Identification of Dominant-Negative Mutants of the ... - Journal of Virology

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Department ofMicrobiology and Immunology, Milton S. Hershey Medical Center, Pennsylvania State. University College ofMedicine, P.O. Box 850, Hershey, ...
Vol. 66, No. 4

JOURNAL OF VIROLOGY, Apr. 1992, P. 2261-2267

0022-538X/92/042261-07$02.00/0 Copyright © 1992, American Society for Microbiology

Identification of Dominant-Negative Mutants of the Herpes Simplex Virus Type 1 Immediate-Early Protein ICPO PETER C. WEBER* AND BRIAN WIGDAHL Department of Microbiology and Immunology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, P.O. Box 850, Hershey, Pennsylvania 17033 Received 31 October 1991/Accepted 9 January 1992

ICPO is a 110,000-molecular-weight immediate-early protein of herpes simplex virus type 1 (HSV-1) which is encoded by three exons. It has been shown to function as a promiscuous transactivator of a variety of different HSV-1 and non-HSV-1 promoters in transient expression assays. Analysis of mutations which truncated the carboxy-terminal end of this 775-amino-acid (aa) protein demonstrated that a polypeptide which contained only aa 1 to 553 still possessed significant transactivation potential. Additional carboxy-terminal truncations which sequentially removed aa 245 to 553 and thus the remainder of the third exon resulted in the eventual loss of transactivation capability in these mutants. However, further analysis of these truncated derivatives demonstrated that they behaved as dominant-negative mutants to the wild-type polypeptide. Moreover, one of the mutants was found to act as a promiscuous repressor, in that it could dramatically inhibit a variety of HSV-1 promoters, non-HSV-1 promoters, and heterologous transactivator proteins in transient expression assays, despite having lost almost the entire third exon. These results indicate that a domain encoded by the first two exons probably interacts with, and can effectively titrate, the unknown cellular factor(s) through which ICPO mediates transactivation. Expression of herpes simplex virus type 1 (HSV-1) genes is temporally regulated at the level of transcription, resulting in the appearance of three classes of viral transcripts: immediate-early, early, and late (28, 29). Five immediateearly genes have been identified and are expressed shortly after infection in the absence of de novo viral protein synthesis. These genes code for the proteins ICP4 (molecular weight of 175,000 [175K]), ICPO (110K), ICP22 (68K), ICP27 (63K), and ICP47 (12K). Studies with temperaturesensitive and deletion mutant viruses have been used to demonstrate that ICP4 is required throughout infection for expression of early and late genes (9, 48), ICP27 is required for late gene expression (51), ICP22 is required for late gene expression in some cell lines but not in others (47, 54), and ICPO and ICP47 appear to be dispensable for virus replication in cell culture, although replication of ICPO deletion mutant viruses is somewhat impaired at low multiplicities of infection (33, 52, 58). Cloned immediate-early genes have also been used in transient expression assays to gain a better understanding of the role these proteins play in HSV-1 gene regulation. These studies have shown that the ICP4 and ICPO proteins are potent and promiscuous transactivators of HSV-1 early and late gene promoters (10, 20, 41, 49, 56) as well as non-HSV-1 promoters (10, 38, 39, 42, 43, 55), while the ICP27 protein acts to modulate the stimulatory activity of the ICP4 and ICPO proteins (11, 36, 50, 55). Although a clear understanding of the complexities of HSV-1 gene regulation is not yet at hand, results from mutational analyses of early and late gene promoters (7, 14, 26, 27, 31, 44, 57, 69) and from in vitro transcription systems (1, 35, 67) indicate that the immediate-early proteins probably mediate their stimulatory effects indirectly by modifying host cell factors involved in transcriptional activation. Several experimental observations have suggested that ICPO is the immediate-early protein which plays a critical

