected cells.

Fig. 6. Stable mRNA species synthesized from the EBB region of HSV-1 DNA. Blots of poly(A)-containing cytoplasmic RNA were prepared with samples isolated from uninfected KB cells (lanes l), and KB cells infected with 20 PFU per cell of HSV-1 for 12 hr with no drugs (late or gamma RNA, lanes 2), 6 hr in the presence of 100 pg/mI of cytosine arabinoside (early or beta RNA, lanes 3), or 4 hr in the presence of 200 &g/ml of cycloheximide (immediate early or alpha RNA, lanes 4). RNA purified from 50 cg of total cytoplasmic RNA was hybridized to a probe (p&mHI-E) that consists of the cloned BumHI-E DNA fragment (panel A), or a pEBB probe (panel B). The markers are as in Fig. 4 and 5.

79 Discussion Hybridization of a region of HSV-1 DNA containing the sequence [GGC],, to human 28s rRNA and rDNA can occur due to the presence of homologous sequence stretches in the ribosomal nucleic acid (Parks et al., 1986). Such hybridization can complicate the detection of viral nucleic acids in cell and tissue samples. We now have shown another source of authentic hybridization between HSV-1 DNA and cellular nucleic acids: the sequence [GGC], was dispersed throu~out the genomes of rn~~an species and expressed in stable mRNA. The comparisons between human cells and those of lower mammals indicate that, in general, GGC repeats were probably more prevalent in the human genome. Consistent with this notion, GGC repeats are not found in the 28s rDNAs of rodents (Goldman et al., 1983) and are less prevalent even in the rDNA of some nonhuman primates (Gonzalez et al., 1985). Thus, the acquisition of GGC repeats by the human 28s ribosomal gene, and other human genes, may be relatively recent in evolutionary terms. The minimum length of simple sequence repeats required to give a strong hybridization signal is probably greater than 20 to 25 base pairs (Tautz and Renz, 1984). A region of human 285 rDNA that contains seven copies of GGC hybridizes weakly to EBB DNA despite the relative abundance of this gene in the human genome (Parks et al., 1986). The intensity of the hyb~dization signals due to heterodisperse DNA and RNA sequences is the product of three unknowns: (1) the number of GGC repeats; (2) the degree of mismatch; and (3) the copy number of the nucleic acid. These unknowns preclude any further speculation on the nature of the GGC repeats present in the cellular nucleic acids. We observed that nonspecific DNAs used as carrier DNAs in blot hybridization experiments were not equivalent. Whereas salmon sperm DNA as carrier allowed the detection of GGC sequences in the cellular nucleic acids, calf thymus DNA did not (Fig. 2, Panel A). This result presumably was due to blocking of the hybridization by the higher content of GGC repeats in the unlabeled calf thymus DNA. Such differences most likely are responsible for discrepancies in the detection of simple repeated sequences in cellular nucleic acid samples by different investigators using the same herpesvirus probes, but different carrier DNAs (Heller et al., 1982; Arrand et al., 1983). We did not detect any RNAs that hybridized to EBB DNA sequences in infected cells but not uninfected cells. If any HSV-1 RNAs were made from this region, they must have accumulated to levels below the background in uninfected cells. GGC- or CCG-containing viral RNAs with mobilities corresponding to lengths of less than 300 nucleotides would not have been detected in our blot hybridization experiments. Thus our experiments do not address the possibility that EBB DNA sequences may be expressed as a small RNA. In the course of examining whether the EBB region of HSV-1 DNA was expressed as abundant stable mRNA, we assayed stable mRNA from adjacent regions of the genome as well. Tr~sc~ption maps have been pub~shed previously for the HSV-1 DNA regions we studied (Anderson et al., 1979, 1980; Watson et al.,

80 1979; Holland et al., 1979). An abundant mRNA maps to the IE-1 region defined by fragments KB and EC (Anderson et al., 1979; Watson et al., 1979). This mRNA probably corresponds to the 2.5kb species we detected. Other less abundant KBand EC-specific RNAs in HSV-l-infected cells also may correspond to mature mRNAs seen by Chou and Roizman (1986). The use of DNA probes of low complexity and labeled to high specific activities has allowed us to detect mRNAs from a region of the genome, defined by the EB and ED fragments, to which no mRNAs had been assigned previously. This region encoded at least six abundant mRNAs during HSV-1 infection. Several pathogenic properties of the virus have been mapped to a portion of the genome that includes the repeated sequences in the EB fragment (Centifano-Fitzgerald et al., 1982; Thompson and Stevens, 1983; Becker et al., 1986). Further analysis of viral gene expression from this region might reveal whether mRNAs we observed have a role in pathogenicity of viral variants.

Acknowledgments We thank M.J. Tevethia, S.S. Tevethia, and B. Wigdahl for kindly providing cell lines and virus used in the designated experiments. We also thank Antoinette Konski, Pamela Gesford and Clarence Colle for technical assistance, Tim Grierson for photography, and Melissa Clement for editorial assistance. Kathy Selvaggi and Zhao-Yin Fang made valuable contributions to this project. We thank Fred Rapp for his continuing support. This work was supported by Public Health Service grants CA09124, CA16498 (to R.W.H.), and CA27503 from the National Institutes of Health and by grant IN-109G (to D.J.S.) from the American Cancer Society.

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received

25 August

1986)