Transcriptional activation by the human herpesvirus-8-encoded interferon regulatory factor

Florence Roan1,2, James C. Zimring1, Stephen Goodbourn3 and Margaret K. Offermann1,4

Winship Cancer Center, Emory University, 1365-B Clifton Road NE, Atlanta, GA 30322, USA1
Program in Biochemistry, Cell and Developmental Biology2 and Department of Medicine4, Emory University, Atlanta, GA 30322, USA
Division of Biochemistry, Department of Cellular and Molecular Sciences, St George's Hospital Medical School, University of London, London SW17 0RE, UK3

Author for correspondence: Margaret Offermann (at Winship Cancer Center). Fax +1 404 778 3965. e-mail mofferm{at}emory.edu


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human herpesvirus-8 (HHV-8), a gammaherpesvirus that is thought to be the viral aetiologic agent of Kaposi's sarcoma and primary effusion lymphoma, encodes a homologue to cellular interferon regulatory factors (IRFs). The HHV-8 IRF homologue (vIRF; ORF K9) has previously been shown to inhibit gene induction by interferons and IRF-1 and to transform NIH3T3 cells or Rat-1 cells. Additionally, expression of antisense to vIRF in BCBL-1 cells results in the repression of certain HHV-8 genes, suggesting that vIRF may also positively regulate gene expression. We demonstrate that vIRF activates transcription when directed to DNA by the GAL4 DNA-binding domain. GAL-vIRF truncation constructs that individually are incapable of activating transcription can cooperate in transactivation when coexpressed in HeLa cells, suggesting that multiple regions of vIRF are involved in transactivation. These studies broaden the potential mechanisms of action of vIRF to include transcriptional activation as well as transcriptional repression.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Human herpesvirus-8 (HHV-8), the probable viral aetiologic agent of Kaposi's sarcoma and primary effusion lymphoma, encodes a homologue to cellular interferon regulatory factors (IRFs) (Moore et al., 1996 ; Russo et al., 1996 ), a family of transcription factors involved in growth regulation and in antiviral and inflammatory responses (Fujita et al., 1988 , 1989 ;Harada et al., 1993 , 1994 ; Reis et al., 1992 ; Taniguchi et al., 1995 ). The HHV-8-encoded IRF (vIRF; ORF K9) inhibits responses to interferons (Gao et al., 1997 ; Zimring et al., 1998 ) as well as IRF-1-mediated transactivation (Zimring et al., 1998 ), and stable expression of vIRF in NIH3T3 or Rat-1 cells results in cellular transformation (Gao et al., 1997 , Li et al., 1998 ).

While vIRF functions in part like IRF-2, a cellular oncogene that represses expression of interferon-inducible genes by competing with the transactivator IRF-1 for binding to DNA (Harada et al., 1989 , 1993 ; Nguyen et al., 1995 ; Tanaka et al., 1993 ; Taniguchi et al., 1995 ), vIRF does not require its putative DNA-binding domain to inhibit gene induction by IRF-1, and recombinant vIRF does not bind an IRF element by gel shift analysis or alter the ability of IRF-1 to bind (Zimring et al., 1998 ). Additionally, while basal and inducible levels of interferon-stimulated gene expression are increased in BCBL-1 cells expressing antisense to vIRF (Li et al., 1998 ), these cells exhibit a significant decrease in the expression of specific HHV-8 genes (Li et al., 1998 ), suggesting that vIRF may also play a role in the induction of certain genes.

