Howard Hughes Medical Institute and Departments of Microbiology and Medicine, University of California, San Francisco, CA 94143-0414, USA1
Author for correspondence: Don Ganem. Fax +1 415 476 0939. e-mail ganem{at}socrates.ucsf.edu
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Abstract |
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Introduction |
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In herpesviruses, activation (or reactivation) of the virus lytic cycle initiates a temporally regulated cascade of gene expression, ultimately resulting in the release of infectious virions (Honess & Roizman, 1974 ). Lytic cycle genes can be categorized roughly into three classes: immediate-early (IE), delayed early (DE) and late genes. IE gene products direct the expression of DE genes, including components of the virus replication machinery. After replication of the viral genome, late genes, generally structural proteins, are expressed and virions are packaged and released. KSHV gene expression is no exception to this paradigm (Sun et al., 1999
). In KSHV, the ORF50 protein (a homologue of the EBV IE R protein) has been shown to trigger reactivation from latency in B cell lines derived from PEL and direct expression of KSHV DE genes (Lukac et al., 1998
, 1999
; Sun et al., 1998
). KSHV assembly protein (ORF17.5) and small viral capsid antigen (ORF65), the homologues of which in herpes simplex virus (HSV) and EpsteinBarr virus (EBV) are classified as late genes, have been shown to behave with late kinetics in that they are induced by activators such as TPA but their induction is blocked by inhibitors of the viral polymerase (Lin et al., 1997
; Unal et al., 1997
). Thus, viral DNA replication seems to constitute an important temporal boundary between early and late gene expression in KSHV.
The prevailing model of herpesvirus late gene regulation is based on work done in HSV. Initial experiments, where late promoters were removed from the virus context to either plasmid vectors or the host genome, showed that such constructs were expressed with early kinetics (Dennis & Smiley, 1984 ; Homa et al., 1986
; Silver & Roizman, 1985
). Further experiments showed that unreplicated viral genomes cannot serve as templates for late gene expression, despite the probable presence of all trans-acting factors via a separate viral genome replicated in trans (Mavromara-Nazos & Roizman, 1987
), highlighting the necessity for DNA replication in cis. Once a lytic origin was provided in cis to a late promoter on a plasmid vector, proper late gene regulation could be observed in infected cells (Johnson & Everett, 1986a
). Several groups subsequently demonstrated that minimal late promoter regions (TATA box+cap site) were sufficient to reproduce late gene regulation in infected cells when a lytic origin was present in cis (Flanagan et al., 1991
; Homa et al., 1986
, 1988
; Johnson & Everett, 1986b
). However, such a model is doubtless an oversimplification, as further studies have shown that structural features other than the TATA box are able to influence late gene expression (Kibler et al., 1991
; Mavromara-Nazos & Roizman, 1989
; Steffy & Weir, 1991
).
The nature of the dependence upon viral DNA replication for late gene expression may vary between herpesviruses. Recent studies of EBV, a gammaherpesvirus more closely related to KSHV, showed that EBV late promoter regions driving a CAT reporter gene exhibited late kinetics in infected cells even when the plasmid lacked an oriLyt in cis (Serio et al., 1997 ). It was subsequently shown that the region responsible for conferring late gene regulation in this context was the minimal core promoter, bearing a unique TATA box variant (TATTAAA) (Serio et al., 1998
). These results suggested that DNA replication in cis might not be a strict requirement for late gene expression in all herpesviruses.
Sparked by these results, we set out to define the sequence requirements for properly regulated expression of KSHV late genes. In this work, we show that KSHV late promoters, unlike their EBV counterparts, do not reproduce correct late gene regulation when removed from the context of the viral genome. KSHV late gene expression therefore seems likely to require replication of the viral genome in cis.
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Methods |
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pPr/AP and pPr/AP rev were constructed by cloning the 1313 nt HindIIIEcoRV fragment from genomic positions 31663 to 32976 in the forward or reverse orientations, respectively, into pGL3-basic that had been digested with SmaI. The HindIII site was blunt-ended with Klenow polymerase.
pAP TTG was constructed by changing the initiating ATG of the AP ORF (nt 31687) to TTG by PCR mutagenesis.
pGL3-gB has been described previously (Lukac et al., 1998 ). pGL3-MCP was constructed by cloning the 1935 nt XmaINcoI fragment from genomic positions 32362 to 31666 into pGL3-basic that had been digested with the same enzymes.
pGL3 -30 AP was constructed by PCR amplification of the pGL3 AP plasmid with primers AP -30 (5' GAGCCACGCGTATTTAAAGGCCAGC) and pGL3rev (5' CTTCATAGCCTTATGCAGTTGCTCTC). The PCR product was digested with MluI and NarI and cloned into the corresponding sites in pGL3-basic.