*

Corresponding author. 2261

role in the process of HSV-1 reactivation from latent infections in sensory ganglia. ICPO is required for de novo virus production after transfection of infectious viral DNA, a situation which is analogous to the process of in vivo reactivation (3). Furthermore, ICPO deletion viruses have an impaired ability to reactivate from latent infections in mice (6, 32), and ICPO is absolutely required for reactivation from latency in vitro (25, 70). Finally, there is a clear correlation between the ability of defined mutations in the ICPO gene to abolish transactivation in transient assays and their ability to impede virus replication when inserted into the viral genome (15). Taken together, these results suggest that ICPO provides a major transcriptional boost which is required by HSV-1 for efficient reactivation from the latent state. The ICPO gene encodes one of the few spliced transcripts in the HSV-1 genome; its protein-coding sequences are contained in three exons (46). Interestingly, the amino acid sequence of ICPO has been observed to contain several stretches of overrepresented amino acids (4, 17, 46). These include a cysteine-rich region in exon 2 (amino acids [aa] 116 to 157) which was originally postulated to constitute a zinc finger DNA binding domain but has recently been shown to represent a novel motif found in a number of diverse nuclear proteins (18). It is this region of the polypeptide which is the most sensitive to the effects of linker insertion mutations (3, 4, 12, 13) and which is highly conserved in the ICPO homologs of other herpesviruses (5, 46, 60), implying that the cysteine-rich domain is critical for ICPO function. ICPO also contains a serine-rich tract from aa 554 to 591 which is hypothesized to be the major phosphorylation site for the protein (46). Deletion analysis has demonstrated that this portion of the molecule is not required for promoter transactivation (13). In addition, ICPO possesses two regions, aa 1 to 115 and aa 233 to 243, which are rich in acidic amino acid residues. The activation domains of transcriptional regulators such as GAL4, GCN4, and VP16 have been shown to be composed of highly acidic regions (30, 34, 59), so that a

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similar function might be envisioned for these two sections of the ICPO polypeptide. Recently, however, the acidic region at aa 233 to 243 was shown to be dispensable for ICPO function (16). Finally, ICPO contains a large proline-rich region (20% proline content) at aa 241 to 551 which may be functionally homologous to the proline-rich activation domains of transcription factors such as CTF-1, C/EBP, OTF-2, and AP-2 (19, 21, 37, 45, 66). What little is known about structure-function relationships in the ICPO protein has been accumulated through several mutagenesis studies employing transient expression assays. It is clear from these analyses that the amino acid sequences encoded by the second exon are absolutely essential for ICPO function (3, 4, 12, 13), while the carboxy-terminal sequences of the protein may mediate synergistic transactivation with ICP4 in some transfection systems (13) but not in others (4). Additionally, the region of ICPO from aa 475 to 548, and particularly the sequence of highly basic residues from aa 500 to 506, is thought to be important for nuclear localization (13). However, the means by which these various domains of ICPO contribute to promiscuous transactivation are far from clear; several possibilities include interactions with the basic transcriptional machinery of the host cell, reassembly of host cell chromatin into a more activated form, or recompartmentalization of transcription complexes in the nucleus (for a review, see reference 17). A mutational analysis of the ICPO gene was therefore carried out in an attempt to map putative host cell factor interaction domains in ICPO. These studies resulted in the identification of the first dominant-negative mutants of this regulatory protein. Further characterization of these mutants has implicated the first two exons as playing a critical role in interacting with the unknown cellular factor(s) through which ICPO mediates transactivation. MATERIALS AND METHODS Cell culture, transfection procedures, and CAT assays. Vero cells were used in all transfection experiments and were grown in minimum essential medium supplemented with 5% fetal calf serum. Transfection of plasmid DNA was performed by calcium phosphate precipitation as described previously (56), except that the transfected cells were not subjected to glycerol or dimethyl sulfoxide shock, in accordance with the procedure of Everett (14). Equimolar amounts (approximately 2 ,ug) of each plasmid were transfected, and all concentrations were verified by agarose gel electrophoresis prior to transfection. In most cases, two cesium chloride gradient-purified plasmid DNA stocks were tested for each construct, and at least two transfections were carried out for each stock. At 48 h after transfection, cells were lysed and extracts were prepared by a detergent extraction procedure (48a). Briefly, the cells were washed three times in TM buffer (2 mM MgCl2, 20 mM Tris-HCl [pH 7.5]), lysed for 5 min with 0.5 ml of lysis buffer (0.1% Triton X-100, 0.25 M Tris-HCl [pH 8.0]), scraped into microcentrifuge tubes, and spun for 5 min. The supernatants were heated at 60°C for 10 min and then spun again for 5 min. A 50-,ul sample of each supernatant was used in chloramphenicol acetyltransferase (CAT) assays; the reactions were incubated at 37°C and included 70 ,ul of Tris-HCl (0.25 M, pH 8.0), 2 ,ll of [14C]chloramphenicol (NEN-DuPont), and 5 ,ul of n-butyryl coenzyme A (5 mg/ml; Pharmacia). CAT activity was quantitated by a liquid scintillation counting assay (48a). Briefly, each CAT reaction was extracted with 300 ,ul of xylene by vortexing for 30 s and centrifuging for 3 min.

