Download Transcriptional activation by the human herpesvirus-8

Journal
of General Virology (1999), 80, 2205–2209. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Transcriptional activation by the human
herpesvirus-8-encoded interferon regulatory factor
Florence Roan,1, 2 James C. Zimring,1 Stephen Goodbourn3 and Margaret K. Offermann1, 4
1
Winship Cancer Center, Emory University, 1365-B Clifton Road NE, Atlanta, GA 30322, USA
Program in Biochemistry, Cell and Developmental Biology2 and Department of Medicine4, Emory University, Atlanta, GA 30322, USA
3
Division of Biochemistry, Department of Cellular and Molecular Sciences, St George’s Hospital Medical School, University of London,
London SW17 0RE, UK
2, 4
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
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
Author for correspondence : Margaret Offermann (at Winship Cancer
Center). Fax j1 404 778 3965. e-mail mofferm!emory.edu
0001-6323 # 1999 SGM
Plasmid constructs. GAL-vIRF was created by digesting pcDNA
3.1(j)vIRF (Zimring et al., 1998) with NcoI, blunting, and then cutting
with XbaI to clone full-length vIRF into the plasmid pCObGAL147
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 12 Jun 2017 06:34:19
CCAF
F. Roan and others
(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 5h and
3h ends, respectively. PCR products were digested with BamHI and EcoRI
and cloned into pcDNA3.1(j) (Invitrogen). Following in vitro translation
(Promega TT 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 UASGALCAT contains four tandem GAL4 DNA-binding elements linked to the
CAT reporter gene. The plasmid pEFLacZ contains the β-galactosidase
gene under the control of the eukaryotic elongation factor-1α promoter.
GAL-VP16 is an expression vector containing the GAL4 DBD fused inframe, upstream of the transactivation domain of the herpes simplex virus
transactivator VP16.
Cell culture, transfections and reporter assays. HeLa cells
were cultured at 37 mC in Eagle’s MEM (Cellgro) supplemented with 10 %
foetal calf serum (Life Technologies), 2 mM -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 0n5 µg UASGAL-CAT, 1 µg
total of either empty plasmid (pcDNA3), GAL-VP16, or GAL-vIRF
(either 1 µg of a single plasmid or 0n5 µg each of two GAL-vIRF plasmids)
and 0n5 µg pEFLacZ. Cells were harvested approximately 48 h posttransfection using a detergent-based lysis (Promega).
CAT activity was determined by phase extraction of butyrylated
["%C]chloramphenicol as previously described (Kingston & Sheen, 1997)
and was adjusted to relative levels of β-galactosidase to correct for
differences in transfection efficiencies. Relative amounts of β-galactosidase
were determined by comparison to a standard curve generated using
recombinant β-galactosidase (Promega). Cell lysates and recombinant βgalactosidase were incubated at 37 mC with an equal volume of 2i βgalactosidase assay buffer [100 mM sodium phosphate buffer, pH 7n3,
2 mM MgCl , 100 mM β-mercaptoethanol, 1n33 mg\ml o-nitrophenyl β#
-galactopyranoside (ONPG)], and absorbance was measured at 405 nm.
Results
To determine whether vIRF could function as a transcriptional activator, we tested the ability of the fusion construct
GAL-vIRF (Fig. 1 A) 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 UASGALCAT into HeLa cells resulted in a strong induction of UASGALCAT, which was comparable to the induction of CAT activity
by GAL-VP16 (Fig. 1 B), 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
CCAG
Fig. 1. vIRF drives transcription when directed to DNA. (A) Diagram of
GAL4 fusion constructs GAL DBD, GAL-vIRF and GAL-VP16. (B) UASGALCAT (0n5 µg) plus pEFLacZ (0n5 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 β-galactosidase activity. These data are from a
representative experiment performed in triplicatepSD.
the GAL4 DNA-binding domain, strongly activates transcription.
A series of C- and N-terminal vIRF truncation mutants were
generated and linked to the GAL4 DNA-binding domain (Fig.
2 A) 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 GALvIRF 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. GALvIRF (1–260) and GAL-vIRF (1–160) ; Fig. 2 B]. 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. 2 B), 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. 2 B], 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
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 12 Jun 2017 06:34:19
Transcriptional activation by HHV-8 vIRF
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 0n5 µg UASGAL-CAT plus 0n5 µg pEFLacZ into HeLa cells. Cell lysates were prepared 48 h post-transfection
and assayed for CAT activity. CAT activity was normalized to β-galactosidase activity to correct for differences in transfection
efficiency. (C) One µg of empty vector or the indicated GAL-vIRF truncation mutant or 0n5 µg each of two expression vectors
for the indicated GAL-vIRF truncation mutants were transfected into HeLa cells with 0n5 µg UASGAL-CAT and 0n5 µg pEFLacZ.
Cell lysates were prepared 48 h post-transfection and assayed for CAT activity, which was then normalized to β-galactosidase
activity. These data are from representative experiments performed in triplicatepSD.
or C terminus of vIRF abolishes the transcriptional activity of
GAL-vIRF.
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
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 12 Jun 2017 06:34:19
CCAH
F. Roan and others
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 Cterminal 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. 2 B), transfection of these expression vectors
together into HeLa cells restored the ability to induce UASGALCAT to levels comparable to that of full-length GAL-vIRF (Fig.
