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J Virol, March 1998, p. 2089-2096, Vol. 72, No. 3
Howard Hughes Medical Institute and
Department of Microbiology and Immunology, University of California
Medical Center, San Francisco, California 94143-0414
Received 15 August 1997/Accepted 10 December 1997
Hepatitis delta virus (HDV) encodes two isoforms of its principal
gene product, hepatitis delta antigen (HDAg). These two forms play
distinctive and complementary roles in viral replication. Here we
report that the large (LHDAg), but not the small (SHDAg), isoform of
HDAg has the capacity to activate the expression of cotransfected genes
driven by a variety of promoters, including the pre-S, S, and C
promoters of hepatitis B virus. Mutational analysis of the C-terminal
19 amino acids unique to LHDAg shows that changing prolines to alanines
in the two PXXP motifs in this region specifically ablates the
activation function without abolishing another activity of LHDAg,
namely, its ability to inhibit HDV RNA synthesis. However, C-terminal
truncations that also disrupt these PXXP motifs only slightly
diminished the activation function, indicating that the proline
mutations were not acting by inactivating potential SH3 interactions
that could be mediated by these motifs. Mutation of the
isoprenylated cysteine to serine decreases but does not abolish the
activation activity, and overexpression of SHDAg does
not interfere with the transactivation function of LHDAg. Although the
mechanism and biological significance of this activity of LHDAg remain
unknown, the presence of this activity serves as yet another marker
that functionally distinguishes this protein from the closely related
isoform SHDAg.
Hepatitis delta virus (HDV) is an
RNA virus that requires coinfection with hepatitis B virus (HBV) to
complete its life cycle. The helper function supplied by HBV is limited
to the provision of envelope proteins (hepatitis B surface antigens)
for the completion of HDV assembly (28, 29, 31). HDV RNA
replication is independent of its HBV helper (19). In fact,
the presence of HDV suppresses HBV replication in vivo (30,
39). Nonetheless, clinical studies have shown that HDV infection
can be associated with more severe hepatitis than HBV alone and is
often implicated in cases of fulminant hepatitis (4, 32).
The genome of HDV is a circular, single-stranded RNA of about 1,700 nucleotides (nt), of which approximately 70% are self-complementary (for a review, see references 20 and
21). This self-complemetarity allows the genome to
form an unbranched rod-like structure. A unique functional protein,
hepatitis delta antigen (HDAg), is encoded by the genome (3,
38), and two isoforms of this protein are produced during
infection. The canonical small form of HDAg (SHDAg) is 195 amino acids
(aa) long; it harbors an N-terminal coiled-coil domain responsible for
oligomerization (37), a central domain responsible for
binding to the RNA genome (7, 23), a nuclear localization
signal (2, 7), and a C-terminal glycine- and proline-rich
region with an uncertain function. This form of HDAg is essential for
viral RNA replication, although it is not itself a polymerase. Host RNA
polymerase II is thought to supply the polymerase function for
replication (15, 26). During viral replication, an RNA
editing event occurs at the UAG termination codon of SHDAg, allowing
readthrough of another 19 aa (Fig. 1) to
generate the large isoform of the protein, LHDAg (25). Since LHDAg contains all of the domains of SHDAg, it too can form multimers with itself and with the SHDAg isoform, bind HDV RNA (as a homo- or
heteromultimer), and be localized to the nucleus.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Activation of Heterologous Gene Expression by the
Large Isoform of Hepatitis Delta Antigen
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (15K):
[in a new window]
FIG. 1.
Sequence of the 19 aa unique to the C terminus of LHDAg.
The PXXP motifs are underlined. Below are shown the amino acid changes
present in the mutants employed in this study. The positions of the
termination codons introduced into the truncation mutants are indicated
by asterisks.