J. VIROL.

The xylene phase was then back-extracted in a similar fashion with 100 ,ul of Tris-HCI (0.25 M, pH 8.0). A 200-pA sample of the final xylene phase, which contains only butyrylated chloramphenicol products, was then added to the scintillation fluid and counted. Construction of plasmids. Reporter constructs used in this work included the promoter for the HSV-1 late gene encoding glycoprotein C fused to the chloramphenicol acetyltransferase (CAT) gene (pgC-CAT); the promoter for the HSV-1 immediate-early gene encoding ICP4, including a VP16responsive TAATGARAT element, fused to the CAT gene (pTAAT-CAT); and the early promoter of simian virus 40 (SV40) fused to the CAT gene (pSV40-CAT). The construction of pgC-CAT has been described previously (56). pTAAT-CAT was constructed in two steps: first, the 0.2-kb SstII-BamHI fragment of pFH100 (65), containing the ICP4 promoter, was inserted into the SstII and BamHI sites of the vector sequences of pTnSAlsv (62); then, the 1.6-kb BgiIIHindIIl fragment of pgC-CAT containing the CAT gene was inserted into the BamHI and HindlIl sites of this plasmid. pSV40-CAT was constructed in four steps: first, the GAL4 sequences of pSG424 (53) contained in a 0.5-kb BglII-BamHI fragment were deleted to create pSG424A; next, the 1.6-kb BglII-HindIII fragment of pgC-CAT containing the CAT gene was inserted into the BamHI and HindIll sites of pUC18; the CAT gene was then transferred out of this plasmid as a 1.6-kb KpnI-HindIII fragment and inserted into the KpnI and HindIII sites of the vector pGEM7; finally, the CAT gene was placed downstream of the SV40 early promoter in pSG424A as a 1.6-kb I7pnI-SstI fragment. pVP16 contains the wild-type VP16 gene and was constructed by inserting the 4.2-kb BglII-PstI fragment of pSG22 (24) into the BamHI and PstI sites of pUC19. All carboxy-terminal truncating mutations of ICPO were made by using the vector pSG424 (53), which contains a polylinker with numerous cloning sites, an adjacent region containing stop codons in all three reading frames that truncates any inserted protein-coding sequences, and an SV40 polyadenylation signal. pSG424-ICPO contains all 775 amino acids of the wild-type ICPO gene and was constructed by inserting the 4.6-kb HindIII-HpaI fragment of pIGA15 (20) into the HindIII and SmaI sites of pSG424. pHKT contains aa 1 to 212 of ICPO and was constructed by inserting the 2.3-kb HindIII-KnI fragment of pIGA15 into the HindIII and KpnI sites of pSG424. pHXT contains aa 1 to 105 of ICPO and was constructed by inserting the 2.0-kb HindIII-XhoI fragment of pIGA15 into the HindIII and SalI sites of pSG424. pD48T, pE56T, pE45T, pE37T, pE28T, pE18T, pESiT, pD7T, pE29T, pD6T, pE53T, pE14T, pE31T, and pD19T contain aa 1 to 541, 1 to 509, 1 to 474, 1 to 428, 1 to 406, 1 to 394, 1 to 374, 1 to 370, 1 to 343, 1 to 341, 1 to 322, 1 to 290, 1 to 278, and 1 to 245 of ICPO, respectively, and were constructed by inserting the smaller of two KjpnIEcoRI fragments (ranging in size from 0.2 to 1.2 kb) of pllOD48/1, pllOE56, pllOE45-1, pllOE37, pllOE28-1, pllOE18, pllOE51-1, pllOD7, pllOE29-1, pllOD6, pllOE53-1, pllOE14-1, pllOE31-1, and pllOD19 (12, 13), respectively, into the KIpnI and EcoRI sites of pHKT. pKAT contains aa 1 to 553 of ICPO and was constructed in three steps: first, the vector pUC19-0.SHK was created by inserting the 0.5-kb HindIII-KpnI fragment of pSG424 into the HindIII and KpnI sites of pUC19; next, the 1.2-kb KpnIAatII fragment of pIGA15 was inserted into the KpnI and SmaI sites of pUC19-0.SHK; finally, this insert was removed as a KIpnI-EcoRI fragment and placed into the KpnI and EcoRI sites of pHKT. pA6T and pA4T contain aa 1 to 549

