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Previous Article | Next Article 
Journal of Virology, December 2006, p. 12171-12186, Vol. 80, No. 24
0022-538X/06/$08.00+0 doi:10.1128/JVI.00990-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, University of Pennsylvania School of Dental Medicine,1 The Wistar Institute, Philadelphia, Pennsylvania 191042
Received 12 May 2006/ Accepted 22 September 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. The revelation of these two functions of RTA, together
with its role in initiation of a transcriptional cascade that leads to
transcription of all viral lytic genes, shows that RTA is a critical
initiator and regulator of KSHV lytic DNA replication and viral
propagation. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Lytic DNA replication of herpesviruses differs from latent DNA replication in at least three aspects. First, latent DNA replication initiates at ori-P and proceeds bidirectionally, while viral lytic replication initiates from a distinct origin (ori-Lyt) and proceeds via a rolling circle mechanism. The lytic rolling circle DNA replication results in the generation of concatemeric genomic DNA which is subsequently cleaved into monomeric units and packaged into virions. Second, latent DNA replication depends on host cellular DNA polymerase and accessory factors, while viral lytic replication utilizes its own DNA polymerase and other factors. Third, latent DNA replication occurs in synchrony with the host cell to maintain a stable number of viral DNA episomes in each cell, while viral DNA is amplified 100- to 1,000-fold in lytic replication.
Two duplicated copies of the lytic DNA replication origin [referred to as ori-Lyt (L) and ori-Lyt (R)] were recently identified in the KSHV genome (2, 20). They are located in the KSHV genome between open reading frames K4.2 and K5 and between K12 and ORF71, respectively. These two ori-Lyt regions share an almost identical 1.1-kb core component sequence and 600-bp GC-rich repeats that are represented as 20-bp and 30-bp tandem arrays (20). A systematic mutational analysis of the core component domain of a KSHV ori-Lyt defined several crucial elements in ori-Lyt (31). The first component contains eight C/EBP binding motifs that organize as four spaced C/EBP palindromes. Substitution mutagenesis of these C/EBP motifs showed that these C/EBP palindromes are required for binding of K8 protein in ori-Lyt DNA and for DNA replication. The second component is an 18-bp AT palindrome, which is believed to facilitate the localized unwinding of double-stranded DNA during the initiation of replication. The third component is a previously unidentified sequence and is required for DNA replication. The fourth component consists of an ORF50/replication and transcription activator (RTA) responsive element (RRE) and a TATA box. The presence of a functional RRE and a downstream TATA box suggests that this region serves as an ORF50/RTA-responsive promoter and that a transcription event may be necessary for ori-Lyt-dependent DNA replication. Using a luciferase reporter system, we demonstrated that the region of the RRE and TATA box indeed constitutes an RTA-dependent promoter. A polyadenylated RNA of 1.4 kb was identified downstream of the promoter (31).
Herpesviral
ori-Lyt-dependent DNA replication relies on
virally encoded proteins. The proteins essential
for DNA replication have been identified in herpes simplex
virus type 1 (HSV-1), cytomegalovirus (CMV), Epstein-Barr virus (EBV),
and KSHV (9,
23,
32,
33). Six core replication
proteins are conserved among all herpesviruses, and they are the DNA
polymerase (POL), the polymerase processivity factor (PPF), the
single-stranded DNA binding protein (SSB), and a trivalent
helicase-primase complex which consists of a helicase (HEL), a primase
(PRI), and a primase-associated factor (PAF). Additional proteins, such
as origin binding proteins, regulatory proteins, and auxiliary factors,
were identified and found to be unique to each individual virus
(7,
8,
10,
28). In terms of KSHV,
six core replication proteins and two regulatory proteins, namely, RTA
and K8, were demonstrated to be necessary and sufficient for KSHV
ori-Lyt-dependent DNA replication
(1). RTA binds to a
consensus RRE in ori-Lyt, and K8 associates with the
ori-Lyt DNA through interaction with C/EBP molecules
bound on a cluster of C/EBP binding motifs
(31).