2 C). Transfection of GAL-vIRF (1–160), which alone had no
transactivating activity (Fig. 2 B), 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. 2 C). 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. 2 C).
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 β-galactosidase activity. While the mean c.p.m.\βgalactosidase 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
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 GALvIRF 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, transCCAI
activation 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 interferonregulated 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-1mediated transactivation but not NF-κB-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.
This work was supported by NIH grant RO1 CA67382 to M. K. O.
and a Wellcome Trust University Award to S. G.
References
Carey, M., Kakidani, H., Leatherwood, J., Mostashari, F. & Ptashne, M.
(1989). An amino-terminal fragment of GAL4 binds DNA as a dimer.
Journal of Molecular Biology 209, 423–432.
Flint, J. & Shenk, T. (1997). Viral transactivating proteins. Annual
Review of Genetics 31, 177–212.
Fujita, T., Sakakibara, J., Sudo, Y., Miyamoto, M., Kimura, Y. &
Taniguchi, T. (1988). Evidence for a nuclear factor(s), IRF-1, mediating
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 12 Jun 2017 06:34:19
Transcriptional activation by HHV-8 vIRF
induction and silencing properties to human IFN-beta gene regulatory
elements. EMBO Journal 7, 3397–3405.
Fujita, T., Reis, L. F., Watanabe, N., Kimura, Y., Taniguchi, T. & Vilcek,
J. (1989). Induction of the transcription factor IRF-1 and interferon-beta
mRNAs by cytokines and activators of second-messenger pathways.
Proceedings of the National Academy of Sciences, USA 86, 9936–9940.
Gao, S. J., Boshoff, C., Jayachandra, S., Weiss, R. A., Chang, Y. &
Moore, P. S. (1997). KSHV ORF K9 (vIRF) is an oncogene which
inhibits the interferon signaling pathway. Oncogene 15, 1979–1985.
Goodbourn, S. (1996). Eukaryotic gene transcription. In Frontiers in
Molecular Biology, pp. 292. New York : IRL Press.
Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia,
A., Miyata, T. & Taniguchi, T. (1989). Structurally similar but functionally
distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of
IFN and IFN-inducible genes. Cell 58, 729–739.
Harada, H., Kitagawa, M., Tanaka, N., Yamamoto, H., Harada, K.,
Ishihara, M. & Taniguchi, T. (1993). Anti-oncogenic and oncogenic
potentials of interferon regulatory factors-1 and -2. Science 259, 971–974.
Harada, H., Takahashi, E., Itoh, S., Harada, K., Hori, T. A. & Taniguchi,
T. (1994). Structure and regulation of the human interferon regulatory
factor 1 (IRF-1) and IRF-2 genes : implications for a gene network in the
interferon system. Molecular and Cellular Biology 14, 1500–1509.
Kingston, R. & Sheen, J. (1997). Phase-extraction assay for CAT
activity. In Current Protocols in Molecular Biology, pp. 9.7.5–9.7.11. Edited
by F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, G. J. Seidman,
J. A. Smith & K. Struhl. New York : John Wiley.
Li, M., Lee, H., Guo, J., Neipel, F., Fleckenstein, B., Ozato, K. & Jung,
J. U. (1998). Kaposi’s sarcoma-associated herpesvirus viral interferon
regulatory factor. Journal of Virology 72, 5433–5440.
Marmorstein, R., Carey, M., Ptashne, M. & Harrison, S. C. (1992).
DNA recognition by GAL4 : structure of a protein–DNA complex.
Nature 356, 408–414.
Moore, P. S., Boshoff, C., Weiss, R. A. & Chang, Y. (1996). Molecular
mimicry of human cytokine and cytokine response pathway genes by
KSHV. Science 274, 1739–1744.
Nguyen, H., Mustafa, A., Hiscott, J. & Lin, R. (1995). Transcription
factor IRF-2 exerts its oncogenic phenotype through the DNA
binding\transcription repression domain. Oncogene 11, 537–544.
Reis, L. F., Harada, H., Wolchok, J. D., Taniguchi, T. & Vilcek, J. (1992).
Critical role of a common transcription factor, IRF-1, in the regulation of
IFN-beta and IFN-inducible genes. EMBO Journal 11, 185–193.
Russo, J. J., Bohenzky, R. A., Chien, M. C., Chen, J., Yan, M.,
Maddalena, D., Parry, J. P., Peruzzi, D., Edelman, I. S., Chang, Y. &
Moore, P. S. (1996). Nucleotide sequence of the Kaposi sarcoma-
associated herpesvirus (HHV8). Proceedings of the National Academy of
Sciences, USA 93, 14862–14867.
Tanaka, N., Kawakami, T. & Taniguchi, T. (1993). Recognition DNA
sequences of interferon regulatory factor 1 (IRF-1) and IRF-2, regulators
of cell growth and the interferon system. Molecular & Cellular Biology 13,
4531–4538.
Taniguchi, T., Harada, H. & Lamphier, M. (1995). Regulation of the
interferon system and cell growth by the IRF transcription factors. Journal
of Cancer Research & Clinical Oncology 121, 516–520.
Zimring, J. C., Goodbourn, S. & Offermann, M. K. (1998). Human
herpesvirus 8 encodes an interferon regulatory factor (IRF) homolog that
represses IRF-1-mediated transcription. Journal of Virology 72, 701–707.
Received 18 March 1999 ; Accepted 7 May 1999
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 12 Jun 2017 06:34:19
CCAJ