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tanstivaion of

pgC-CAT (fold iaease)

pSG424-ICPO

pro-rich

8.5

(AA 1-775)

pKAT (AA 1-553)

pHKT

H

(AA 1-212)

pHXT

HKcNj

(AA 1-I1)

FIG. 1. Transactivation potential of plasmids encoding wild-type and mutant derivatives of the HSV-1 immediate-early protein ICPO. The ability of each plasmid to transactivate the reporter construct pgC-CAT in cotransfection assays is indicated on the right by the increase in CAT activity over that of pgC-CAT transfected alone. Plasmids were constructed as described in Materials and Methods. Open boxes represent the three protein-coding exons of ICPO, lines represent transcripts and introns, and the shaded regions represent the proline (pro)-rich region at aa 241 to 551.

and 1 to 424 of ICPO, respectively, and were constructed in two steps: first, the 1.2- and 0.8-kb KpnI-HindIII fragments

of pllOA6 and p110A4 (12, 13), respectively, were inserted into the Kpmnl and HindIII sites of the vector pGEM7; then, the inserts were removed as KpnI-SstI fragments and placed into the Kpnl and SstI sites of pHKT. pHNT contains aa 1 to 312 of ICPO and was constructed by inserting the 2.7-kb HindIII-NruI fragment of pIGA15 into the HindIII and SmaI sites of pSG424. In all plasmids, the coding sequences for the GAL4 gene in pSG424 were replaced by ICPO DNA.

RESULTS AND DISCUSSION Sequential deletion of coding sequences derived from the third exon of ICPO (aa 245 to 775) results in the progressive loss of transactivation potential. A previous analysis of TnSgenerated carboxy-terminal truncations in the ICPO polypeptide demonstrated that a mutant derivative encoding only aa 1 to 553 still mediated significant transactivation of the HSV-1 glycoprotein C gene promoter in transient expression assays (63). To rule out any possible contribution of the inserted transposon DNA in this activity, an analogous mutant which was devoid of all TnS sequences was constructed. This construct, pKAT, was found to have a level of transactivation, about 40 to 50% of the wild-type ICPO in pSG424-ICPO, comparable to that of the original TnS insertion mutant (Fig. 1). Interestingly, the pKAT mutant retained a large proline-rich region (20% proline residues) found in the native ICPO at aa 241 to 551. Deletion of this region in plasmids pHKT and pHXT abolished all transactivation capabilities (Fig. 1). The requirement of these sequences for ICPO function suggests that they might be functionally homologous to the proline-rich activation domains found in a number of transcriptional regulatory proteins (19, 21, 37, 45, 66). To further examine the role of the proline-rich region in transactivation by ICPO, a number of carboxy-terminal truncations which spanned this domain were introduced into the pKAT plasmid (Fig. 2A). In agreement with the behavior of mutants pHKT and pHXT (Fig. 1), the gradual deletion of the proline-rich coding sequences in this panel of mutants

