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Herpes simplex virus expressing Epstein-Barr virus nuclear antigen 1
VIROLOGY
148,337-348
(1986)
Herpes Simplex Virus Expressing MARY HUMMEL,
Epstein-Barr
Virus Nuclear Antigen 1
MINAS ARSENAKIS, ANDREW MARCHINI, BERNARD ROIZMAN, AND ELLIOTT KIEFF’
The Marjorie
B. Km&r Vim! Oncology Laboratwies, The University 910 East 58th Street, Chicago, Illinois 60637
LINDA
LEE,
of Chicago,
Received July 8, 1985; accepted October 19, 1985 DNA. fragments containing an open reading frame known to encode most or all of the EBNAl protein of Epstein-Barr virus (EBV) were fused in the proper transcriptional orientation to the promoter regulatory domain, capping site, and a portion of the 5’ transcribed noncoding sequences of the HSV-1 (~4 gene of herpes simplex virus 1 (HSV-1). In these constructs 20, 130, or 385 bp of EBV DNA and 28 bp of HSV-1 DNA separated the a4 cap site from a putative initiator codon of the EBNAl gene. The chimeric o4-EBNAl genes were introduced into L cells or recombined into the viral genome using the thymidine kinase selection system. The three chimeric gene constructs resident in the L cell clones expressed a protein indistinguishable from authentic EBNAl with respect to electrophoretic and immunologic properties indicating that the ATG at the beginning of the EBV open reading frame initiated translation of the bonafide EBNAl protein. The chimeric a4EBNAl genes resident in L cells were induced by HSV-1 infection and were regulated as a genes. The chimeric (~4-EBNAl gene recombined into the viral genome was also regulated as an u gene. The recombinant viruses were stable and expressed 50- to loo-fold more EBNAl than is ordinarily expressed in human lymphocytes carrying the EBV genome. EBNAl did not alter the program of HSV-1 protein expression. The utility of the vector is discussed. 0 1986 Academic Press. 1ne. INTRODUCTION
Epstein-Barr virus (EBV) and herpes simplex virus-l (HSV-1) are human herpesviruses with similar virion morphology, genome size, and programs of replication in permissively infected cells and similar size capsid proteins (for review see Kieff et ccZ.,1982). Although there is neither antigenie relatedness nor extensive DNA homology between these two viruses, there is considerable similarity in the nucleotide sequences of their terminal direct repeats and in the amino acid sequence of some of their genes, including ribonucleotide reductase, DNA polymerase, and a glycoprotein (Baer et al, 1984; Gibson et aL, 1984; Matsuo et al., 1984). Thus, at least a part of the genomes of these two herpesviruses arose from a common progenitor. i Author to whom requests for reprints addressed.
should be
There are, however, many biological differences that might make recombinants between the two viruses of interest. While HSV replicates efficiently in a wide variety of cells in culture, the host range of EBV is limited to human and some primate B cells; and there is usually no virus replication (Gerber et al, 1969; Henle et al, 1967; Pope et al, 1968). Because EBV replicates poorly in cultured cells, genetic analyses to define functions of viral genes have not been feasible. Expression of EBV genes by a heterologous virus vector such as HSV1 in a wide variety of cells may enable such analyses to be done. Whereas EBV can efficiently transform the cells it infects into immortalized cell lines, HSV transforms cells at a very low frequency. Therefore the immortalizing genes of EBV could be studied against a low background of spontaneous or HSV-1 induced transformation. EBV DNA and four viral encoded gene products can be detected in EBV-trans337
0042-6822/86 $3.00 Copyright All rights
Q 1986 by Academic Press, Inc. of reproduction in any form reserved.
338
HUMMEL
ET AL.
formed cells (Hennessy and Kieff, 1983, in the a4 gene (Post et aL, 1981). The virus 1985; Hennessy et aL, 1983, 1984, 1985). stocks were grown in HEp-2 cells. All virus These proteins are thought to be important titration, plaque purification, and stocks of either singly or in combination, in altering recombinant viruses were made in Vero cell the growth properties of EBV-transformed cultures. The human 143tk- cells (Camto the pione-Piccardo et aZ., 1979) were used for cells in vitro, and in contributing malignancies associated with EBV infecTK- virus selection. tion: Burkitt’s lymphoma and nasophaCloning the EBNAl gene into a TK seryngeal carcinoma. The role of these EBV lection vector. The 2.9-kbp BamHI-Hind111 genes in growth transformation could be subfragment of BamHI K (pDK225) was established by analysis of the effects of isolated by electroelution from an agarose conditional mutations in these genes. Also, gel slice (Dambaugh et ak, 1980). This fraghigher level expression of these genes ment was partially digested with Sau3A would facilitate the biochemical characand the products were cloned into the terization of the proteins they encode. BamHI and Hind111 sites of pRB334 (Fig. HSV could be a useful vector for studying l), a derivative of pBR322 which has the EBV genes since it has a broad host range HSV-1 TKPvuII fragment, the HSV-1 a4 and current studies suggest that it could promotor, a BamHI site 3’ to the (~4 procarry at least 25 kbp of foreign DNA. Furmotor, and the pBR322 Hind111 site 3’ to ther, several classes of HSV promotors the BamHI site (Post et al., 1982). Clones have been defined so that EBV genes could were isolated which varied in their insert length depending on the Sau3A site which be expressed at different levels and at different stages of HSV infection. Moreover, delined the 5’ end of the insert. Clone S2.9 the stability and long-term potential for contained the entire 2926-bp BamHI to expression of HSV genes resident in cell Hind111 fragment whereas S2.6 and S2.5 genomes (Munyon et a& 1971; Persson et contained 2671 and 2561 bp of EBV DNA 5’ to the Hind111 site, respectively (Baer et ak, 1985) suggest that it might be possible al, 1984). These constructs placed the first to establish host range mutants of HSV carrying EBV genes which could replicate codon of the EBNAl open reading frame and express HSV lytic functions only in 413, 158, and 48 bp downstream from the cells stably expressing the HSV gene. In HSV-1 a4 gene transcription initiation site order for HSV to serve as a suitable vector (Mackem and Roizman, 1982; Baer et aL, for the introduction of EBV genes, it is 1984). necessary that these genes be stable in Transfection into L cells. Ltk- cells(3 X lo6 HSV and not interfere with its replication. cells per dish) were transfected with 50, We now report the results of a series of 100, or 150 ng of S2.9, S2.6, S2.5, or pRB334 experiments in which the EBV EBNAl DNAs and 20 pg of salmon sperm DNA gene, a gene expressed in EBV-immortalcarrier by calcium phosphate precipitation ized lymphoblastoid cells, was recombined (Graham and van der Eb, 1973). After 2 into the HSV genome. The EBV gene is ex- days in nonselective medium, cells expressed from the HSV genome at high lev- pressing TK activity were selected by els, but does not interfere with HSV rep- growth in HAT medium (lop4 M hypoxanlication. thine; 1.6 X 10m5M thymidine; 4.4 X lo-’ M aminopterin). Two weeks after transMATERIALS AND METHODS fection two or three foci were picked from Vimses and cells. Strain F [HSV-l(F)] is each dish. The average transfection effithe prototype HSV-1 strain used in our ciency was 580 cell foci/hg DNA. The TK+ laboratories (Ejercito et al, 1968). Like cell lines carrying the (u4-EBNAl genes most HSV-1 isolates passaged a limited were designated by the length of the EBV number of times in cell culture it carries a insert in the transfected DNA and a unique ts lesion in a4 gene. ts502A305 is a double number, e.g., L2.9-1, etc. mutant containing a 700-bp deletion in the Analyses of the L ceUclones carrying the thymidine kinase (TK) gene and a ti lesion &EBNA 1 chime& genesfor EB V Protein
EBV
NUCLEAR
and DNA. For protein analysis, cells were washed in PBS and resuspended in 6% SDS, 2 n-&f /3-mercaptoethanol, 140 mM Tris, pH 7.0,28% glycerol, 0.07% bromophenol blue, briefly sonicated to reduce viscosity and boiled for 5 min. Proteins were electrophoretically separated on SDS-polyacrylamide bisacrylamide gels and transferred to nitrocellulose (Burnette, 1981). Total protein was visualized in some cases by staining replicate nitrocellulose filters with amido black. EBNAl was detected by immunostaining with B. Thorpe sera (1:40 dilution) which had been preabsorbed with extracts of an EBV negative lymphoblastoid cell line (Hennessy and Kieff, 1983). HSV-1 a4 and a0 proteins were detected with a mixture of monoclonal antibodies, H640 and H1083 (kindly provided by Lenore Pereira) diluted 1:lO and 150, respectively (Ackerman et aL, 1984). IgG-binding sites were identified with ‘%I-labeled Staphykmccus aureus protein A (New England Nuclear, Boston, Mass.). For DNA analysis, cells were lysed in situ in 4.0 ml of 10 mM EDTA, 50 mM Tris, pH 7.4, 1% SDS, and 100 pg/ml proteinase K (Sigma, St. Louis, MO.). The mixture was heated at 55” for 30 min and DNA was separated by two successive isopycnic bandings in neutral cesium chloride (Heller et aL, 1981). DNA was digested with a 2.5fold excess of restriction enzyme for 4 hr at 3’7” along with phage X DNA (New England Biolabs, Beverly, Mass.) to monitor the extent of digestion. Five micrograms of cell DNA or an aliquot of plasmid DNA equivalent to one plasmid copy per diploid genome was loaded onto each lane of a 0.4% agarose gel. After electrophoresis and a 20min depurination in 0.25 M HCl, gels were blotted (Southern, 1975) onto Genescreen Plus (New England Nuclear). DNA blots were prehybridized in 25 m&l Tris, pH 7.5, 0.02%polyvinyl-pyrrolidone and Ficoll400, 1% SDS, 6 X SSC (pH 7.5) and 250 pg/ml denatured salmon sperm DNA, for 5-6 hr and hybridized for 18 hr at 76’ with 2 X lo7 cpm r2P]GTP-labeled cRNA per milliliter. Labeled cRNA was transcribed by SP6 polymerase from the AvaI-Hind111 fragment of EBV BamHI K which had been cloned into pSP64 (Zinn et aL, 1983).