In this paper, we demonstrate that vIRF, when directed to DNA by fusing it to a GAL4 DNA-binding domain, has the ability to drive transcription. Using vIRF truncation mutants linked in-frame to the GAL4 DNA-binding domain, we show that some GAL-vIRF truncation constructs are incapable individually of activating transcription but can cooperate in transactivation when expressed together in HeLa cells. The ability of GAL-vIRF to transactivate suggests that vIRF could be a potent regulator of viral and cellular responses through the induction of viral or cellular gene expression.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Plasmid constructs.
GAL-vIRF was created by digesting pcDNA 3.1(+)vIRF (Zimring et al., 1998 ) with NcoI, blunting, and then cutting with XbaI to clone full-length vIRF into the plasmid pCObGAL147 (Zimring et al., 1998 ), which had been cut with EcoRI, blunted, and re-cut with XbaI. This places vIRF in-frame, downstream of sequences encoding the GAL4 DNA-binding domain (amino acids 1 to 147). To generate a series of C- and N-terminal vIRF truncation mutants linked to the GAL4 DNA-binding domain, coding regions for vIRF truncation mutants were generated via PCR amplification from BCBL-1 DNA using Pfu polymerase and primers of 32 to 42 nucleotides complementary to the indicated vIRF coding regions with BamHI and EcoRI sites at the 5' and 3' ends, respectively. PCR products were digested with BamHI and EcoRI and cloned into pcDNA3.1(+) (Invitrogen). Following in vitro translation (Promega TnT system) and SDS–PAGE analysis to confirm that vIRF products of the appropriate sizes were generated from these plasmids, these vIRF coding regions were cloned into pCObGAL147 downstream of the GAL4 DNA-binding domain (GAL DBD). The reporter UASGAL-CAT contains four tandem GAL4 DNA-binding elements linked to the CAT reporter gene. The plasmid pEFLacZ contains the ß-galactosidase gene under the control of the eukaryotic elongation factor-1{alpha} promoter. GAL-VP16 is an expression vector containing the GAL4 DBD fused in-frame, upstream of the transactivation domain of the herpes simplex virus transactivator VP16.

{blacksquare} Cell culture, transfections and reporter assays.
HeLa cells were cultured at 37 °C in Eagle's MEM (Cellgro) supplemented with 10% foetal calf serum (Life Technologies), 2 mM l-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin and passaged every 48 to 72 h. HeLa cells were split into six-well plates approximately 24 h prior to transfection and were transfected at 60 to 80% confluency using FuGENE 6 (Boehringer Mannheim) according to the manufacturer's protocol. DNA, totalling 2 µg per well, consisted of 0·5 µg UASGAL-CAT, 1 µg total of either empty plasmid (pcDNA3), GAL-VP16, or GAL-vIRF (either 1 µg of a single plasmid or 0·5 µg each of two GAL-vIRF plasmids) and 0·5 µg pEFLacZ. Cells were harvested approximately 48 h post-transfection using a detergent-based lysis (Promega).

CAT activity was determined by phase extraction of butyrylated [14C]chloramphenicol as previously described (Kingston & Sheen, 1997 ) and was adjusted to relative levels of ß-galactosidase to correct for differences in transfection efficiencies. Relative amounts of ß-galactosidase were determined by comparison to a standard curve generated using recombinant ß-galactosidase (Promega). Cell lysates and recombinant ß-galactosidase were incubated at 37 °C with an equal volume of 2x ß-galactosidase assay buffer [100 mM sodium phosphate buffer, pH 7·3, 2 mM MgCl2, 100 mM ß-mercaptoethanol, 1·33 mg/ml o-nitrophenyl ß-d-galactopyranoside (ONPG)], and absorbance was measured at 405 nm.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
To determine whether vIRF could function as a transcriptional activator, we tested the ability of the fusion construct GAL-vIRF (Fig. 1A) to induce the reporter UASGAL-CAT, which contains four tandem GAL4 DNA-binding elements linked to the CAT reporter gene. Cotransfection of an expression vector for GAL-vIRF and the reporter UASGAL-CAT into HeLa cells resulted in a strong induction of UASGAL-CAT, which was comparable to the induction of CAT activity by GAL-VP16 (Fig. 1B), a fusion construct containing the transactivation domain from the herpes simplex virus transcription factor VP16 (Fig. 1 A). Transfection of UASGAL-CAT alone or UASGAL-CAT with an expression vector for the GAL4 DNA-binding domain (GAL DBD) did not result in CAT activity (Fig. 1 B). Thus vIRF, when directed to DNA by the GAL4 DNA-binding domain, strongly activates transcription.