Despite these similarities, the two HDAgs have very distinct functions (22) and play complementary roles in HDV replication, which takes place largely in the nuclei of infected cells (34). While SHDAg activates HDV RNA replication, LHDAg is a trans-dominant inhibitor of this process (8). By contrast, LHDAg, but not SHDAg, is capable of interacting with the HBV envelope proteins to mediate envelopment of the HDV ribonucleoprotein in viral assembly (6). This interaction has been shown to require farnesylation of a cysteine residue found in the C-terminal 19 aa unique to LHDAg (27, 16). Furthermore, it has been shown recently that only LHDAg is phosphorylated in cells (1).
In this report, we describe yet another activity of LHDAg that further differentiates it from the related isoform SHDAg, i.e., the ability to activate gene expression in trans.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Plasmids.
All wild-type (WT) and mutant HDAg coding regions
were cloned into the KpnI and XbaI sites of
pcDNA3 (Invitrogen). The complete LHDAg open reading frame was first
cloned into pBluescript II SK(
) (Stratagene) to generate pHDL. All
LHDAg mutants were prepared by PCR with pHDL as the template.
)CAT was constructed by two PCR rounds. In the first round, N-myc5' paired
with primer
5'-GTGTGCGGAGAGGGGGGACAATAGCCA-3'
and primer 5'-TGGCTATTGTCCCCCCTCTCCGCACAC-3'
paired with N-myc3' were used in two independent PCRs in which
N-myc2-CAT was the template. The PCR products were then pooled. The
second PCR round was performed with N-myc5' and N-myc3' as the primer.
The final PCR products were cloned into the XhoI and
XbaI sites of pCAT, which was described previously
(36). 3Sp1CAT was generated by annealing the sense oligonucleotide CAG CTC GAG ATT GCC CCC GCC CTC ATT GCC CCC GCC CTC ATT
GCC CCC GCC CTC TAG ATA C to the antisense oligonucleotide GTA TCT AGA
GGG CGG GGG CAA TGA GGG CGG GGG CAA TGA GGG CGG GGG CAA TCT CGA GCT G,
which contains three Sp1 binding sites. The double-stranded
oligonucleotide was cut with XhoI and XbaI and cloned into pCAT. 3Sp1mCAT was constructed in the same way with the
sense oligonucleotide CAG CTC GAG ATT GTC CCC
CCT CTC ATT GTC CCC
CCT CTC ATT GTC CCC
CCT CTC TAG ATA C and the antisense oligonucleotide GTA TCT AGA GAG GGG
GGA CAA TGA GAG GGG GGA
CAA TGA GAG GGG GGA CAA TCT CGA GCT
G, which contain three mutated Sp1 binding sites.
The nucleotide sequence of the fragments from PCR was confirmed by
conventional dideoxy sequencing.
Plasmid pDL452 was a gift from D. Lazinski (Tufts University)
(unpublished data). The promoter constructs (hsp70 TATA series and
simian virus 40 [SV40] early series) were the gift of D. Lukac and J. Alwine (University of Pennsylvania) and R. Kingston (Harvard Medical
School) and have been described elsewhere (24, 33). pPreS1CAT, pSCAT, and pUCAT9 were the gift of T. S. B. Yen
(University of California, San Francisco). pPreS1CAT and pUCAT9 have
been described previously (17, 40). pSCAT was obtained by
cloning the HBV fragment spanning nt 3125 to 33 in pCAT. Luciferase
reporter vector pGL2-promoter (SV40 promoter) was obtained from
Promega.