ANTIGEN
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Induction of &EBNAl gene expression by HSV iqfectzk Cells (3 X 106) were infected (10 PFU/cell) with ts502A305. After 1 hr absorption at 0”, the cells were incubated for various times at 39” and harvested for protein analysis. Zero time points were taken by harvesting the cells immediately after adding virus. Mock-infected cells were treated the same way as infected cultures except that no virus was added. Cmtmtion of HSV-1 vectors currying the chimeric c&EBVAl gene. The 3761-bp PwuII fragment from plasmid S2.5 and the 4125-bp fragment from plasmid S2.9 containing the coding sequence of the EBNAl gene fused in the proper transcriptional orientation to the promotor-regulatory sequenceof the (~4gene of HSV-I were each cloned into the XbaI site of plasmid pRB3166 and the resulting plasmids were designated as pRB2.5 and pRB2.9, respectively. The plasmid pRB3166 was specifically constructed to facilitate the insertion of foreign genes into HSV-1 DNA. The vector contains the HSV-1 BamHI Q fragment in which the 490-bp BglII-Sac1 fragment carrying the 5’ noncoding and coding sequences of the TK gene was replaced by a polylinker containing an XbaI site (Shih et aL, 1984). To construct the recombinant HSV-1 viruses carrying the (YI-EBNAl chimeric genes, pRB2.5 or pRB2.9 DNAs were cut with the restriction enzyme XhoI, which excises the HSV-1 BamHI Q sequences carrying the EBV DNA insert from the plasmid, and ethanol precipitated. DNA (0.5 pg) was cotransfected with 0.5 pg of intact HSV-l(F) DNA into rabbit skin cells, as previously described (Mocarski et aL, 1980). TK- recombinants arising by recombination of BamHI Q sequences flanking the inserted (w4-EBNAl chimeric gene were selected by plating the progeny of the transfection on 143 TK- cells in the presence of 100 pg of BudR per milliliter of medium. After three sequential plaque purification in the presence of BudR, the viral recombinants were grown in Vero cells and their DNA was analysed by restriction enzyme and Southern blot analysis for ar4EBNAl chimeric gene insertion. The re-
HUMMEL
340
combinant viruses carrying the 2.5 and 2.9 kb a4 EBNAl chimeric genes were designated as recombinants R2.5 and R2.9, respectively. Preparation of illfected cell lysates. Vero cells (4 X 10”) were infected with 20-40 PFU per cell and incubated for 20 hr at either 33 or 39”. Infected cell proteins were labeled from 1.5 hr postinfection with [?S]methionine (50 &i/ml of medium; sp act 900 mCi/mmol; New England Nuclear). At 20 hr postinfection, the medium was discarded, the cell monolayers were washed with cold PBS, and then lysed in 2% SDS, 5 mJ4 @-mercaptoethanol, and 50 mAf TrisHCl (pH 7.0) and were stored at -80” until needed. RESULTS
Earlier studies have shown that the EBNAl gene is very likely translated from a 1928-bp open reading frame encoded by a 2-kb exon at the 3’ end of a 3.7-kb RNA (Baer et al, 1984; Heller et d, 1982; Hennessy et aL, 1984;Hennessy and Kieff, 1983; Kieff et aL, 1984; Weigel and Miller, 1985). Specifically, the single potential splice acceptor site, which is probably the 5’ end of the exon, is followed by stop codons and a potential initiating ATG (Baer et aL, 1984). Furthermore, the BamHI K fragment, which begins 385 bp 5’ to the open reading frame, produced a protein which was identical in size to EBNAl when expressed in mammalian cells from plasmids containing SV40 or polyoma virus early promotors (Hearing et aL, 1984; Robert et aL, 1984; Summers et aL, 1982; Baer et aL, 1984). Among the HSV-1 promoters, that of the a4 gene has been extensively characterized (Post et aL, 1981; Mackem and Roizman, 1982; Kristie and Roizman, 1984) and used to express non-HSV genes inserted into the cellular (Post et al, 1982, Herz and Roizman, 1983) and viral genome (Shih et aL, 1984). The choice of the cu4promoter to express EBNAl protein was predicated on the observation that (Ygenes are the first viral genes to be transcribed and expressed in HSV-1 infected cells, and therefore the EBNAl gene would be expressed and its effect on HSV-1 replication would be mea-
ET AL.
sured during the earliest stages of HSV-1 replication. A characteristic of the (Ypromotors is that they are expressed at low but detectable levels when resident in the cellular genome and are induced to a much higher level of expression by an atram inducing factor (aTIF) encoded by the HSV genome and introduced into the cell during infection (Post et aL, 1981; Batterson and Roizman, 1983,Campbell et al, 1984, Pellett et d, 1985). To test the expression of EBNAl in the environment of the cellular and viral genomes, two sets of a4-EBNAl chimeric genes were constructed. The first was introduced into the cellular genome and the cells were tested for EBNAl constitutive expression and for induction of EBNAl expression by HSV-1 infection. The second set of chimeric genes was recombined into the HSV-1 genome. Expression of agEBNA1 chimeric gene resident in cells umverted to TK+ pherwtype. pRB334 consists of pBR322 carrying a HSV-1 TK gene linked to the promotorregulatory domain, capping site, and 28 bp of the 5’ transcribed noncoding sequences of the (~4gene, and a BamHI cloning site immediately 3’ to the (~4 promotor-regulatory domain (Post et aL, 1982). Because the function of the transcribed sequences 5’ to the EBNAl open reading frame was not known, three different subfragments of the EBV BamHI K fragment were inserted into a BamHI site of pRB334 in the correct transcriptional orientation relative to the a4 promotor. These constructs contained 28 bp of the 5’ transcribed noncoding sequences of the a4 gene 3’ to the capping site and 385,130, or 20 bp of EBV DNA 5’ to the putative initiator ATG. There is no other ATG in the 28 bp of a4 gene (Mackem and Roizman, 1982) or in the 20 bp of EBV DNA (Baer et aL, 1984) 5’ to the putative initiator ATG on the smallest construct. The resultant plasmid constructs are designated S2.9, S2.6, and S2.5, respectively (Fig. 1). TK+ L cell clones were selected following transfection of LTK- cells with each of the recombinant plasmids and designated as L2.9, L2.6, and L2.5. Most L cell clones expressed a protein which was reactive with EBNAl positive human sera. The protein
EBV
S2.812.6l2.5 8.3l0.017.8
NUCLEAR
Kbp
FIG. 1. Construction of a4-EBNAl chimeric genes inserted in pRB334. SadA partial digestion products of the EBV BumHI-Hind111 fragment were cloned into the BumHI and H&l111 sites of pRB334 to generate three constructs, S2.5, S2.6, and S2.9, placing the first methionine codon of the EBNAl open reading frame 48, 153, and 413 bp, respectively, downstream from the a4-cap site. The direction and domain of the EBNAl open reading frame is shown by the open arrow. The IR3 region which encodes a glycine alanine copolymer within EBNAl (Hennessy and Kieff, 1933) is indicated by stippling. The putative polyadenylation signal (Baer et al, 1934) is indicated as PA. Immediately 5’ to the EBNAl open reading frame is a splice acceptor site which is followed by stop codons and a typical initiator ATG (Kozack, 1934). pRB334 vector sequences are indicated by the dashed arrow, B, BarnHI; S, &u&A.
ANTIGEN
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was identical in size to EBNAl in lymphocytes from which the open reading frame was derived (Fig. 2). One cell line, L2.9-12 expressed two immunoreactive proteins, one similar in size to EBNAl and one larger. The amount of EBNAl expressed in the L cell clones varied, but none of the clones made more EBNAl protein than that made in the latently infected growth transformed lymphoblastoid cell line IB4 (Fig. 2). Further, clones transfected with plasmid S2.5, expressed about as much EBNAl as clones transfected with S2.6 or S2.9, even though S2.5 has only 20 bp of EBV DNA sequences upstream to the putative initiator codon. To determine whether the resident EBNAl chimeric gene was expressed under the control of the a4 promotor-regulatory domain, the L cell clones were infected with HSV-1 ts502A305 and incubated at 39”. This mutant carries a temperature-sensitive lesion in the Lu$gene, but by 24 hr infected cells incubated at the nonpermissive (39”) temperature accumulate some /3 and y proteins. EBNAl synthesis was induced by HSV infection. This protein accumulated in increasing amounts up to 5-10 hr after infection at 39” (Fig. 3A). In the L cell line tested (L2.5-6), the accumulation
FIG. 2. Autoradiographic image of electrophoretically separated proteins from protein lysates of L cells carry a4-EBNAl chimeric genes. Proteins from each of the cloned cell lines converted to TK+ phenotype by S2.5, S2.6, or 52.9 were separated on SDS-8.5% polyacrylamide gels, transferred to nitrocellulose, and stained with EBNAl positive human antiserum. TK+ cloned cell lines were designated L2.5, L2.6, or L2.9 according to the length of the EBV insert. The numbers following the dash designate a unique clone. IB4 is a latently infected lymphoblastoid cell line derived by immortalization of neonatal cord blood cells with the B95-8 EBV isolate (King et al, 1980) L334-6 is a clone of TK+ L cells converted to TK+ phenotype with the parental plasmid pRB334 and therefore did not contain EBV DNA sequences.