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Fig. 1. vIRF drives transcription when directed to DNA. (A) Diagram of GAL4 fusion constructs GAL DBD, GAL-vIRF and GAL-VP16. (B) UASGAL-CAT (0·5 µg) plus pEFLacZ (0·5 µg) were transfected into HeLa cells with 1 µg empty vector or 1 µg of expression vector for GAL DBD, GAL-vIRF or GAL-VP16; 48 h post-transfection, cells were harvested, and lysates were assayed for CAT activity. CAT activity was standardized to transfection efficiency as assessed by ß-galactosidase activity. These data are from a representative experiment performed in triplicate±SD.

 
A series of C- and N-terminal vIRF truncation mutants were generated and linked to the GAL4 DNA-binding domain (Fig. 2A) to determine which regions of vIRF were required for transcriptional activation. Expression vectors for each of the GAL-vIRF truncation mutants were cotransfected with UASGAL-CAT into HeLa cells, and CAT activity was determined. Deletion of 89 amino acids from the C terminus of GAL-vIRF [GAL-vIRF (1–360)] abolished the ability of GAL-vIRF to drive transcription, indicating that the C terminus is required for transactivation (Fig. 2 B). Consistent with this, no transcriptional activity was seen when additional amino acids were removed from the C terminus of GAL-vIRF [e.g. GAL-vIRF (1–260) and GAL-vIRF (1–160); Fig. 2B]. Deletion of the N-terminal 151 amino acids of vIRF [GAL-vIRF (152–449)] decreased, but did not eliminate, the ability of GAL-vIRF to transactivate (Fig. 2B), whereas transactivation by GAL-vIRF was completely lost with the truncation of an additional 100 amino acids from the N-terminal end of vIRF [GAL-vIRF (252–449); Fig. 2B], suggesting that a domain or boundary for a domain involved in transactivation resides between amino acids 152 and 252. These data indicate that the outer boundaries of vIRF required for transactivation reside between amino acids 152 and 449, since further deletion of either the N or C terminus of vIRF abolishes the transcriptional activity of GAL-vIRF.



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Fig. 2. Multiple vIRF regions are involved in transactivation and can cooperate to activate transcription. (A) Diagram of GAL-vIRF truncation constructs. (B) One µg of empty vector or expression vector for the indicated GAL-vIRF truncation mutants was cotransfected with 0·5 µg UASGAL-CAT plus 0·5 µg pEFLacZ into HeLa cells. Cell lysates were prepared 48 h post-transfection and assayed for CAT activity. CAT activity was normalized to ß-galactosidase activity to correct for differences in transfection efficiency. (C) One µg of empty vector or the indicated GAL-vIRF truncation mutant or 0·5 µg each of two expression vectors for the indicated GAL-vIRF truncation mutants were transfected into HeLa cells with 0·5 µg UASGAL-CAT and 0·5 µg pEFLacZ. Cell lysates were prepared 48 h post-transfection and assayed for CAT activity, which was then normalized to ß-galactosidase activity. These data are from representative experiments performed in triplicate±SD.

 
Studies of other viral transactivators, such as VP16, have indicated that a region as large as is suggested by these data is likely to contain more than one surface for interactions with the basal transcriptional machinery (for review see Flint & Shenk, 1997 ; Goodbourn, 1996 ). For example, the C-terminal acidic activation domain of VP16 is involved in multiple stages of transcriptional initiation and elongation and contains two distinct subdomains at the C- and N-terminal ends that allow interactions with distinct components of the basal machinery. To determine if vIRF may be similarly constituted, we tested whether individual GAL-vIRF fusions that cannot transactivate on their own can do so when coexpressed with other nonfunctional fusions. This complementation assay may allow functionally distinct subdomains to be identified. This takes advantage of the fact that GAL fusions can dimerize through the GAL DBD (Carey et al., 1989 ; Marmorstein et al., 1992 ) and that our reporter construct contains multiple binding sites for these fusion proteins. Combinations of expression vectors for different GAL-vIRF truncation mutants, each of which was still linked to the GAL DBD at the N terminus, were cotransfected with UASGAL-CAT into HeLa cells. While the C-terminal GAL-vIRF truncation mutant GAL-vIRF (1–260) and the N-terminal GAL-vIRF truncation mutant GAL-vIRF (252–449) were individually incapable of activating transcription (Fig. 2B), transfection of these expression vectors together into HeLa cells restored the ability to induce UASGAL-CAT to levels comparable to that of full-length GAL-vIRF (Fig. 2C). Transfection of GAL-vIRF (1–160), which alone had no transactivating activity (Fig. 2B), together with the weak transactivator GAL-vIRF (152–449), resulted in a superinduction of UASGAL-CAT over that of GAL-vIRF (152–449) alone (Fig. 2C). In contrast, the combination of GAL-vIRF (1–160) with GAL-vIRF (252–449) did not restore transcriptional activity even though these deletion mutants were able to complement distinct vIRF truncations (Fig. 2C).