Cell transfection and CAT and luciferase assays. HepG2, 293, and CCL13 (Chang liver) cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% bovine serum and antibiotics. Cells were transfected by calcium phosphate coprecipitation using 1 µg of reporter plasmid and 4 µg of transactivator plasmids. (For the Western immunoblot assay, 10 µg of DNA was transfected.) At 12 h after transfection, cells were washed with phosphate-buffered saline and cultured for another 24 h, at which time cell extracts were assayed for chloramphenicol acetyltransferase (CAT) activity or HDAg (by immunoblotting with anti-HDAg). For HDV RNA replication analyses, cells were harvested 4 to 6 days after transfection. The CAT assay method used is described in detail elsewhere (36). For the luciferase assay, cells were harvested 48 h after transfection and lysed in lysis buffer containing 25 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 1% Triton X-100, 1% bovine serum albumin, 15% glycerol, and 1 mM dithiothreitol. Cellular extract was measured for luciferase activity in the lysis buffer in the presence of 0.4 mM ATP and 1 mM luciferin.
Northern blot analysis. Total RNA was isolated from cells with RNAzol B (TEL-TEST), electrophoresed through a standard 1% agarose-2.2 M formaldehyde gel, and transferred to a positively charged nylon membrane (Boehringer Mannheim) in 10 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Membranes were hybridized at 72°C with an HDV antigenome-specific riboprobe uniformly labeled with digoxigenin (5). After hybridization, blots were washed at 72°C with 0.1% sodium dodecyl sulfate and 2×, 0.5×, and 0.1× SSC, successively. The probe was detected as previously described (12).
Western immunoblot analysis. Transfected cells were harvested and lysed in a solution containing 50 mM Tris-HCl (pH 7.5), 400 mM NaCl, 0.2% Nonidet P-40, and protease inhibitors. Lysates were fractionated by sodium dodecyl sulfate-polyacrylamide (12.5%) gel electrophoresis and transferred to Immobilon-P membranes (Millipore). The membranes were incubated with an anti-HDAg polyclonal antibody (1:10,000 dilution) for 1 h at room temperature. Immune complexes were detected with horseradish peroxidase-conjugated goat antibodies to rabbit immunoglobulin G (1:7,500 dilution) (Gibco BRL) and enhanced chemiluminescence (Amersham).
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Evidence for transactivation activity of LHDAg. The possibility that LHDAg harbors an activation activity was initially suggested by experiments aimed at identifying cellular factors that might interact with the protein in the yeast two-hybrid assay. In those experiments, a fusion protein containing the DNA binding domain of Gal4p fused to intact LHDAg was expressed in yeast cells. Surprisingly, this Gal4p-LHDAg fusion protein was able to strongly activate transcription of a lacZ reporter gene under the control of a promoter containing multiple Gal4p binding sites, even in the absence of any other interacting activator proteins. No comparable activation was seen when a similar fusion protein containing SHDAg was expressed in the same yeast strain (5). However, since many proteins that do not activate transcription under normal conditions can score in this assay, we decided to conduct further, more rigorous tests of gene activation by LHDAg.
To examine the ability of LHDAg to activate transcription in mamalian cells, plasmids expressing LHDAg or SHDAg were cotransfected into HepG2 cells along with a CAT reporter gene driven by the cellular N-myc2 promoter. Forty-eight hours later, the cells were assayed for CAT activity; the results are expressed as fold CAT activity above that induced in the absence of LHDAg (each value represents the mean result of at least three independent experiments). A plasmid expressing human growth hormone was used as an internal control for transfection efficiency. When the CAT gene was under control of the N-myc2 promoter, CAT activity was increased more than sixfold in the presence of LHDAg (Fig. 2A). In contrast, no activation of the N-myc2 promoter by SHDAg was detected (Fig. 2A). Since immunoblotting with anti-HDAg confirmed the expression of both LHDAg and SHDAg in these experiments (Fig. 2A), the absence of transactivation activity of SHDAg is not due to the absence of the protein. By contrast, a CAT gene under the control of a minimal promoter (TATA box) from adenovirus E1B was not activated by either LHDAg or SHADg (data not shown), suggesting that activation by LHDAg likely operates through factors bound upstream of the TATA box (see also Fig. 2D).