HUMMEL
342 A. Anti - EBNAl
ET AL. B. Anti-HSVa4,aO
,EBNA 1
FIG. 3. Autoradiographic images of EBNAl, a4, and (~0 proteins in lysates of L2.5-6 cell line harvested at different times after infection with fe502A305. The cells were harvested at 0,5,10, and 24 hr postinfection. Proteins were separated on SDS-8.5% polyacrylamide gels transferred to nitrocellulose and reacted with (A) human serum containing antibody to EBNAl or (B) with a mixture of monoclonal antibodies to cu4, and a0 (Ackerman et al, 1934). L334-6 cells are mouse Ltk- cells transfected with the vector alone. Approximately equivalent amounts of protein as determined by staining replicate blots with amido black were loaded in each lane. Uninduced L2.56 cells make approximately the same amount of EBNAl as IB4 cells (Fig. 2). In this example it appears that L2.5-6 cells harvested at the time of infection (0 hr) have less EBNAl than IB4 cells because cellular protein present in the HSV preparations has been added to L2.5-6 cells and this dilutes the amount of EBNAl in the L2.5-6 cells. Comparison of L2.5-6 cells at 0 and 5 hr p.i., shows that the HSV infected cell accumulated lo-fold higher levels of EBNAl. No virus protein was added to mockinfected L2.5-6 cells (M).
of EBNAl paralleled that of the a4 protein and the two genes appeared to be regulated in the same fashion (Fig. 3B). Approximately fivefold higher EBNAl levels were attained in cells incubated at 39” relative to those observed in infected cultures incubated at the permissive temperature (3’7”; Fig. 3A and data not shown). The maximum level of EBNAl accumulating in the infected cells was lo- to 20-fold higher than the amount detected in uninfected cells. However, the accumulated EBNAl protein was not stably associated with the infected cell inasmuch as by 24 hr after infection EBNAl was barely detectable in the experiment shown in Fig. 3A. In other experiments, EBNAl and (~4 proteins accumulated in increasing amounts during incubation at 39” for up to 24 hr. Similar levels of EBNAl induction were seen following HSV infection of clones of L2.6 and
L2.9 cell lines, indicating that EBNAl expression was regulated by the promoterregulatory domain of the a4 gene (Fig. 4). It should be noted that the expression of EBNAl in L2.5, L2.6, and L2.9 clones infected with HSV-1 appears artifactually lower in Figs. 3 and 4 since cell protein has been considerably diluted by the protein added with the preparation of infecting virus. Southern blot hybridizations of the electrophoretically separated restriction digests of each L cell clone DNA with an EBNAl specific, q-labeled probe indicated the presence of one to three copies of the transfected plasmid per cell. The a4EBNAl chimeric portion of the plasmid DNA resident in the cellular genome was in almost all instances identical in size to the (~4-EBNAl chimera contained in the plasmids used for transfection. The L2.9-
EBV NUCLEAR * 2.942
2.9-2 2.6-132.6-11
2.5-9 2.5-6
L334
g(50(15~011501(5o(Is0115
- EBNAl
FIG. 4. Autoradiographic images of EBNAl in lysates of Ltk+ cells infected with ts502A305 at a multiplicity of 5 PFU per cell. The infected cells were harvested for protein at the time of infection and at 5 hr postinfection. Proteins were electrophoretically separated in denaturing 8.5% gels, transferred to nitrocellulose, and stained with human serum containing antibody to EBNAl. L334-6 cells are mouse Ltkcells transfected with the vector alone.
12 cell line (see below) contained one copy of an anomalous, more slowly migrating a4-EBNAl gene sequence (data not shown). Construdian of a HSV-1 vedor exprating EBNAl gene. The procedure for the construction of the HSV-1 vector carrying the chimeric (w4-EBNAl genes is illustrated in Fig. 5. Specifically, intact HSV-l(F) DNA was cotransfected into rabbit skin cells with the DNA of a plasmid containing a TK gene from which the 490-bp BgZII-Sad fragment was replaced with the a4-EBNAl gene. The TK- viruses selected after three sequential plaque purifications were shown to contain an insert in the TK gene by restriction endonuclease digestion of their DNA. The recombinant viruses grew similarly to their parent. As expected, they induced the expression of EBNA in infected Vero cells (Fig. 6). As noted under Materials and Methods, HSV-l(F) carries a ts lesion in the a4 gene (Roizman et al, 1981). The chimeric EBNAl gene was expressed as an LYgene inasmuch as it was overproduced in cells at the nonpermissive temperature and was shut off when cells expressed B and y proteins at
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the permissive temperature (Fig. 6). There was no difference in the HSV-1 proteins expressed from the parent or recombinant virus at the permissive or nonpermissive temperature, or in cells infected with these viruses and labeled with FSJmethionine at different times following removal of cycloheximide added to the medium 30 min before infection and for 5 hr thereafter (Fig. 6 and data not shown). Furthermore, parent or recombinant viruses grew to similar titers in Vero cell cultures. In contrast to the L cell clones carrying the chimeric a4-EBNAl, which upon induction by infection with HSV-1 produced lo- to 20-fold higher levels of EBNAl protein, Vero cells infected with the recombinant virus gene produced 50- to lOO-fold higher levels of EBNAl than is expressed in IB4 cells or in the uninfected recombinant L cell clones. The difference between the 50- to loo-fold higher level of expression of EBNAl from the recombinant virus as opposed to lo- to 20-fold higher levels of expression induced by virus infection of recombinant L cells could reflect gene dosage inasmuch as the multiplicity of infection of Vero cells was 20-40 PFU per cell whereas the L cell clones contained two to three integrated genes per cell. Alternatively, EBNAl gene expression could be affected by the environment of the gene, i.e., its site of integration in host cell or viral DNA. The existence of a clone of L cells expressing two forms of EBNAl, one of which is anomalously large (L2.9-12 cells, Fig. 2), enabled us to investigate the relative expression of EBNAl from viral and cell DNA loci of the same infected cell. L2.9-12 cells, which express both a normal and larger size EBNAl from integrated chimeric genes were infected at the nonpermissive temperature with 20 PFU of recombinant R2.5, which expresses a normal EBNAl. The results (Fig. 7) show that the time course of EBNAl induction parallels that of the (~4gene: at 24 hr postinfection EBNAl accumulated in large quantities from both the integrated copy (higher molecular weight band) and the viral copy of the EBNAl gene. Furthermore, the level of expression from both loci approximated the gene dosage, since there
HUMMEL
344
EBNAI A+---
PO4 B/S
HP A.
.s2.9/!32.5
---if
ET AL.
-\
‘\
P , I’
-\ Sl ‘y/y
/’ hsc
----
B. pRB3166
----c
----_
C. pR82.9/pRB2.5
0 -----
X
--PTK I-__ (1 b
D. HSV-I(F)
-
E. R2.9lR2.5
=--/Y
-pa4-.._
-.___--
ATK
_-I
___---
-__---
b’ a’
a’ c’
co 3
b’ d
d C’
co 3
C
I,
---aATK
I, Pa4
EBNAI
PTK
FIG. 5. Diagrammatic representation of the construction of recombinant HSV-1 viruses carrying a4-EBNAl chimeric genes. (A) Plasmids S2.5 and S2.9 carrying the a4-EBNAl chimeras (see Fig. 1). Tbe direction of gene transcription is indicated by arrow. (B) Plasmid pRB3166 used as the vector for recombination-insertion of the o4-EBNAl chimera into the HSV-1 genome (Shih et al, 1934). (C) Plasmids pRB2.9 and pRB2.5 containing the 4125- and 3’761-bp PvuII fragments (a4-EBNAl) from plasmid S2.9 and S2.5, respectively, cloned into the XbaI site of pRB3166. The arrows indicate the direction of gene transcription. (D) Representation of the prototype arrangement of the HSV1 genome. The open boxes ab and b’a’ represent the inverted repeats flanking the L component whereas the open boxes a% and ea represent the inverted repeats flanking the S component of the HSV-1 genome. The hatched box indicates the position of the TK gene. (E) Sequence arrangement of the recombinant viruses R2.9 and R2.5. The hatched boxes indicate the position of the TK gene and the open boxes the a4-EBNAl chimera. The arrows indicate the direction of transcription. H = Hi&II, P = &II, S = Sot&A, B = BornHI, Sl = S&I, X = XbaI, SC = SacI, Xho = %I, pTK = promotor of the TK gene, pa4 = promotor of the o4 gene, ATK = remaining portion of the structural TK gene after recombination.
is a single cellular copy of an anomalous large a4/EBNAl gene and one cellular copy and 20 viral copies of the normal size a4EBNAl gene per cell. DISCUSSION
The experiments described in this report were done for two reasons, i.e., to exploit chimeric constructs resident in cells or virus vectors as potential sources of large amounts of EBV proteins for studies of their function and to determine the effect of EBNAl on lytic infections by HSV-1. With respect to the first objective, the results presented in this study demonstrate that the EBV EBNAl gene can be stably maintained in the HSV-1 genome as a chi-
merit gene consisting of the 5’ transcribed noncoding and coding sequences of the EBNAl gene fused to the promotor regulatory domain, capping site, and 28 bp of the 5’ transcribed noncoding sequences of the HSV-1 ar4gene. The expression of the chimeric (r4-EBNAl gene does not interfere with HSV gene expression or replication. Furthermore, the recombinant virus expressed 50- to lOO-fold more EBNAl protein in the infected cells than is ordinarily expressed in EBV transformed lymphocytes. Thus, the vector is useful for production of EBNAl protein for studies of its biochemical and immunological properties or for studies of the biologic effects of EBNAl in microinjected lymphocytes. Further, since the HSV-1 a! promo-
EBV A%-METHt0NINE
NUCLEAR
8. Anti-EBMAl
NPNPNP
NPNPNP
GL
EO:B IO.11 EBNAl
FIG. 6. Autoradiographic images of electrophoretically separated proteins from lysates of Vero cells mock infected or infected with ts502A305. Vero cell culture were mock infected or infected with t%502A395 or recombinant viruses R2.5 and R2.9 and maintained at 33” (P) or 39’ (N). The cells were labeled with FSJmethionine (New England Nuclear; 900 mCi/ mmol) 3 to 20 hr postinfection or mock infection. Proteins from lysates of cells harvested at harvested 20 hr postinfection were electrophoretically separated in SDS-lo% polyacrylamide gels, transferred onto nitrocellulose, and stained with human serum containing antibody to EBNAl and ‘%I-labeled protein A (New England Nuclear). (A) Shows both %l- and ?-labeled protein. ~SJMethionine-labeled a4 protein, barely visible in these reproductions, is just above the darker bands of the major capsid protein (infected cell protein No. 5; ICP 5) and ribonucleotide reductase (infected cell protein No. S; ICP 6). (B) Shows relatively enhanced lesI (EBNAl) signal after blocking a% emissions with an interposed piece of film. The mock infection profile was the same at both temperatures.