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eventually resulted in a complete loss of activity (Fig. 2B). Comparable results were obtained by Cai and Schaffer in their analysis of a different set of carboxy-terminal truncation mutants (3). Since mutants which lacked the ability to transactivate still possessed the ability to interfere with the wild-type protein in cotransfection assays (discussed below), this loss in function had to be due to the removal of the proline-rich region rather than the production of improperly folded, unstable, or cytoplasmic polypeptides. However, there is only a limited correlation of the ability of each of these mutants to transactivate the promoter for the HSV-1 glycoprotein C gene with the size of the proline-rich region. For example, progressive truncation from the carboxyterminal end of pKAT seemed to result in two discrete, rather than continuous, reductions in transactivation potential. The first reduction occurred between mutants pD48T and pE56T, the second occurred between mutants pE53T and pHNT, and both transitions involved the loss of only a few proline residues. Furthermore, mutants such as pD7T and pD29T exhibit higher levels of transactivation than mutants pE56T and pE45T but possess far fewer proline residues. Since the strength of an activation domain in a transcription regulator is typically dependent upon the number of its overrepresented amino acid residues (22, 59), this deletion analysis of pKAT suggests that the region of ICPO at aa 241 to 551 does not function as a proline-rich activation domain. Further support for this conclusion comes from the inability to directly demonstrate that the proline-rich region of ICPO can function as a movable transcription activation domain. These experiments involved replacing the acidic activation domain of GAL4 with the proline-rich region of ICPO and replacing the proline-rich region of pKAT with the acidic activation domain of VP16; neither of these chimeric constructs was capable of transactivation (61). It is likely that the proline-rich region mediates some other essential function in the ICPO polypeptide; indeed, the region from aa 475 to 548 has previously been shown to contain one of the signals necessary for proper nuclear localization of the protein in transfection assays (13). In this regard, it is interesting that the pKAT derivatives which contain at least aa 1 to 541 (i.e., pKAT, pA6T, and pD48T), and thus, most of this putative nuclear localization signal, exhibit higher levels of transactivation than the other mutants. Alternatively, the overrepresentation of proline residues may be simply a manifestation of the extremely high GC content found in this portion of the ICPO coding sequences (46). ICPO derivatives containing carboxy-terminal truncations in exon 3 behave as dominant-negative mutants. Several of the carboxy-terminal truncation mutants were tested for their ability to interfere with the activity of the wild-type ICPO protein by cotransfecting equimolar amounts of both plasmids in transient assays; results of some representative experiments are shown in Fig. 3. Both pD19T and pKAT were found to behave as dominant-negative mutants, since the transactivation level observed in cotransfection assays reflected that of the mutant protein rather than that of the wild-type protein. This effect was most dramatic in the pD19T mutant, which completely lacked any transactivation capability when tested by itself (Fig. 2B) and which therefore behaved as a particularly powerful repressor of ICPO activity when tested in cotransfection experiments (Fig. 3). Similar results were obtained with other carboxy-terminal truncation mutants of pKAT (61). Cotransfections with equimolar levels of the wild-type ICPO plasmid or the ICPO null mutant pHXT (Fig. 1) were found to have no inhibitory effects on

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B.

A. A....

ICPO (AA 1-553)

AA 553

AA241

transactivation (fold increase)

11111 111I1

I 1 I II 1111111 11 I 1111111

II

I1111 1

11111

l1111

1

Prole resid

2

4

3 1

pKAT

pA6r pD48T pE56T pE45T pE37T pA4T pE25T

PEIST

mmmm.MENEW-4 IN---I

A

pES1T pDTr

i

pE29T

pD6T pE53T pHNT

pEI4T pE31T pDI9T FIG. 2. Transactivation potential of carboxy-terminal truncation mutants of plasmid pKAT. (A) Carboxy-terminal truncation mutants of plasmid pKAT. The structure of pKAT is shown at the top, with open boxes designating intact exon sequences and dashed lines designating deleted ICPO sequences. An expanded view of aa 241 to 553 is presented at the bottom. The locations of individual proline residues are indicated by vertical lines, and the lengths of the intact sequences present in each carboxy-terminal truncation mutant are designated by horizontal lines. Plasmids were constructed as described in Materials and Methods. (B) Transactivation of pgC-CAT by the carboxy-terminal truncation mutants. The ability of each mutant to transactivate the reporter construct in cotransfection assays is indicated by the increase in CAT activity over that of pgC-CAT transfected alone.