The participation of RTA and K8 in ori-Lyt-dependent DNA replication appears to be the most salient feature in the event. Wu et al. (33) reported that six KSHV core replication proteins could replicate EBV ori-Lyt-containing plasmid in the presence of EBV-encoded protein ZTA. In the process, ZTA functioned as a DNA binding initiator protein responsible for loading core replication machinery complexes onto the EBV ori-Lyt DNA (10). KSHV K8 protein is a distant evolutionary homologue of ZTA and was found to be incorporated into viral replication compartments (VRC) in the nuclei of KSHV-infected primary effusion lymphoma cells (33). In view of this, we wanted to find out if K8 is a functional analog of ZTA of EBV and functions as a DNA binding initiator protein in KSHV ori-Lyt DNA replication. Furthermore, the role of RTA in ori-Lyt-dependent DNA replication was also very intriguing. It may contribute to ori-Lyt replication through its transcriptional activity. It may also contribute to the DNA replication through other mechanisms, such as serving as an origin binding protein (OBP) and DNA replication initiator protein because of its DNA binding nature. In an effort to elucidate the mechanisms underlying the regulation of KSHV ori-Lyt-dependent DNA replication, we investigated the roles of RTA and K8 in viral lytic DNA replication and studied the contribution of the ori-Lyt-associated transcription to viral DNA replication. The results demonstrated a tight coupling of the DNA replication and transcription events. ori-Lyt-dependent DNA replication is inhibited when the RNA transcription is prematurely terminated. The interplay between KSHV DNA replication and transcription has provided a basis for further investigation into the regulation of DNA replication in DNA viruses and eukaryotic cells. Furthermore, RTA and K8 were found to be components of the viral replication compartment, where they interact with core replication proteins and recruit the replication complexes to ori-Lyt DNA.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids and constructs.
Plasmid pOri-A
and its mutants (pOri-
15.7, pOri-16.2, pOri-M12,
pOri-M1256, etc.) were constructed and described previously
(31). pOri-polyA1 and
pOri-polyA3 were generated by inserting a simian virus 40 (SV40) late
poly(A) signal sequence at nucleotides 24271 and 25067 (according to
the numbering of Russo et al.
[27]), respectively, in
the pOri-A plasmid. pOri-polyA2 is a derivative of pOri-polyA1 in which
the polyadenylation consensus sequence AAUAAA has
been deleted by using a PCR-based mutagenesis system, namely, ExSite
(Stratagene). In brief, a pair of phosphorylated oligonucleotides in
opposite directions was used in a high-fidelity PCR with pOri-polyA1
plasmid as a template. After PCR, the template DNA was removed by
complete digestion with DpnI, which does not degrade PCR-synthesized
DNA. The PCR products were self-ligated and used to transform
Escherichia coli competent cells. pOri-TR and
pOri-mTR were generated by deleting the entire fragment of GC-rich
tandem repeats (the PmlI-MulI fragment between nucleotides 24252 and
25069) from pOri-A and by replacing this fragment with a PmlI-MulI
fragment from pGL3 plasmid (between nucleotides 747 and 16) (Promega),
respectively.
pOri-PmU6, pOri-PSV40, and pOri-PCMV were constructed by replacing the RRE-associated promoter region (between nucleotides 24022 and 24252) with mouse U6 (mU6) promoter (315-bp), SV40 early promoter (454-bp), or human CMV (HCMV) immediate-early (IE) promoter (596-bp) sequence, respectively.
pCR3.1-ORF50 was constructed by cloning the cDNA sequence of the ORF50 coding region into pCR3.1 vector (Invitrogen). The construct was described in detail by Lin et al. (20). The expression vectors for Gal4 fusion proteins (pSG-C50 and pSG-424) were described by Lukac et al. (21). The internal deletion mutants of pSG-C50 and pCR3.1 were generated using the ExSite system as described above.