Cell lines and transfections.
BCBL-1 cells were propagated and maintained in RPMI 1640 medium as described by Renne et al. (1996) . Cells were diluted to a density of 2·5x106 cells/ml 1824 h prior to transfection. For Superfect transfection, cells were washed once with PBS and 1x107 cells were resuspended in 6 ml growth medium for each transfection. DNA was diluted in 150 µl unsupplemented RPMI 1640 medium and 20 µl Superfect (Qiagen) was added. Complexes were allowed to form for 10 min at room temperature and then 1 ml of growth medium was mixed with the DNASuperfect complexes which were then added to the cells. Twenty-four h post-transfection, cells were split for chemical treatment. Cells were harvested 48 h post-induction.
BJAB cells were propagated and maintained in RPMI 1640 medium as described by Renne et al. (1996) . For electroporation, cells were washed once with PBS and resuspended in unsupplemented medium. Cells (1x106) were aliquotted in 0·4 µl volumes to electroporation cuvettes (0·42 cm) containing 20 µg DNA. Cells were electroporated at 960 µF and 250 mV and subsequently transferred to 10 ml fresh medium. Superfect transfection was carried out as described above.
293 cells were maintained and propagated in Dulbeccos modified Eagles medium H16 supplemented with 10% FCS, 2 mM glutamine and antibiotics. For transfections, cells were plated at 2x105 cells per 60 mm dish and grown overnight. On the day of transfection, cells were fed 3 h before DNA addition. DNA was precipitated by using the calcium phosphate procedure. Twenty-four h after DNA addition, cells were washed with PBS and fresh medium with or without TPA was added. Cells were harvested 48 h after addition of DNA.
Preparation of RNA and cDNA cloning.
BCBL-1 cells were diluted to a density of 3x105 cells/ml and induced with 20 ng/ml TPA or mock treated. Twenty-four h post-induction, cells were harvested and RNA was isolated by using RNAzol B (Tel-Test) following the manufacturers directions.
mRNA from induced BCBL-1 cells was primed with poly(dT) and cloned into Lambda Zap (Stratagene) by R. Renne (UCSF, San Francisco, USA). The library was screened with a ClaISacI fragment from the assembly protein ORF by using a Random DNA Prime kit (Amersham).
RNase protection assay.
The riboprobe plasmid was constructed by PCR amplification of a 256 nt region spanning genomic positions 3151131736 with primers 5' TGGAATTCAACTGACAGAGGTGTGG and 5' TCGGGTACCGGGGAACATCTGGCC. The PCR product was digested with KpnI and EcoRI and cloned into the corresponding sites in pBSII SK-. The riboprobe plasmid was linearized with EcoRI and transcribed with T7 RNA polymerase in the presence of [32P]UTP. Gel-purified riboprobes (4x105 or 8x104 c.p.m.) were hybridized to 10 µg total RNA overnight at 42 °C. RNase digestion was performed with a 1:100 dilution of the RNase A/RNase T1 solution from the RPA II kit (Ambion). Digested RNAs were precipitated and separated on an 8% denaturing acrylamide gel. The gel was exposed to Kodak BioMax film for 18 h.
Primer extension.
Primer extension was performed as described by Zhong et al. (1996) . Ten µg total RNA from BCBL-1 cells, induced with TPA for 48 h or uninduced, was hybridized with primer PE1 (5' GGGATGGATATGATATCCTCTTGAC) or PE2 (5' GGATGCCGACCGGGAATTGGCTGGC). Samples were separated on an 8% denaturing acrylamide gel. The gel was exposed to Kodak BioMax film for 4 days.
Luciferase assay.
Cells were washed twice with 1 ml PBS. After the addition of 0·2 ml reporter lysis buffer (Promega), cells were harvested by scraping, vortexed and spun briefly to pellet cellular debris. Aliquots (20 and 50 µl) were analysed by luciferase or -galactosidase assay, respectively, according to the manufacturers instructions (Promega).