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140 (relative to the cap site of the
mRNA) maintained the transactivation activity of LHDAg, while deletions
to 84 reduced activation by 5.7-fold (Fig. 2C). A computer search of
the sequence from 140 to 84 revealed the presence of binding sites
for Sp1 and for members of the GATA family of transcription factors
(Fig. 2B). To determine if Sp1 might be a target of activation, we
mutated the Sp1 site in this promoter and tested the ability of the
mutant to be activated; this lesion reduced LHDAg induction twofold
(Fig. 2C). From the fact that constructs with inactive Sp1 binding
sites continue to show a residual level of activation (Fig. 2C), we infer that additional factors can also be up-regulated by LHDAg (see
below).
To provide a direct test of the ability of Sp1 to be activated in
trans (directly or indirectly) by LHDAg, we cotransfected LHDAg or SHDAg into HepG2 cells together with a vector containing three
Sp1 binding sites upstream of the minimal E1B TATA box-driven CAT gene
(Fig. 2D). CAT activity was measured 48 h after transfection. As
expected, the presence of SHDAg had no effect on CAT activity, whereas
LHDAg strongly activated the CAT gene linked to the three Sp1 sites
(over 20-fold) (Fig. 2D). The specificity of Sp1 activation by LHDAg
was further demonstrated by introducing mutations into the Sp1 sites.
Control experiments with nuclear extracts showed that oligonucleotides
bearing these mutant Sp1 sites do not bind Sp1 in vitro (data not
shown). Correspondingly, transcription of a CAT gene linked to the
mutant Sp1 sites was not up-regulated by LHDAg (Fig. 2D). This result
confirms that Sp1 is one target of activation by LHDAg.
To explore the generality of this activation response, we examined if
it can be observed in cell lines other than HepG2. Accordingly, we
cotransfected the 3×Sp1E1B CAT reporter gene with the plasmids expressing SHDAg and LHDAg into 293 (human embryonal kindey) and CCL13
(human liver) cells and assayed for CAT activity. In both cell lines,
LHDAg activated CAT gene expression as strongly as in HepG2 cells (Fig.
2D); as in HepG2 cells, SHDAg was inactive in this assay in both lines.
The fact that CAT genes with differing 5' regions respond differently
to LHDAg makes it unlikely that the prime target of this activation
resides within CAT sequences. However, to exclude the possibility of
reporter-specific artifacts, we also examined the ability of LHDAg to
activate a luciferase reporter, in this case driven by an SV40
promoter. As shown in Fig. 2E, this reporter was reproducibly
up-regulated fourfold, an amount similar to that observed with CAT
reporters driven by promoters containing SV40 components (Fig.
3). No effect of SHDAg on luciferase
expression was observed.
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Effect of LHDAg on various promoters. To assess the range of potential transcription factor targets of LHDAg, we tested two series of promoters in transient transfection assays. The hsp70 minimal promoter and the TATA box of the SV40 early promoter were each paired with a single copy of a variety of upstream stimulatory elements (USEs), including CAAT, Ap1, Sp1, ATF, and a nonsense sequence (none), all driving CAT reporter genes (for detailed sequences, see references 24 and 33). Each of these plasmids was cotransfected into HepG2 cells with constructs expressing either LHDAg or SHDAg, and the transfected cells were assayed for CAT activity. As shown in Fig. 3, the increase in CAT activity caused by LHDAg was higher with the promoters based on the hsp70 minimal promoter than those based on the SV40 early promoter for the same USE. When different USEs were linked to the same promoter and tested for response to LHDAg, there was a hierarchy of responses, in the following order: ATF > Sp1 = CAAT > AP1. These data affirm that Sp1 is not the only factor that can be activated by LHDAg expression. By contrast, preliminary experiments failed to show activation of Oct-1 and HNF4 (data not shown), suggesting that LHDAg is not an indiscriminate activator of all upstream activators.