6 .8

5 a

.u

4 3

2

pHXT

pD19T

pKAT

pSG424-ICPO

counsfected ICPO mutant plasmid

FIG. 3. Dominant-negative properties of carboxy-termninal truncation mutants of plasmid pKAT. Each of the plasmids indicated at the bottom was cotransfected with pgC-CAT (0) or with pgC-CAT and the wild-type ICPO gene in pSG424-ICPO (-). The ability of each plasmid or combination of plasmids to transactivate the reporter construct is indicated by the increase in CAT activity over that of pgC-CAT transfected alone.

ICPO transactivation (Fig. 3). These results demonstrate that the interference observed was a specific effect mediated by pKAT and its derivatives and was not simply due to a promoter competition phenomenon. The pD19T construct was examined for its ability to inhibit a wide variety of HSV-1 promoters, non-HSV-1 promoters, and HSV-1 transactivator proteins in transient expression assays. In every experiment carried out to date, this mutant protein has behaved as a powerful repressor of gene expression. The transactivation capabilities of ICPO as well as another HSV-1 regulatory protein, VP16, were reduced to below basal levels in the presence of the pD19T mutant; similarly, the high constitutive activity of the SV40 early promoter was abolished when it was cotransfected with pD19T (Table 1). Identical results were obtained with a second non-HSV-1 promoter, that of the HIV long terminal repeat region, as well as with a third HSV-1 transactivator protein, ICP4 (61). In all of these experiments, pSG424-ICPO stimulated gene activation and pHXT had no effect. Thus, just as the wild-type ICPO protein behaves as a promiscuous transactivator of gene expression in transient assays, the dominant-negative mutant of ICPO in pD19T behaved as a promiscuous repressor. The mechanism by which the dominant-negative mutants of ICPO described in this work mediate their interference with the wild-type protein is unclear. The ability of the pD19T mutant to act as a generalized suppressor of gene expression strongly suggests that ICPO mediates its promiscuous transactivation by interacting with some very general

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TABLE 1. Promiscuous repression mediated by the pD19T mutant in transient expression assays' Affected promoter or transactivator and transfected plasmid(s)

CAT enzyme activity (U, 104)

Transactivation by VP16 42.2 pTAAT-CAT ........................................ 213.4 pTAAT-CAT + pVP16 + pHXT ....................... pTAAT-CAT + pVP16 + pSG424-ICPO ..............1,114.1 24.6 pTAAT-CAT + pVP16 + pD19T .......................

Expression of the SV40 early promoter

pSV40-CAT pSV40-CAT pSV40-CAT

+ + +

pHXT ...................................... pSG424-ICPO ............................ pD19T .....................................

115.8 621.5 17.7

a Equimolar amounts of plasmid DNAs were transfected into Vero cells, and CAT assays were performed with cellular extracts as described in Materials and Methods. n-Butyryl-['4CJchloramphenicol products were extracted from CAT reactions and quantitated by liquid scintillation. Counts were converted to units of CAT enzyme activity by the preparation of a standard curve with purified CAT enzyme (Promega); values represent the total CAT activity in a 60-mm dish of transfected cells.

component of the host cell transcription machinery and that this mutant acts to effectively compete with ICPO (and other regulatory proteins) for the binding of this factor. As proposed by others (17), the cellular target contacted by ICPO could be one of the basic transcription factors which facilitates the initiation of transcription by RNA polymerase II (2), a component of chromatin which mediates reassembly into an activated state (8, 68), or a factor involved in the recompartmentalization of transcription complexes in the nucleus. The pD19T mutant may act to irreversibly bind up the available cellular target protein molecules to prevent interactions with ICPO or other regulatory proteins, since effective inhibition of ICPO transactivation is observed even at 1/20 molar concentrations of pD19T in transient expression assays (64). Alternatively, the pD19T mutant may localize in a region of the nucleus which is distinct from that of the wild-type ICPO protein (13); this would act to sequester the cellular target and make it inaccessible for interactions with other proteins. These possibilities are under investigation at the present time. Remarkably, the pD19T mutant is capable of interacting with the cellular target required for ICPO transactivation, in spite of having lost all but four amino acids of the third exon; this amounts to a deletion of over two-thirds of the wild-type coding sequences. These results indicate that the domain of ICPO which interacts with the putative cellular target for this protein is encoded by the first two exons. Not surprisingly, this region corresponds to the most mutation-sensitive region of the molecule in several mutagenesis studies (3, 4, 12, 13). Furthermore, the ICPO homologs found in other herpesviruses, including varicella-zoster virus, pseudorabies virus, and bovine herpesvirus 4, are considerably smaller than ICPO and show sequence homology only to this same region (5, 46, 60). It is interesting that, like the pD19T mutant, the varicella-zoster virus ICPO homolog manifests repression rather than transactivation characteristics in transient expression assays (40). Moreover, a derivative of ICPO with dominant mutant properties similar to that of pD19T can potentially be created by a failure to splice out the intron 2 sequences; translation of this message results in a derivative of ICPO, called ICPOR, which contains all 241 amino acids encoded by exons 1 and 2 plus an additional 21 amino acids derived from translation into the unspliced second intron (64). These observations raise the interesting possibility that