tors function in a variety of cells, the recombinant HSV can be used to introduce an active EBNAl gene into a variety of cell types, including lymphocytes. Since HSV replicates in most cell types and kills cells, only transient effects of EBNAl could be studied with the unmodified vector. However, a vector modified by uv inactivation of lytic HSV replication or by deletion of an essential HSV (Yor fi gene could be used to efficiently induce EBNAl expression without HSV replication and cell death. The second objective rests on the homology of several HSV-1 genes to open
ANTIGEN
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reading frames in the EBV genome, suggesting that these viruses may be more closely related than originally thought, and on the apparent function of the EBNAl gene. Specifically, recent studies indicate that one important function of the EBNAl gene is to maintain EBV DNA as an episome (Yates et aL, 1985). Further, 1 kb of EBV DNA was defined which in ti permits perpetuation of DNA episomes in cells expressing only the EBV EBNAl gene in trum (Yates et aL, 1984,1985). It has been reported that HSV-1 is also retained as an episome in a latent state in the dorsal root ganglia (Fraser et a& 1984) and conceivably might have similar cis and trans acting genetic elements. If there was homology between the EBV c&acting site and an analogous site in HSV DNA, EBNAl expression might have been expected to interfere with HSV replication. The absence of the interA.Anti- EBNAl
8. Anti-HSVa4,oO e E
R2.512.~12 5 0 1024
ICP4 ICPO EBNAl
FIG. ‘7. Autoradiographic images of electrophoretically separated EBNAl protein from the L2.9-12 cell lysates infected with the R2.5 recombinant virus. (A) The TK+ cell clone L2.9-12, which expressed an anomalously large EBNAl protein and a normal size EBNAl protein, was infected with R2.5, an EBNAlexpressing recombinant and maintained at the nonpermissive temperature (39”). Infected cell proteins were separated on an 8% acrylamide gel and transferred onto nitrocellulose for immunostaining with EBNAl reactive human serum (A) or with monoclonal antibody against HSV-1 (~4 and Cueproteins (Ackerman et d, 1934).
346
HUMMEL
ference reported here may reflect the overriding induction by HSV-1 (Y proteins of the /3 genes and subsequent HSV-1 DNA synthesis or the absence of a c&r-acting site recognized by EBNAl. The observation that EBNAl can be stably maintained and expressed suggests that it might be feasible to maintain replication defective HSV-1 mutants in nonpermissive cells or subgenomic components of HSV-1 DNA in an episomal state by creating recombinants with the EBNAl gene and the EBV cis-acting sequence. Such episomal DNA structures could be very useful for the study of the expression and function of both HSV and EBV genes expressed in the latent states. Since the HSV-1 genome can carry up to 25 kbp of foreign sequences (Poffenberger et aL, 1983) the insertion of several EBV genes ordinarily expressed late in infection is feasible. HSV-l/EBV recombinant viruses could also be utilized to overcome the difficulty in studying genes involved in productive EBV infection. It may be possible to exploit the existing library of HSV conditional lethal mutants and the common evolutionary origin of EBV and HSV to achieve genetic complementation. The similarities between EBV and HSV ribonucleotide reductase, DNA polymerase, and glycoprotein genes suggest that this approach may be feasible.
ET AL. P. L., FARRELL,P. J., GIBSON,T. G., HATFULL, G., HUDSON,G. S., SATCHWELL,S. C., SEGUIN,C., TUFFNELL, P. S., and BARRELL, B. G. (1984). DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London) 310,207-211. BATTERSON,W., and ROIZMAN,B. (1983). Characterization of the herpes simplex virion-associated factor responsible for induction of alpha genes. J. Firol 46,371-377. BURNET~E,W. (1981). Western blotting: Electrophoretie transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. And Biochem 112,195203. CAMPBELL,M. E. M., PALFREYMAN,J. W., and PRESTON, C. M. (1984). Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription. J. lkfd Biol 180, 1-19. CAMPIONE-PICCARDO, J., RAWLS,W. E., and BACCHE~TI, S. (1979).Selective assay for herpes simplex viruses expressing thymidine kinase. J. vird 31,281-287. DAMBAUGH,T., BEISEL, C., HUMMEL, M., KING, W., FENNEWALD,s.. CHEUNG,A., HELL&R, M., RAABTRAUB,N., and KIEFF, E. (1980). Epstein Barr Virus (B95-8) DNA. VII. Molecular cloning and detailed mapping. Pmt. Natl. Ad Sci USA 77,2999-3003. DAMBAUGH,T., HENNESSY,K., FENNEWALD,S., and KIEFF, E. (1985). The EBV genome and its expression in latent infection. In “The Epstein Barr Virus, Recent Advances” (M. Epstein and B. Achong, eds.), pp. 69-98. Heinemann, London, England. EJERCITO,P., KIEFF, E., and ROIZMAN,B. (1968). Characterization of herpes simplex virus strains differing in their effects on social behavior of infected cells. J. Gen Viral 2,357-364.
ACKNOWLEDGMENTS These studies were aided by Grants CA 17281, CA 08494,and CA 19264from the National Cancer Institute, the United States Public Health Service, and by Grants MV 321 and MV2 from the American Cancer Society. M.H., and A.M. were supported by National Research Service Awards AI 07182, and GM 0’7197, respectively. M.A. is a fellow of the Damon RunyonWalter Winchell Cancer Fund, DRG 01’7.E.K. is the recipient of a Faculty Research Award from the American Cancer Society. REFERENCES AC-, M., BRAUN,D., PEREIRA,L., and ROIZMAN, B. (1984).Characterization of Herpes Simplex Virus 1 alpha proteins 0,4 and 27 with monoclonal antibodies. J. &oL 52,108-118. BAER,R., BANKER, A. T., BIGGIN, M. D., DEININGER,
GERBER,P., WHANG-PENG,J., and MONROE,J. (1969). Transformation and chromosome changes induced by Epstein-Barr virus in normal human leukocyte cultures. Proc. Natl. Acud Sci USA 63,740-747. GIBSON,T., STOCKWELL,P., GINSBURG,M., and BARRELL,B. (1984). Homology between two EBV early genes and HSV ribonucleotide reductase and 38K genes. Nucleic Acids Res. 12.5087-5099. GRAHAM,F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. virdoglv 52,456-467. HEARING,J., NICOLAS,J., and LEVINE, A. (1984). Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes. Proc Nat1 Ad Set USA 81. 43734377. HELLER,M., DAMBAUGH,T., and KIEFF, E. (1981). Epstein-Barr virus DNA. IX. Variation among viral DNAs from producer and nonproducer infected cells. J. I&o1 38,632-648. HEZLER, M., VAN SANTEN,V., and KIEFF, E. (1982).
EBV NUCLEAR Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections. J. ViroL 44,311-320. HENLE, W., DIEHL, V., KOHN, G., ZURHAUSEN,H., and HENLE, G. (1967). Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. S&ence (Washington, D. C.) 157,1064-1065. HENNESSY,K., FENNEWALD,S., HUMMEL,M., COLE,T., and KIEFF, E. (1984). A membrane protein encoded by EBV in latent growth transforming infection. Proc NatL Acad Sci USA 81,7207-7211. HENNESSY,K., FENNEWALD,S., and KIEFF, E. (1985). A third viral nuclear protein in lymphoblasts immortalized by Epstein Barr Virus. Proc Nati Ad Sci USA 82,5944-5948.
HENNESSY,K., HELLER,M., VANSANTEN,V., and KIEFF, E. (1983).Simple repeat array in Epstein-Barr virus DNA encodes part of the Epstein-Barr nuclear antigen. Science (Washingtan, D. C.) 220,1396-1398. HENNESSY,K., and KIEFF, E. (1983). One of two Epstein-Barr virus nuclear antigens contains a glytine-alanine copolymer domain, Proc Natl Ad Sci. USA 80,5665-5669.
HENNESSY,K., KIEFF, E. (1985). A second nuclear protein is encoded by Epstein Barr Virus in latent infection. Science (Washingtun, D. C.) 227,1238-1240. HERZ,C., and ROIZYAN,B. (1983). The a promoter regulator-ovalbumin chimeric gene resident in human cells is regulated like the authentic a4 gene after infection with herpes simplex virus 1 mutants in a4 gene. CeU33,145-151. JENKINS,F. J., CASADABAN,M., and ROIZMAN,B. (1985). Application of the mini Mu phage for target sequence specific insertional mutagenesis of the Sci herpes simplex virus genome. Proc. NatL Ad
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domains of herpes simplex virus 1 genes. Proc Natl Sci. USA 81,4065-4069. MACKEM,S., and ROIZMAN,B. (1982). Structural features of the Herpes Simplex Virus alpha gene 4,0 and 27 promoter-regulatory sequences which confer alpha regulation on chimeric thymidine kinase genes. J. vird 44,939-949. MATSUO,T., HELLER, M., PETTI, L., C’SHIRO, E., and KIEFF, E. (1984). Persistence of the entire EpsteinBarr virus genome integrated into human lymphocyte DNA. Science (Wushinghm, D. C.) 226, 13221325. MOCARSKI,E. S., POST,L. E., and ROIZMAN,B. (1980). Molecular engineering of the herpes simplex virus genome: Insertion of a second L-S junction into the genome causes additional genome inversions. Cell 22,243-255. MUNYON,W., KRAISELBURD,E., DOWIS,D., and MANN, J. (1971).Transfer of thymidine kinase to thymidine kinaseless L cells by infection with ultraviolet-irradiated herpes simplex virus. J. ViroL 7,813-820. PERSSON, R. H., BACCHFITI,S., and SMILEY,J. R. (1985). Cells that constitutively express the herpes simplex virus immediate-early protein ICP4 allow efficient activation of viral delayed-early genes in trans. J. Ad
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54.414421.