Transient-transfection DNA replication assay. To transfect BCBL-1 cells, 5 µg pOri-A or its mutant plasmids and 5 µg pCR3.1-ORF50 (or pCR3.1 vector) were mixed with 107 cells in OPTI-MEM medium (GIBCO-BRL) and electroporated (200 V, 960 µF) with a Genepulser II (Bio-Rad, Hercules, CA). Electroporated cells were then transferred to RPMI 1640 medium supplemented with 10% serum and grown for 72 h.
Extrachromosomal DNAs were prepared from cells by using the Hirt DNA extraction method as follows. Cells were lysed in 700 µl lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 0.6% sodium dodecyl sulfate [SDS]). Chromosomal DNA was precipitated at 4°C overnight by adding 5 M NaCl to a final concentration of 0.85 M. Cell lysates were centrifuged at 4°C at 14,000 rpm for 30 min. The supernatant containing extrachromasomal DNA was subjected to phenol-chloroform extraction, followed by ethanol precipitation. The DNA was treated with RNase A at 25°C for 30 min and then with proteinase K at 50°C for 30 min. DNA (5 µg) was digested with KpnI/SacI or KpnI/SacI/DpnI (New England Biolabs). The DNAs were separated by electrophoresis on 1% agarose gels and transferred onto GeneScreen membranes (Perkin Elmer, Boston, MA). The Southern blots were hybridized with 32P-labeled pBluescript plasmid in 5x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 2x Denhardt's solution, 1% SDS, and 50 µg/ml denatured salmon sperm DNA at 68°C.
Northern blotting and hybridization. Total RNA was isolated from transfected 293 cells with Trizol reagent (Gibco-BRL) and separated by electrophoresis in 1% agarose-6% formaldehyde gel in 20 mM morpholinepropanesulfonic acid (MOPS) buffer, pH 7.0. Each lane was loaded with mRNA from 2 x 107 cells. The RNA was transferred to a Nytran membrane (Schleicher & Schuell, Keene, N.H.) and hybridized with a single-stranded 32P-labeled probe. The probe was prepared by asymmetric PCR with PmlI-digested pOri-A plasmid as the template and the oligonucleotide OriT-1 (5'-TGTTTATTTCAAGAGCCTATGCTCG-3', nucleotides 25428 to 25403) as the primer. The labeling reaction was performed in 15 µl of reaction solution (1x Taq polymerase buffer, which consisted of 16.67 µM [each] dATP, dGTP, and dTTP, 1.67 µM dCTP, 5 µl of [32P]dCTP [800 Ci/mmol, 10 µCi/µl; Amersham], 100 ng of DNA, 20 pmol of primer, and 2.5 U of Taq polymerase). The PCR was initiated with a denaturing step of 2 min at 94°C, followed by 15 cycles of sequential steps of 1 min at 94°C, 1 min at 50°C, and 3 min at 74°C. Finally, the reaction was extended for 10 min at 74°C. The RNA loading equivalence was controlled by probing with ß-actin cDNA. A 0.24- to 9.5-kb RNA ladder (Gibco-BRL) was included in each agarose-formaldehyde gel and detected in Northern blots by hybridization with labeled DNA.
Reporter plasmids and luciferase assays. Reporter plasmids (pK8-DE250 and its derivatives, 13F and its mutants) were constructed and described previously (30, 31). Ten micrograms luciferase reporter construct, 1 µg pRL-TK plasmid (Promega), and 3 µg pCR3.1-ORF50 (or pCR3.1 empty vector) were mixed with 107 BJAB or BCBL-1 cells in OPTI-MEM medium (GIBCO-BRL) and electroporated (250 V, 960 µF) with a Genepulser II (Bio-Rad, Hercules, CA). Electroporated cells were then transferred to RPMI 1640 medium supplemented with 10% serum and grown for 48 h. The pRL-TK plasmid was included as an internal control that constitutively expresses Renilla luciferase. A dual luciferase reporter assay system (Promega) was used to examine the responsiveness of the promoters to ORF50/Rta. Transfected cells were washed once with 1x PBS and suspended in 400 µl of 1x passive lysis buffer. Cells were broken by one freeze-thaw cycle and centrifuged in a mirocentrifuge for 1 min. Supernatants were assayed for firefly luciferase and Renilla luciferase activities by use of a TD-20/20 luminometer with a dual auto injector (Turner Designs). The luciferase assays were carried out according to the manufacturer's instruction (Promega).