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Results |
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KSHV late promoters taken out of the virus context are not dependent upon virus replication
In order to study the transcriptional regulation of KSHV late genes, we set out to identify regions of the AP promoter that could reproduce late gene regulation in a heterologous context. A 700 bp SacIEcoRV fragment and a 1·3 kb HindIIIEcoRV fragment were cloned into a luciferase reporter plasmid in both orientations (Fig. 2a) and tested for their ability to drive luciferase expression. When tested in 293 cells, both pPr/AP and pAP were able to direct the expression of luciferase up to 29·2- and 89·9-fold over the promoterless, parent reporter vector, pGL3-basic (Fig. 2b
). This activity was dependent upon orientation, as the same fragments cloned in the reverse direction, pPr/AP rev and pAP rev, were much less active (1·8- and 15·4-fold) in the same assay.
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To determine whether this 12-fold TPA induction reflected faithful reproduction of late gene regulation, we transfected pAP TTG into BCBL-1 cells under four different conditions: chemical inducer alone (ionomycin or TPA), viral polymerase inhibitor ganciclovir (GCV) alone, inducer and inhibitor combined (ionomycin/GCV or TPA/GCV) or no treatment. Luciferase activity of untreated cells was set to 1. Fig. 3(b) shows that pAP TTG activation was not inhibited by GCV. Moreover, activation of this reporter construct was not entirely dependent upon viral cofactors, as pAP TTG was also activated modestly by TPA and ionomycin in BJAB cells, an uninfected B cell line (Fig. 3c
), and behaved identically with regard to GCV in this background.
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Minimal promoter constructs are also insufficient to direct gene expression with late gene kinetics
Serio et al. (1998) showed that, for EBV late gene regulation, the minimal core of a late promoter was able to confer late gene kinetics on a reporter construct. Their observation that upstream enhancers could override the temporal control provided by a core EBV late promoter led us to consider whether the KSHV promoter regions we tested may have been too large, encompassing enhancer regions that would mask the ability of a KSHV core late promoter to confer regulated late gene expression. Moreover, many of the EBV late promoters have an unusual TATA element (TATTAAA) (Serio et al., 1998
). Inspection of the promoter regions of various KSHV late promoters showed that a subset of the late promoters, including AP, also had unusual TATA elements (TATTTAAA) reminiscent of the EBV late promoter TATA motif identified by the Miller group (Serio et al., 1998
). To test this hypothesis, we created a deletion mutant of pAP TTG, truncating the upstream region to -30 from the start site. Fig. 4
shows that, unlike EBV, this minimal promoter region was unresponsive to viral polymerase inhibitors in BCBL-1 cells following lytic induction.
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Discussion |
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The presence of a candidate variant TATA motif, restricted to a subset of late promoters, suggested a possible explanation for our inability to reproduce late gene regulation; that our promoter regions incorporated upstream elements that do not normally play a role in the regulation of late genes but, in our assay, when the region is taken out of its native context, would override the genuine regulation that is directed by minimal late gene promoters during the natural virus lytic cycle. Our results, however, show that a core late gene promoter, while responsive to TPA, is not responsive to viral polymerase inhibitors, confirming our experience that late gene expression in KSHV differs from that in EBV.
Our data suggest that late gene expression in KSHV may follow more closely the HSV model of late gene regulationnamely a dependence upon virus replication in cis to facilitate control of late gene expression. Our results are reminiscent of early HSV experiments, which showed that HSV late promoters, when transferred to the host genome, behave as early promoters.
One possible model for the requirement of DNA synthesis in cis is that replication alters an inhibitory state such as methylation in promoter regions, restricted access of promoters packaged in nucleosomes or repression of transcription initiation by negative-regulatory proteins bound to the promoter. Replication of the viral genome would result in promoter regions free of inhibitory elements due either to the generation of DNA that has not been post-transcriptionally modified (i.e. methylated) or to the displacement of inhibitors (histones or negative-regulatory proteins) by the virus replication machinery. For any of these scenarios, the act of replication is central to the ability to express late genes. If this is so, further study of late gene regulation will be dependent upon the identification of the KSHV lytic origin.
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Received 20 January 2000;
accepted 14 April 2000.