POFFENBERGER, K. L., TABARES,E., and ROIZMAN,B. (1983). Characterization of a viable, noninverting herpes simplex virus 1 genome derived by insertion and deletion of sequences at the junction of components L and S. Prop NatL Acad Sci USA 80,26902694. POPE,J., HORNE,M., and SCOTT,W. (1968). Transformation of fetal human leukocytes in vitro by filtrates of a human leukaemic cell line containing herpes-like virus. Int. J. Cancer 3,857-866. USA 82,4773-4777. POST,L., MACKEM,S., and ROIZMAN,B. (1981). Regulation of alpha genes of herpes simplex virus: KIEFF, E., DAMBAUGH,T., HENNESSY,K., FENNEWALQ Expression of chimeric genes produced by fusion of S., HELLER,M., MATSUO,T., and HUMMEL,M. (1984). thymidine kinase with alpha gene promoters. Cell Latent infection and growth transformation by Epstein Barr virus. In “The Role of Viruses in Hu24,555-565. man Cancer” (Giraldo and Beth, eds.), pp. 103-118. POST,L. E., NORRILD,B., SIMPSON,T., and ROIZMAN, B. (1982).Chicken ovalbumin gene fused to a Herpes Elsevier, New York. Simplex virus alpha promoter and linked to a thyKIEFF, E., DAMBAUGH, T., KING, W., HELLER, M., midine kinase gene is regulated like a viral gene. CHEUNG,A., VAN SANTEN,V., HUMMEL,M., BEISEL, C., and FENNEWALD,S. (1982). Biochemistry of EpMoL Cell BioL 2.233-240. stein-Barr virus. In “The Herpesviruses” (B. Roiz- ROBERT,M., SHEDD,D., WEIGEL, R., FISCHER,D., and MILLER,G. (1984). Expression in COS-1 cells of Epman, ed.), Vol. 1, pp. 105-150. Plenum, New York. stein-Barr virus nuclear antigen from a complete KING, W., POWE:LL,A., RAAB-TRAUB,N., HAWKE, M., gene and a deleted gene. J. ViroL 50,822~831. and KIEFF, E. (1980). Epstein Barr virus RNA. V. ROIZMAN,B., BAT~ERSON, W., and KNIPE, D. M. (1981). Viral RNA in restringently infected, growth transThe potential significance of late herpes simplex formed cells. J. Viral 36, 506-518. virus functions expressed early in the replication: KOZACK,M. (1984). Compilation and analysis of seThe tale of a temperature sensitive mutant. In quences upstream from the translational start site “Perspectives in Virology XI,” pp. 77-92. Liss. New in eukaryotic RNAs. Nucleic Acids Res. 12,857-872. York. KRISTIE, T. M., and ROIZYAN,B. (1984). Separation of sequences defining basal expression from those SHIH, M.-F., ARSENAKIS,M., TIOLLAIS, P., and ROIZMAN, B. (1984). Expression of hepatitis B Virus S conferring gene recognition within the regulatory
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gene by herpes simplex virus type 1 vectors carrying alpha and beta regulated gene chimeras. PWX NaU Ad
Sci 81,5867-58’70.
SO~RN, E. M. (1975).Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mdec. Bid 98,503-517. SUMMERS,W., GROGAN,E., SHEDD, D., ROBERT,M., LIU, C.-R., and MILLER, G. (1982). Stable expression in mouse cells of nuclear neoantigen after transfer of a 3.4-megadalton cloned fragment of EpsteinBarr virus DNA. Proc N&L Acad Sci USA 79,56885692.
WEIGEL,R., and MILLER, G. (1985). Latent and viral replicative transcription in vivo from the BamHI
ET AL. K fragment of Epstein-Barr virus DNA. J. Vim! 54,591~598. YATES,J., WARREN,N., REISYAN, D., and SUGDEN,B. (1984).A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Prcc Natl Ad Sci USA 81,3806-3810. YATES,J., WARREN,N., and SUGDEN,B. (1985). Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells. Nature (London) 313,812-815. ZINN, K., DIMAIO, D., and MANIATIS, T. (1983). Identification of two distinct regulatory regions adjacent to the human @-interferon gene. CeU34,865-879.
148,337-348
(1986)
Herpes Simplex Virus Expressing MARY HUMMEL,
Epstein-Barr
Virus Nuclear Antigen 1
MINAS ARSENAKIS, ANDREW MARCHINI, BERNARD ROIZMAN, AND ELLIOTT KIEFF’
The Marjorie
B. Km&r Vim! Oncology Laboratwies, The University 910 East 58th Street, Chicago, Illinois 60637
LINDA
LEE,
of Chicago,
Received July 8, 1985; accepted October 19, 1985 DNA. fragments containing an open reading frame known to encode most or all of the EBNAl protein of Epstein-Barr virus (EBV) were fused in the proper transcriptional orientation to the promoter regulatory domain, capping site, and a portion of the 5’ transcribed noncoding sequences of the HSV-1 (~4 gene of herpes simplex virus 1 (HSV-1). In these constructs 20, 130, or 385 bp of EBV DNA and 28 bp of HSV-1 DNA separated the a4 cap site from a putative initiator codon of the EBNAl gene. The chimeric o4-EBNAl genes were introduced into L cells or recombined into the viral genome using the thymidine kinase selection system. The three chimeric gene constructs resident in the L cell clones expressed a protein indistinguishable from authentic EBNAl with respect to electrophoretic and immunologic properties indicating that the ATG at the beginning of the EBV open reading frame initiated translation of the bonafide EBNAl protein. The chimeric a4EBNAl genes resident in L cells were induced by HSV-1 infection and were regulated as a genes. The chimeric (~4-EBNAl gene recombined into the viral genome was also regulated as an u gene. The recombinant viruses were stable and expressed 50- to loo-fold more EBNAl than is ordinarily expressed in human lymphocytes carrying the EBV genome. EBNAl did not alter the program of HSV-1 protein expression. The utility of the vector is discussed. 0 1986 Academic Press. 1ne. INTRODUCTION
Epstein-Barr virus (EBV) and herpes simplex virus-l (HSV-1) are human herpesviruses with similar virion morphology, genome size, and programs of replication in permissively infected cells and similar size capsid proteins (for review see Kieff et ccZ.,1982). Although there is neither antigenie relatedness nor extensive DNA homology between these two viruses, there is considerable similarity in the nucleotide sequences of their terminal direct repeats and in the amino acid sequence of some of their genes, including ribonucleotide reductase, DNA polymerase, and a glycoprotein (Baer et al, 1984; Gibson et aL, 1984; Matsuo et al., 1984). Thus, at least a part of the genomes of these two herpesviruses arose from a common progenitor. i Author to whom requests for reprints addressed.
should be
There are, however, many biological differences that might make recombinants between the two viruses of interest. While HSV replicates efficiently in a wide variety of cells in culture, the host range of EBV is limited to human and some primate B cells; and there is usually no virus replication (Gerber et al, 1969; Henle et al, 1967; Pope et al, 1968). Because EBV replicates poorly in cultured cells, genetic analyses to define functions of viral genes have not been feasible. Expression of EBV genes by a heterologous virus vector such as HSV1 in a wide variety of cells may enable such analyses to be done. Whereas EBV can efficiently transform the cells it infects into immortalized cell lines, HSV transforms cells at a very low frequency. Therefore the immortalizing genes of EBV could be studied against a low background of spontaneous or HSV-1 induced transformation. EBV DNA and four viral encoded gene products can be detected in EBV-trans337
0042-6822/86 $3.00 Copyright All rights
Q 1986 by Academic Press, Inc. of reproduction in any form reserved.
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ET AL.