The data in Fig. 2(B, C) are from separate experiments done in triplicate, and CAT activity is reported in c.p.m. standardized to relative ß-galactosidase activity. While the mean c.p.m./ß-galactosidase varied between these two experiments due to minor differences in cell density and transfection efficiency, the relative levels of transactivation seen with different truncation mutants were consistent. For example, the reduction in mean reporter activity between GAL-vIRF (152–449) compared to full-length GAL-vIRF in Fig. 2(B) and 2(C) was 81 and 71%, respectively.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Previous studies have established vIRF as an inhibitor of interferon signalling and IRF-1-mediated transcriptional activation (Gao et al., 1997 ; Li et al., 1998 ; Zimring et al., 1998 ). The current studies demonstrate that vIRF, when directed to DNA, also activates transcription. Mapping of vIRF transactivation domains via deletion mutants suggests that more than one distinct subdomain of vIRF is involved in transactivation since transactivation-negative GAL-vIRF mutants can partially or fully reconstitute the activity of other GAL-vIRF constructs. Since the studies reported in this paper use a GAL-vIRF fusion, whether vIRF itself has a DNA target at which it transactivates remains unknown. However, transactivation resulting from nonspecific interactions is unlikely since vIRF does not contain any large stretches of charged amino acids. Studies are currently under way to identify vIRF DNA-binding elements and targets of gene induction by vIRF.

When antisense to vIRF was used to block vIRF expression in BCBL-1 cells, the expression of transcripts from the HHV-8 T1.1, ORF K8 and vIL-6 genes was significantly lower than in untransfected BCBL-1 or BCBL-1/anti-vIL-6 cells, while the HHV-8 sVCA and cellular actin transcripts were unaffected (Li et al., 1998 ). The selective repression of specific HHV-8 gene expression in cells expressing antisense to vIRF suggests that vIRF may indeed be involved in the upregulation of certain genes, although it does not address whether this upregulation is due to direct transcriptional activation by vIRF. The data presented here suggest that vIRF can function as a transcriptional activator; thus the changes in HHV-8 gene expression resulting from the expression of antisense to vIRF might be independent of the effects of vIRF on interferon-regulated processes. While it is possible that the ability of vIRF to activate transcription may reflect interactions with transcriptional machinery that result in the sequestration of proteins important in mediating transactivation by certain transcription factors such as IRF-1, transcriptional repression and activation by vIRF act, at least in part, through distinct mechanisms. A vIRF truncation mutant lacking the N-terminal 151 amino acids (vIRF 152–449; NTvIRF) retains wild-type ability to inhibit IRF-1-mediated transactivation (Zimring et al., 1998 ), yet deletion of the same N-terminal region of vIRF significantly reduces transactivation by GAL-vIRF. Furthermore, transcriptional repression by vIRF has some specificity with respect to enhancer elements since vIRF is able to inhibit IRF-1-mediated transactivation but not NF-{kappa}B-mediated transactivation (Zimring et al., 1998 ).

The level of transcriptional activation seen with GAL-vIRF is comparable to that of GAL-VP16, which contains the transactivation domain from the herpes simplex virus protein VP16, a prototype of strong transcriptional activators. Thus, in addition to the potential of vIRF to block the antiviral and growth regulatory signalling pathways of interferons, the ability of vIRF to transactivate raises the possibility that vIRF may contribute to cellular transformation and regulation of the virus life-cycle through the induction of cellular or viral genes.


   Acknowledgments
 
This work was supported by NIH grant RO1 CA67382 to M.K.O. and a Wellcome Trust University Award to S.G.


   References
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Introduction
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Results
Discussion
References
 
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Received 18 March 1999; accepted 7 May 1999.