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herpesviruses utilize repressor proteins as a means of downregulating gene expression during infection. Indeed, the dominant-negative mutant of ICPO encoded by pD19T has been found to behave as a powerful suppressor of HSV-1 replication (64). Such a repression mechanism may play an important role in the determination of host range or even in the establishment of latent infections in vivo. The proline-rich region appears to be capable of restoring some transactivation function to the domain encoded by the first two exons, but additional carboxy-terminal sequences (aa 554 to 775) are required for complete restoration of activity (Fig. 1). Thus, the third exon encodes a domain(s) which is essential for converting the interaction between the amino-terminal region of ICPO and its cellular target into a transactivation phenomenon. The deletion of these sequences in pD19T therefore causes the promiscuous transactivator protein ICPO to be converted into a promiscuous repressor. Additionally, the carboxy-terminal region of ICPO apparently serves to alter the conformation or availability of the target binding domain encoded by the first two exons, since deletion of aa 554 to 775 in pKAT (and its deletion mutants) results in a derivative which can effectively sequester this factor from the wild-type ICPO. It may be that the carboxy-terminal sequences which prevent the formation of such a high-affinity interaction domain play a vital role in preventing squelching (23) of other ongoing transcriptional processes. Studies aimed at further defining these functional domains in ICPO are currently under way. ACKNOWLEDGMENTS We thank M. Fieo, D. Siever, and R. Sarisky for technical assistance; M. Ptashne and especially R. Everett for sending plasmids; and R. Everett for critically reading the manuscript. This study was supported by NIH grants AI29961 (P.C.W.) and CA27503 (B.W.) and funds from the Pennsylvania State University, the Pennsylvania Research Corporation, and the CIBA-GEIGY Corporation (P.C.W.). REFERENCES 1. Abmayr, S. M., J. L. Workman, and R. G. Roeder. 1988. The pseudorabies immediate early protein stimulates in vitro transcription by facilitating TFIID:promoter interactions. Genes Dev. 2:542-553. 2. Buratowski, S., S. Hahn, L. Guarente, and P. A. Sharp. 1989. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell 56:549-561. 3. Cai, W., and P. A. Schaffer. 1989. Herpes simplex virus type 1 ICPO plays a critical role in the de novo synthesis of infectious virus following transfection of viral DNA. J. Virol. 63:45794589. 4. Chen, J., X. Zhu, and S. Silverstein. 1991. Mutational analysis of the sequence encoding ICPO from herpes simplex virus type 1. Virology 180:207-220. 5. Cheung, A. K. 1991. Cloning of the latency gene and the early protein 0 gene of pseudorabies virus. J. Virol. 65:5260-5271. 6. Clements, G. B., and N. D. Stow. 1989. A herpes simplex virus type 1 mutant containing a deletion within immediate early gene 1 is latency-competent in mice. J. Gen. Virol. 70:2501-2506. 7. Coen, D. M., S. P. Weinheimer, and S. L. McKnight. 1986. A genetic approach to promoter recognition during trans induction of viral gene expression. Science 234:53-59. 8. Croston, G. E., L. A. Kerrigan, L. M. Lira, D. R. Marshak, and J. T. Kadonaga. 1991. Sequence-specific antirepression of histone Hi-mediated inhibition of basal RNA polymerase II transcription. Science 251:643-649. 9. DeLuca, N. A., A. M. McCarthy, and P. A. Schaffer. 1985. Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J. Virol. 56:558-570.

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