formed cells (Hennessy and Kieff, 1983, in the a4 gene (Post et aL, 1981). The virus 1985; Hennessy et aL, 1983, 1984, 1985). stocks were grown in HEp-2 cells. All virus These proteins are thought to be important titration, plaque purification, and stocks of either singly or in combination, in altering recombinant viruses were made in Vero cell the growth properties of EBV-transformed cultures. The human 143tk- cells (Camto the pione-Piccardo et aZ., 1979) were used for cells in vitro, and in contributing malignancies associated with EBV infecTK- virus selection. tion: Burkitt’s lymphoma and nasophaCloning the EBNAl gene into a TK seryngeal carcinoma. The role of these EBV lection vector. The 2.9-kbp BamHI-Hind111 genes in growth transformation could be subfragment of BamHI K (pDK225) was established by analysis of the effects of isolated by electroelution from an agarose conditional mutations in these genes. Also, gel slice (Dambaugh et ak, 1980). This fraghigher level expression of these genes ment was partially digested with Sau3A would facilitate the biochemical characand the products were cloned into the terization of the proteins they encode. BamHI and Hind111 sites of pRB334 (Fig. HSV could be a useful vector for studying l), a derivative of pBR322 which has the EBV genes since it has a broad host range HSV-1 TKPvuII fragment, the HSV-1 a4 and current studies suggest that it could promotor, a BamHI site 3’ to the (~4 procarry at least 25 kbp of foreign DNA. Furmotor, and the pBR322 Hind111 site 3’ to ther, several classes of HSV promotors the BamHI site (Post et al., 1982). Clones have been defined so that EBV genes could were isolated which varied in their insert length depending on the Sau3A site which be expressed at different levels and at different stages of HSV infection. Moreover, delined the 5’ end of the insert. Clone S2.9 the stability and long-term potential for contained the entire 2926-bp BamHI to expression of HSV genes resident in cell Hind111 fragment whereas S2.6 and S2.5 genomes (Munyon et a& 1971; Persson et contained 2671 and 2561 bp of EBV DNA 5’ to the Hind111 site, respectively (Baer et ak, 1985) suggest that it might be possible al, 1984). These constructs placed the first to establish host range mutants of HSV carrying EBV genes which could replicate codon of the EBNAl open reading frame and express HSV lytic functions only in 413, 158, and 48 bp downstream from the cells stably expressing the HSV gene. In HSV-1 a4 gene transcription initiation site order for HSV to serve as a suitable vector (Mackem and Roizman, 1982; Baer et aL, for the introduction of EBV genes, it is 1984). necessary that these genes be stable in Transfection into L cells. Ltk- cells(3 X lo6 HSV and not interfere with its replication. cells per dish) were transfected with 50, We now report the results of a series of 100, or 150 ng of S2.9, S2.6, S2.5, or pRB334 experiments in which the EBV EBNAl DNAs and 20 pg of salmon sperm DNA gene, a gene expressed in EBV-immortalcarrier by calcium phosphate precipitation ized lymphoblastoid cells, was recombined (Graham and van der Eb, 1973). After 2 into the HSV genome. The EBV gene is ex- days in nonselective medium, cells expressed from the HSV genome at high lev- pressing TK activity were selected by els, but does not interfere with HSV rep- growth in HAT medium (lop4 M hypoxanlication. thine; 1.6 X 10m5M thymidine; 4.4 X lo-’ M aminopterin). Two weeks after transMATERIALS AND METHODS fection two or three foci were picked from Vimses and cells. Strain F [HSV-l(F)] is each dish. The average transfection effithe prototype HSV-1 strain used in our ciency was 580 cell foci/hg DNA. The TK+ laboratories (Ejercito et al, 1968). Like cell lines carrying the (u4-EBNAl genes most HSV-1 isolates passaged a limited were designated by the length of the EBV number of times in cell culture it carries a insert in the transfected DNA and a unique ts lesion in a4 gene. ts502A305 is a double number, e.g., L2.9-1, etc. mutant containing a 700-bp deletion in the Analyses of the L ceUclones carrying the thymidine kinase (TK) gene and a ti lesion &EBNA 1 chime& genesfor EB V Protein
EBV
NUCLEAR
and DNA. For protein analysis, cells were washed in PBS and resuspended in 6% SDS, 2 n-&f /3-mercaptoethanol, 140 mM Tris, pH 7.0,28% glycerol, 0.07% bromophenol blue, briefly sonicated to reduce viscosity and boiled for 5 min. Proteins were electrophoretically separated on SDS-polyacrylamide bisacrylamide gels and transferred to nitrocellulose (Burnette, 1981). Total protein was visualized in some cases by staining replicate nitrocellulose filters with amido black. EBNAl was detected by immunostaining with B. Thorpe sera (1:40 dilution) which had been preabsorbed with extracts of an EBV negative lymphoblastoid cell line (Hennessy and Kieff, 1983). HSV-1 a4 and a0 proteins were detected with a mixture of monoclonal antibodies, H640 and H1083 (kindly provided by Lenore Pereira) diluted 1:lO and 150, respectively (Ackerman et aL, 1984). IgG-binding sites were identified with ‘%I-labeled Staphykmccus aureus protein A (New England Nuclear, Boston, Mass.). For DNA analysis, cells were lysed in situ in 4.0 ml of 10 mM EDTA, 50 mM Tris, pH 7.4, 1% SDS, and 100 pg/ml proteinase K (Sigma, St. Louis, MO.). The mixture was heated at 55” for 30 min and DNA was separated by two successive isopycnic bandings in neutral cesium chloride (Heller et aL, 1981). DNA was digested with a 2.5fold excess of restriction enzyme for 4 hr at 3’7” along with phage X DNA (New England Biolabs, Beverly, Mass.) to monitor the extent of digestion. Five micrograms of cell DNA or an aliquot of plasmid DNA equivalent to one plasmid copy per diploid genome was loaded onto each lane of a 0.4% agarose gel. After electrophoresis and a 20min depurination in 0.25 M HCl, gels were blotted (Southern, 1975) onto Genescreen Plus (New England Nuclear). DNA blots were prehybridized in 25 m&l Tris, pH 7.5, 0.02%polyvinyl-pyrrolidone and Ficoll400, 1% SDS, 6 X SSC (pH 7.5) and 250 pg/ml denatured salmon sperm DNA, for 5-6 hr and hybridized for 18 hr at 76’ with 2 X lo7 cpm r2P]GTP-labeled cRNA per milliliter. Labeled cRNA was transcribed by SP6 polymerase from the AvaI-Hind111 fragment of EBV BamHI K which had been cloned into pSP64 (Zinn et aL, 1983).
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Induction of &EBNAl gene expression by HSV iqfectzk Cells (3 X 106) were infected (10 PFU/cell) with ts502A305. After 1 hr absorption at 0”, the cells were incubated for various times at 39” and harvested for protein analysis. Zero time points were taken by harvesting the cells immediately after adding virus. Mock-infected cells were treated the same way as infected cultures except that no virus was added. Cmtmtion of HSV-1 vectors currying the chimeric c&EBVAl gene. The 3761-bp PwuII fragment from plasmid S2.5 and the 4125-bp fragment from plasmid S2.9 containing the coding sequence of the EBNAl gene fused in the proper transcriptional orientation to the promotor-regulatory sequenceof the (~4gene of HSV-I were each cloned into the XbaI site of plasmid pRB3166 and the resulting plasmids were designated as pRB2.5 and pRB2.9, respectively. The plasmid pRB3166 was specifically constructed to facilitate the insertion of foreign genes into HSV-1 DNA. The vector contains the HSV-1 BamHI Q fragment in which the 490-bp BglII-Sac1 fragment carrying the 5’ noncoding and coding sequences of the TK gene was replaced by a polylinker containing an XbaI site (Shih et aL, 1984). To construct the recombinant HSV-1 viruses carrying the (YI-EBNAl chimeric genes, pRB2.5 or pRB2.9 DNAs were cut with the restriction enzyme XhoI, which excises the HSV-1 BamHI Q sequences carrying the EBV DNA insert from the plasmid, and ethanol precipitated. DNA (0.5 pg) was cotransfected with 0.5 pg of intact HSV-l(F) DNA into rabbit skin cells, as previously described (Mocarski et aL, 1980). TK- recombinants arising by recombination of BamHI Q sequences flanking the inserted (w4-EBNAl chimeric gene were selected by plating the progeny of the transfection on 143 TK- cells in the presence of 100 pg of BudR per milliliter of medium. After three sequential plaque purification in the presence of BudR, the viral recombinants were grown in Vero cells and their DNA was analysed by restriction enzyme and Southern blot analysis for ar4EBNAl chimeric gene insertion. The re-
HUMMEL
340
combinant viruses carrying the 2.5 and 2.9 kb a4 EBNAl chimeric genes were designated as recombinants R2.5 and R2.9, respectively. Preparation of illfected cell lysates. Vero cells (4 X 10”) were infected with 20-40 PFU per cell and incubated for 20 hr at either 33 or 39”. Infected cell proteins were labeled from 1.5 hr postinfection with [?S]methionine (50 &i/ml of medium; sp act 900 mCi/mmol; New England Nuclear). At 20 hr postinfection, the medium was discarded, the cell monolayers were washed with cold PBS, and then lysed in 2% SDS, 5 mJ4 @-mercaptoethanol, and 50 mAf TrisHCl (pH 7.0) and were stored at -80” until needed. RESULTS
Earlier studies have shown that the EBNAl gene is very likely translated from a 1928-bp open reading frame encoded by a 2-kb exon at the 3’ end of a 3.7-kb RNA (Baer et al, 1984; Heller et d, 1982; Hennessy et aL, 1984;Hennessy and Kieff, 1983; Kieff et aL, 1984; Weigel and Miller, 1985). Specifically, the single potential splice acceptor site, which is probably the 5’ end of the exon, is followed by stop codons and a potential initiating ATG (Baer et aL, 1984). Furthermore, the BamHI K fragment, which begins 385 bp 5’ to the open reading frame, produced a protein which was identical in size to EBNAl when expressed in mammalian cells from plasmids containing SV40 or polyoma virus early promotors (Hearing et aL, 1984; Robert et aL, 1984; Summers et aL, 1982; Baer et aL, 1984). Among the HSV-1 promoters, that of the a4 gene has been extensively characterized (Post et aL, 1981; Mackem and Roizman, 1982; Kristie and Roizman, 1984) and used to express non-HSV genes inserted into the cellular (Post et al, 1982, Herz and Roizman, 1983) and viral genome (Shih et aL, 1984). The choice of the cu4promoter to express EBNAl protein was predicated on the observation that (Ygenes are the first viral genes to be transcribed and expressed in HSV-1 infected cells, and therefore the EBNAl gene would be expressed and its effect on HSV-1 replication would be mea-
ET AL.