Effect of LHDAg mutations on transactivation activity. LHDAg and SHDAg are identical in sequence, save for the additional 19 aa present at the C terminus of LHDAg. Since SHDAg does not possess the transactivation activity displayed by LHDAg, it seemed likely that this activity was endowed by this 19-aa C-terminal extension. To explore this possibility, we constructed several classes of LHDAg mutants, including both truncations and amino acid substitutions. In a preliminary inspection of the sequence of this region, two particular features that pointed to the potential for protein-protein interactions attracted our attention. First, we noted the presence of two PXXP motifs (Fig. 1); in some proteins, such motifs have been implicated in interactions with other proteins harboring SH3 domains (9). The second and more well-established feature was the CXXX motif responsible for the known farnesylation of LHDAg at cysteine 211 (27) that is essential for interactions with HBsAg (16). Special attention was paid to these two features in the design of mutations in this region.
The mutations we constructed are summarized in Fig. 1. Three lesions were specifically designed to target the PXXP motifs. Mutations in LHDAgP1m and LHDAgP2m consist of replacement of prolines with alanines in the first and second PXXP motifs, respectively (Fig. 1), while LHDAgP1P2m bears mutations in both PXXP motifs (Fig. 1). Mutant Lcys211 changes Cys 211 to serine, thereby ablating isoprenylation. The remaining mutations introduce stop codons after codons 8, 13, 16, and 18 of this 19-codon region (Fig. 1) and are designated S+8, S+13, S+16, and S+18, respectively. Each mutant was transfected into HepG2 cells and tested for both transactivation of the 3×Sp1CAT reporter and for the expression of the mutant proteins (as assayed by immunoblotting with anti-HDAg). As shown in Fig. 4C, all of the mutants expressed similar levels of LHDAg proteins and had electrophoretic mobilities consonant with their predicted chain lengths.
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Effect of SHDAg on transactivation activity of LHDAg. During viral replication, SHDAg and LHDAg coexist in cells and their relative stoichiometries are important in determining function. For example, the inhibitory effects of LHDAg on viral RNA replication can be overcome by overexpression of the SHDAg isoform. To determine if expression of SHDAg can influence the transactivation activity of LHDAg, HepG2 cells were transfected with a fixed quantity of the LHDAg expression plasmid and increasing amounts of an SHDAg-expressing construct, and the ability to activate CAT expression from a cotransfected reporter gene was assayed. Over an eightfold range of relative SHDAg-to-LHDAg plasmid concentrations, no significant effect on the level of transactivation was observed (Fig. 5).
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Effect of LHDAg on HBV promoters. Since HBV is the natural helper virus of HDV, we wanted to know what effect expression of LHDAg would have on HBV promoters. pPreS1CAT, pSCAT, and pUCAT9 are plasmids in which the CAT gene is under the control of the HBV pre-S, S, and C promoters, respectively (17, 40). Each of these plasmids was cotransfected with the LHDAg expression vector into HepG2 cells, and extracts were assayed for CAT activity. As shown in Fig. 6, all of the HBV promoters tested were activated by LHDAg; again, SHDAg expression was without effect.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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These studies document that, at least in acutely transfected cell cultures, LHDAg, but not SHDAg, displays a novel activity, the ability to activate gene expression in trans. Activation is independent of the reporter function used and occurs in several cell types and on a wide variety of natural and synthetic promoters but is not observed on minimal (basal) promoter elements, suggesting that it operates principally on accessory rather than basal transcription factors.
We know little of the mechanism by which this activation occurs. The yeast two-hybrid result that initially drew our attention to this phenomenon could be interpreted to mean that LHDAg can interact directly or indirectly with the basal transcriptional machinery once targeted to DNA, and certainly binding to accessory transcription factors might be one way to achieve such targeting in vivo. However, since many proteins with no transcriptional roles in vivo can score in two-hybrid assays, we are reluctant to place too much emphasis on this result as a clue to the mechanism. Many alternative mechanisms can be envisioned, including much more indirect ones. For example, LHDAg might function from a cytoplasmic location (e.g., the cytosolic face of the endoplasmic reticulum) to initiate a signal transduction cascade that ultimately converges on nuclear transcription factors. Such a proposal would be formally similar to certain models proposed for the activation of gene expression by the HBV X protein (11). Additional in vivo and in vitro studies are required to address the mechanistic issues raised here.