sured during the earliest stages of HSV-1 replication. A characteristic of the (Ypromotors is that they are expressed at low but detectable levels when resident in the cellular genome and are induced to a much higher level of expression by an atram inducing factor (aTIF) encoded by the HSV genome and introduced into the cell during infection (Post et aL, 1981; Batterson and Roizman, 1983,Campbell et al, 1984, Pellett et d, 1985). To test the expression of EBNAl in the environment of the cellular and viral genomes, two sets of a4-EBNAl chimeric genes were constructed. The first was introduced into the cellular genome and the cells were tested for EBNAl constitutive expression and for induction of EBNAl expression by HSV-1 infection. The second set of chimeric genes was recombined into the HSV-1 genome. Expression of agEBNA1 chimeric gene resident in cells umverted to TK+ pherwtype. pRB334 consists of pBR322 carrying a HSV-1 TK gene linked to the promotorregulatory domain, capping site, and 28 bp of the 5’ transcribed noncoding sequences of the (~4gene, and a BamHI cloning site immediately 3’ to the (~4 promotor-regulatory domain (Post et aL, 1982). Because the function of the transcribed sequences 5’ to the EBNAl open reading frame was not known, three different subfragments of the EBV BamHI K fragment were inserted into a BamHI site of pRB334 in the correct transcriptional orientation relative to the a4 promotor. These constructs contained 28 bp of the 5’ transcribed noncoding sequences of the a4 gene 3’ to the capping site and 385,130, or 20 bp of EBV DNA 5’ to the putative initiator ATG. There is no other ATG in the 28 bp of a4 gene (Mackem and Roizman, 1982) or in the 20 bp of EBV DNA (Baer et aL, 1984) 5’ to the putative initiator ATG on the smallest construct. The resultant plasmid constructs are designated S2.9, S2.6, and S2.5, respectively (Fig. 1). TK+ L cell clones were selected following transfection of LTK- cells with each of the recombinant plasmids and designated as L2.9, L2.6, and L2.5. Most L cell clones expressed a protein which was reactive with EBNAl positive human sera. The protein
EBV
S2.812.6l2.5 8.3l0.017.8
NUCLEAR
Kbp
FIG. 1. Construction of a4-EBNAl chimeric genes inserted in pRB334. SadA partial digestion products of the EBV BumHI-Hind111 fragment were cloned into the BumHI and H&l111 sites of pRB334 to generate three constructs, S2.5, S2.6, and S2.9, placing the first methionine codon of the EBNAl open reading frame 48, 153, and 413 bp, respectively, downstream from the a4-cap site. The direction and domain of the EBNAl open reading frame is shown by the open arrow. The IR3 region which encodes a glycine alanine copolymer within EBNAl (Hennessy and Kieff, 1933) is indicated by stippling. The putative polyadenylation signal (Baer et al, 1934) is indicated as PA. Immediately 5’ to the EBNAl open reading frame is a splice acceptor site which is followed by stop codons and a typical initiator ATG (Kozack, 1934). pRB334 vector sequences are indicated by the dashed arrow, B, BarnHI; S, &u&A.
ANTIGEN
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was identical in size to EBNAl in lymphocytes from which the open reading frame was derived (Fig. 2). One cell line, L2.9-12 expressed two immunoreactive proteins, one similar in size to EBNAl and one larger. The amount of EBNAl expressed in the L cell clones varied, but none of the clones made more EBNAl protein than that made in the latently infected growth transformed lymphoblastoid cell line IB4 (Fig. 2). Further, clones transfected with plasmid S2.5, expressed about as much EBNAl as clones transfected with S2.6 or S2.9, even though S2.5 has only 20 bp of EBV DNA sequences upstream to the putative initiator codon. To determine whether the resident EBNAl chimeric gene was expressed under the control of the a4 promotor-regulatory domain, the L cell clones were infected with HSV-1 ts502A305 and incubated at 39”. This mutant carries a temperature-sensitive lesion in the Lu$gene, but by 24 hr infected cells incubated at the nonpermissive (39”) temperature accumulate some /3 and y proteins. EBNAl synthesis was induced by HSV infection. This protein accumulated in increasing amounts up to 5-10 hr after infection at 39” (Fig. 3A). In the L cell line tested (L2.5-6), the accumulation
FIG. 2. Autoradiographic image of electrophoretically separated proteins from protein lysates of L cells carry a4-EBNAl chimeric genes. Proteins from each of the cloned cell lines converted to TK+ phenotype by S2.5, S2.6, or 52.9 were separated on SDS-8.5% polyacrylamide gels, transferred to nitrocellulose, and stained with EBNAl positive human antiserum. TK+ cloned cell lines were designated L2.5, L2.6, or L2.9 according to the length of the EBV insert. The numbers following the dash designate a unique clone. IB4 is a latently infected lymphoblastoid cell line derived by immortalization of neonatal cord blood cells with the B95-8 EBV isolate (King et al, 1980) L334-6 is a clone of TK+ L cells converted to TK+ phenotype with the parental plasmid pRB334 and therefore did not contain EBV DNA sequences.
HUMMEL
342 A. Anti - EBNAl
ET AL. B. Anti-HSVa4,aO
,EBNA 1
FIG. 3. Autoradiographic images of EBNAl, a4, and (~0 proteins in lysates of L2.5-6 cell line harvested at different times after infection with fe502A305. The cells were harvested at 0,5,10, and 24 hr postinfection. Proteins were separated on SDS-8.5% polyacrylamide gels transferred to nitrocellulose and reacted with (A) human serum containing antibody to EBNAl or (B) with a mixture of monoclonal antibodies to cu4, and a0 (Ackerman et al, 1934). L334-6 cells are mouse Ltk- cells transfected with the vector alone. Approximately equivalent amounts of protein as determined by staining replicate blots with amido black were loaded in each lane. Uninduced L2.56 cells make approximately the same amount of EBNAl as IB4 cells (Fig. 2). In this example it appears that L2.5-6 cells harvested at the time of infection (0 hr) have less EBNAl than IB4 cells because cellular protein present in the HSV preparations has been added to L2.5-6 cells and this dilutes the amount of EBNAl in the L2.5-6 cells. Comparison of L2.5-6 cells at 0 and 5 hr p.i., shows that the HSV infected cell accumulated lo-fold higher levels of EBNAl. No virus protein was added to mockinfected L2.5-6 cells (M).
of EBNAl paralleled that of the a4 protein and the two genes appeared to be regulated in the same fashion (Fig. 3B). Approximately fivefold higher EBNAl levels were attained in cells incubated at 39” relative to those observed in infected cultures incubated at the permissive temperature (3’7”; Fig. 3A and data not shown). The maximum level of EBNAl accumulating in the infected cells was lo- to 20-fold higher than the amount detected in uninfected cells. However, the accumulated EBNAl protein was not stably associated with the infected cell inasmuch as by 24 hr after infection EBNAl was barely detectable in the experiment shown in Fig. 3A. In other experiments, EBNAl and (~4 proteins accumulated in increasing amounts during incubation at 39” for up to 24 hr. Similar levels of EBNAl induction were seen following HSV infection of clones of L2.6 and
L2.9 cell lines, indicating that EBNAl expression was regulated by the promoterregulatory domain of the a4 gene (Fig. 4). It should be noted that the expression of EBNAl in L2.5, L2.6, and L2.9 clones infected with HSV-1 appears artifactually lower in Figs. 3 and 4 since cell protein has been considerably diluted by the protein added with the preparation of infecting virus. Southern blot hybridizations of the electrophoretically separated restriction digests of each L cell clone DNA with an EBNAl specific, q-labeled probe indicated the presence of one to three copies of the transfected plasmid per cell. The a4EBNAl chimeric portion of the plasmid DNA resident in the cellular genome was in almost all instances identical in size to the (~4-EBNAl chimera contained in the plasmids used for transfection. The L2.9-
EBV NUCLEAR * 2.942
2.9-2 2.6-132.6-11
2.5-9 2.5-6
L334
g(50(15~011501(5o(Is0115
- EBNAl
FIG. 4. Autoradiographic images of EBNAl in lysates of Ltk+ cells infected with ts502A305 at a multiplicity of 5 PFU per cell. The infected cells were harvested for protein at the time of infection and at 5 hr postinfection. Proteins were electrophoretically separated in denaturing 8.5% gels, transferred to nitrocellulose, and stained with human serum containing antibody to EBNAl. L334-6 cells are mouse Ltkcells transfected with the vector alone.
12 cell line (see below) contained one copy of an anomalous, more slowly migrating a4-EBNAl gene sequence (data not shown). Construdian of a HSV-1 vedor exprating EBNAl gene. The procedure for the construction of the HSV-1 vector carrying the chimeric (w4-EBNAl genes is illustrated in Fig. 5. Specifically, intact HSV-l(F) DNA was cotransfected into rabbit skin cells with the DNA of a plasmid containing a TK gene from which the 490-bp BgZII-Sad fragment was replaced with the a4-EBNAl gene. The TK- viruses selected after three sequential plaque purifications were shown to contain an insert in the TK gene by restriction endonuclease digestion of their DNA. The recombinant viruses grew similarly to their parent. As expected, they induced the expression of EBNA in infected Vero cells (Fig. 6). As noted under Materials and Methods, HSV-l(F) carries a ts lesion in the a4 gene (Roizman et al, 1981). The chimeric EBNAl gene was expressed as an LYgene inasmuch as it was overproduced in cells at the nonpermissive temperature and was shut off when cells expressed B and y proteins at
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the permissive temperature (Fig. 6). There was no difference in the HSV-1 proteins expressed from the parent or recombinant virus at the permissive or nonpermissive temperature, or in cells infected with these viruses and labeled with FSJmethionine at different times following removal of cycloheximide added to the medium 30 min before infection and for 5 hr thereafter (Fig. 6 and data not shown). Furthermore, parent or recombinant viruses grew to similar titers in Vero cell cultures. In contrast to the L cell clones carrying the chimeric a4-EBNAl, which upon induction by infection with HSV-1 produced lo- to 20-fold higher levels of EBNAl protein, Vero cells infected with the recombinant virus gene produced 50- to lOO-fold higher levels of EBNAl than is expressed in IB4 cells or in the uninfected recombinant L cell clones. The difference between the 50- to loo-fold higher level of expression of EBNAl from the recombinant virus as opposed to lo- to 20-fold higher levels of expression induced by virus infection of recombinant L cells could reflect gene dosage inasmuch as the multiplicity of infection of Vero cells was 20-40 PFU per cell whereas the L cell clones contained two to three integrated genes per cell. Alternatively, EBNAl gene expression could be affected by the environment of the gene, i.e., its site of integration in host cell or viral DNA. The existence of a clone of L cells expressing two forms of EBNAl, one of which is anomalously large (L2.9-12 cells, Fig. 2), enabled us to investigate the relative expression of EBNAl from viral and cell DNA loci of the same infected cell. L2.9-12 cells, which express both a normal and larger size EBNAl from integrated chimeric genes were infected at the nonpermissive temperature with 20 PFU of recombinant R2.5, which expresses a normal EBNAl. The results (Fig. 7) show that the time course of EBNAl induction parallels that of the (~4gene: at 24 hr postinfection EBNAl accumulated in large quantities from both the integrated copy (higher molecular weight band) and the viral copy of the EBNAl gene. Furthermore, the level of expression from both loci approximated the gene dosage, since there
HUMMEL
344
EBNAI A+---
PO4 B/S
HP A.
.s2.9/!32.5
---if
ET AL.
-\
‘\
P , I’
-\ Sl ‘y/y
/’ hsc
----
B. pRB3166
----c
----_
C. pR82.9/pRB2.5
0 -----
X
--PTK I-__ (1 b
D. HSV-I(F)
-
E. R2.9lR2.5
=--/Y
-pa4-.._
-.___--
ATK
_-I
___---
-__---
b’ a’
a’ c’
co 3
b’ d
d C’
co 3
C
I,
---aATK
I, Pa4
EBNAI
PTK
FIG. 5. Diagrammatic representation of the construction of recombinant HSV-1 viruses carrying a4-EBNAl chimeric genes. (A) Plasmids S2.5 and S2.9 carrying the a4-EBNAl chimeras (see Fig. 1). Tbe direction of gene transcription is indicated by arrow. (B) Plasmid pRB3166 used as the vector for recombination-insertion of the o4-EBNAl chimera into the HSV-1 genome (Shih et al, 1934). (C) Plasmids pRB2.9 and pRB2.5 containing the 4125- and 3’761-bp PvuII fragments (a4-EBNAl) from plasmid S2.9 and S2.5, respectively, cloned into the XbaI site of pRB3166. The arrows indicate the direction of gene transcription. (D) Representation of the prototype arrangement of the HSV1 genome. The open boxes ab and b’a’ represent the inverted repeats flanking the L component whereas the open boxes a% and ea represent the inverted repeats flanking the S component of the HSV-1 genome. The hatched box indicates the position of the TK gene. (E) Sequence arrangement of the recombinant viruses R2.9 and R2.5. The hatched boxes indicate the position of the TK gene and the open boxes the a4-EBNAl chimera. The arrows indicate the direction of transcription. H = Hi&II, P = &II, S = Sot&A, B = BornHI, Sl = S&I, X = XbaI, SC = SacI, Xho = %I, pTK = promotor of the TK gene, pa4 = promotor of the o4 gene, ATK = remaining portion of the structural TK gene after recombination.
is a single cellular copy of an anomalous large a4/EBNAl gene and one cellular copy and 20 viral copies of the normal size a4EBNAl gene per cell. DISCUSSION
The experiments described in this report were done for two reasons, i.e., to exploit chimeric constructs resident in cells or virus vectors as potential sources of large amounts of EBV proteins for studies of their function and to determine the effect of EBNAl on lytic infections by HSV-1. With respect to the first objective, the results presented in this study demonstrate that the EBV EBNAl gene can be stably maintained in the HSV-1 genome as a chi-
merit gene consisting of the 5’ transcribed noncoding and coding sequences of the EBNAl gene fused to the promotor regulatory domain, capping site, and 28 bp of the 5’ transcribed noncoding sequences of the HSV-1 ar4gene. The expression of the chimeric (r4-EBNAl gene does not interfere with HSV gene expression or replication. Furthermore, the recombinant virus expressed 50- to lOO-fold more EBNAl protein in the infected cells than is ordinarily expressed in EBV transformed lymphocytes. Thus, the vector is useful for production of EBNAl protein for studies of its biochemical and immunological properties or for studies of the biologic effects of EBNAl in microinjected lymphocytes. Further, since the HSV-1 a! promo-
EBV A%-METHt0NINE
NUCLEAR
8. Anti-EBMAl
NPNPNP
NPNPNP
GL
EO:B IO.11 EBNAl
FIG. 6. Autoradiographic images of electrophoretically separated proteins from lysates of Vero cells mock infected or infected with ts502A305. Vero cell culture were mock infected or infected with t%502A395 or recombinant viruses R2.5 and R2.9 and maintained at 33” (P) or 39’ (N). The cells were labeled with FSJmethionine (New England Nuclear; 900 mCi/ mmol) 3 to 20 hr postinfection or mock infection. Proteins from lysates of cells harvested at harvested 20 hr postinfection were electrophoretically separated in SDS-lo% polyacrylamide gels, transferred onto nitrocellulose, and stained with human serum containing antibody to EBNAl and ‘%I-labeled protein A (New England Nuclear). (A) Shows both %l- and ?-labeled protein. ~SJMethionine-labeled a4 protein, barely visible in these reproductions, is just above the darker bands of the major capsid protein (infected cell protein No. 5; ICP 5) and ribonucleotide reductase (infected cell protein No. S; ICP 6). (B) Shows relatively enhanced lesI (EBNAl) signal after blocking a% emissions with an interposed piece of film. The mock infection profile was the same at both temperatures.
tors function in a variety of cells, the recombinant HSV can be used to introduce an active EBNAl gene into a variety of cell types, including lymphocytes. Since HSV replicates in most cell types and kills cells, only transient effects of EBNAl could be studied with the unmodified vector. However, a vector modified by uv inactivation of lytic HSV replication or by deletion of an essential HSV (Yor fi gene could be used to efficiently induce EBNAl expression without HSV replication and cell death. The second objective rests on the homology of several HSV-1 genes to open
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reading frames in the EBV genome, suggesting that these viruses may be more closely related than originally thought, and on the apparent function of the EBNAl gene. Specifically, recent studies indicate that one important function of the EBNAl gene is to maintain EBV DNA as an episome (Yates et aL, 1985). Further, 1 kb of EBV DNA was defined which in ti permits perpetuation of DNA episomes in cells expressing only the EBV EBNAl gene in trum (Yates et aL, 1984,1985). It has been reported that HSV-1 is also retained as an episome in a latent state in the dorsal root ganglia (Fraser et a& 1984) and conceivably might have similar cis and trans acting genetic elements. If there was homology between the EBV c&acting site and an analogous site in HSV DNA, EBNAl expression might have been expected to interfere with HSV replication. The absence of the interA.Anti- EBNAl
8. Anti-HSVa4,oO e E
R2.512.~12 5 0 1024
ICP4 ICPO EBNAl
FIG. ‘7. Autoradiographic images of electrophoretically separated EBNAl protein from the L2.9-12 cell lysates infected with the R2.5 recombinant virus. (A) The TK+ cell clone L2.9-12, which expressed an anomalously large EBNAl protein and a normal size EBNAl protein, was infected with R2.5, an EBNAlexpressing recombinant and maintained at the nonpermissive temperature (39”). Infected cell proteins were separated on an 8% acrylamide gel and transferred onto nitrocellulose for immunostaining with EBNAl reactive human serum (A) or with monoclonal antibody against HSV-1 (~4 and Cueproteins (Ackerman et d, 1934).
346
HUMMEL
ference reported here may reflect the overriding induction by HSV-1 (Y proteins of the /3 genes and subsequent HSV-1 DNA synthesis or the absence of a c&r-acting site recognized by EBNAl. The observation that EBNAl can be stably maintained and expressed suggests that it might be feasible to maintain replication defective HSV-1 mutants in nonpermissive cells or subgenomic components of HSV-1 DNA in an episomal state by creating recombinants with the EBNAl gene and the EBV cis-acting sequence. Such episomal DNA structures could be very useful for the study of the expression and function of both HSV and EBV genes expressed in the latent states. Since the HSV-1 genome can carry up to 25 kbp of foreign sequences (Poffenberger et aL, 1983) the insertion of several EBV genes ordinarily expressed late in infection is feasible. HSV-l/EBV recombinant viruses could also be utilized to overcome the difficulty in studying genes involved in productive EBV infection. It may be possible to exploit the existing library of HSV conditional lethal mutants and the common evolutionary origin of EBV and HSV to achieve genetic complementation. The similarities between EBV and HSV ribonucleotide reductase, DNA polymerase, and glycoprotein genes suggest that this approach may be feasible.
ET AL. P. L., FARRELL,P. J., GIBSON,T. G., HATFULL, G., HUDSON,G. S., SATCHWELL,S. C., SEGUIN,C., TUFFNELL, P. S., and BARRELL, B. G. (1984). DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London) 310,207-211. BATTERSON,W., and ROIZMAN,B. (1983). Characterization of the herpes simplex virion-associated factor responsible for induction of alpha genes. J. Firol 46,371-377. BURNET~E,W. (1981). Western blotting: Electrophoretie transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. And Biochem 112,195203. CAMPBELL,M. E. M., PALFREYMAN,J. W., and PRESTON, C. M. (1984). Identification of herpes simplex virus DNA sequences which encode a trans-acting polypeptide responsible for stimulation of immediate early transcription. J. lkfd Biol 180, 1-19. CAMPIONE-PICCARDO, J., RAWLS,W. E., and BACCHE~TI, S. (1979).Selective assay for herpes simplex viruses expressing thymidine kinase. J. vird 31,281-287. DAMBAUGH,T., BEISEL, C., HUMMEL, M., KING, W., FENNEWALD,s.. CHEUNG,A., HELL&R, M., RAABTRAUB,N., and KIEFF, E. (1980). Epstein Barr Virus (B95-8) DNA. VII. Molecular cloning and detailed mapping. Pmt. Natl. Ad Sci USA 77,2999-3003. DAMBAUGH,T., HENNESSY,K., FENNEWALD,S., and KIEFF, E. (1985). The EBV genome and its expression in latent infection. In “The Epstein Barr Virus, Recent Advances” (M. Epstein and B. Achong, eds.), pp. 69-98. Heinemann, London, England. EJERCITO,P., KIEFF, E., and ROIZMAN,B. (1968). Characterization of herpes simplex virus strains differing in their effects on social behavior of infected cells. J. Gen Viral 2,357-364.
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