The potential biological significance of this activity is likewise a matter of conjecture. For example, we do not know if activation occurs at levels of LHDAg achieved during infection in vivo. Attempts to address this question in transfected cells have been impeded by technical difficulties. Starting from cloned HDV DNA, it takes days in cell culture before RNA editing generates substantial LHDAg; by this time, most of the input reporter CAT plasmid has disappeared. We have been unable to examine rigorously whether native LHDAg expressed from edited RNA can activate a reporter gene, although we think this highly likely. The larger question is what, if any, functional consequences such transactivation might have. It seems unlikely that such an activity would be required for HDV RNA synthesis, since the latter is dependent upon SHDAg and is achieved prior to peak LHDAg levels. By contrast, induction of HBV envelope protein expression by LHDAg could well serve to enhance HDV assembly, and, as shown in Fig. 6, the HBV pre-S and S promoters are clearly responsive to LHDAg expression.
Irrespective of its role in HDV replication, the activation activity might influence other aspects of the infection, e.g., pathogenesis. Induction of host gene expression by LHDAg could play a role in enhancing pathogenesis; for example, if induction included class I major histocompatibility complex genes, elevated liver cell cytotoxicity might result from enhanced cytotoxic T-lymphocyte action. Induction of HBV gene expression (Fig. 6) could have similar effects in sensitized infected hosts. This activity could contribute to the clear association of HDV with fulminant hepatitis, which is thought to result largely from exaggerated cytotoxic responses to viral antigens. In this formulation, the reduction in HBV markers typically observed in HDV coinfection (30) would be the result of enhanced immune clearance (rather than direct repression of HBV genes by HDV gene products). Alternatively, it is possible that suppression of HBV in HDV-infected cells is mediated by host factors induced by LHDAg or that the inductive effect of LHDAg observed here is countered by a repressive effect of another viral gene product, e.g., SHDAg (39).
Finally, the transactivation function described here might have no function in present-day HDV replication but may simply represent a vestige of the evolutionary history of LHDAg. We recently identified a host gene, termed dipA, whose product interacts with HDAg (5). Characterization of this gene showed that it is distantly related to that for LHDAg, suggesting that present-day HDV may have been derived from the capture of a cellular gene by a primitive, viroid-like replicon. Of note is the fact that dipA also shares limited homology with members of the fra-encoded family of transcription factors (10), and the dipA gene is directly adjacent to fra-1 in the mouse genome (5a), again suggesting a relationship. If these tantalizing evolutionary connections to cellular transcription factors are correct, then the activation function of LHDAg may simply be the vestigial remnant of an activity that was once its central mission but which now is only a secondary or accessory function.
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ACKNOWLEDGMENTS |
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We are grateful to Rob Brazas for many insightful discussions and for helpful comments on the manuscript. We thank David Lazinski for plasmid pDL452; David Lukac, James Alwine, and Robert Kingston for the promoter constructs based on hsp70 and SV40 early promoters; and T.-S. Benedict Yen for pPreS1CAT, pSCAT, and pUCAT9. We thank F. Bergametti, D. Sitterlin, and C. Transy for help with the luciferase assay.
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FOOTNOTES |
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* Corresponding author. Mailing address: Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California Medical Center, San Francisco, CA 94143-0414. Phone: (415) 476-2826. Fax: (415) 476-0939. E-mail: ganem{at}socrates.ucsf.edu.
Present address: U.R.E.G., Institut Pasteur, 75015 Paris,
France.
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Abstract
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Discussion
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J Virol, March 1998, p. 2089-2096, Vol. 72, No. 3
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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