DNA affinity purification and assays. Various biotinylated DNA fragments were synthesized using PCR with pOri-A DNA or its mutants as templates and two oligonucleotides as primers. The two oligonucleotides for 3F DNA fragments were 3F (5'-CGGCAAAGCTAATTTGCATG-3') and Biotin-7R (5'-biotin-ACTGGAATAGGGGCTGCGATGACTC-3'). The oligonucleotides for 9F DNA were 9F (5'-CAATTCTATAATTAAACAAGGTAGAA-3') and Biotin-ID13R (5'-biotin-CGCCACCGAACAACCCCGTGGACAG-3'). The oligonucleotides for 11F DNAs were 11F (5'-TAGGGCCCGATGAGTCATGGGGTT-3') and Biotin24280R (5'-biotin-ACGGGTAAATCCAAGAGATCCGTCCC-3'). The resultant biotinylated PCR fragments were coupled to streptavidin-conjugated magnetic beads (Dynal, Oslo, Norway) and then incubated with nuclear extracts prepared from 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced (and uninduced) BCBL-1 cells for 45 min at 25°C. The bound material was washed four times in D150 buffer (20 mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 150 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.05% NP-40) and then progressively eluted with D300 buffer (same as D150 buffer, except with 300 mM KCl), D500 buffer (with 500 mM KCl), and D1000 buffer (with 1 M KCl). The affinity-purified materials were assayed by Western blotting with different antibodies.
Coimmunoprecipitation assay. Approximately 5 x 107 cells were collected by centrifugation and washed with cold PBS. The cells were suspended in 0.5 ml of immunoprecipitation (IP) buffer (120 mM potassium acetate, 20 mM Tris-acetate [pH 7.9], 5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.1% Nonidet P-40, and one complete protease inhibitor cocktail tablet per 25 ml), placed on ice for 30 min, and sonicated for 5 s. The cell lysates were clarified by centrifugation at 4°C for 10 min. Immunoprecipitation was performed by the addition of 2 µl of monoclonal anti-ORF50 (a gift from Keiji Ueda at Osaka University, Japan) or monoclonal anti-K8 antibody to the cell lysates (0.5 ml) with gentle agitation at 4°C for 60 min. Then, 100 µl of protein G-coated paramagnetic beads (Dynal Biotech, Oslo, Norway) was added and the mixtures were incubated at 4°C overnight. A reaction with 2 µl of mouse immunoglobulin G (IgG) (Sigma) was performed as a control. The beads were then washed five times with cold IP buffer. The precipitates were resuspended in 100 µl of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, boiled for 10 min, and loaded onto SDS-PAGE gels. To eliminate DNA-mediated protein interactions, ethidium bromide (EtBr) was added to aliquots of some cell lysates and maintained at a concentration of 50 or 100 µg/ml throughout the entire IP process, including wash steps.
IFA.
The primary antibodies used in the
immunofluorescence assay (IFA) included anti-K8 monoclonal IgG (1:300
dilution; raised using purified glutathione S-transferase
[GST]-K8 fusion protein), anti-ORF50 monoclonal
antibody (1:300 dilution; provided by Keiji Ueda at Osaka University,
Japan), anti-ORF50 polyclonal antibody (1:300 dilution; provided by
David Lukac at UMDNJ), anti-PPF polyclonal antibody (1:1,000 dilution;
provided by Robert Ricciardi at the University of Pennsylvania),
anti-SSB polyclonal antibody (1:300 dilution; provided by Gary Hayward
at Johns Hopkins), and sheep antibromodeoxyuridine (anti-BrdU) antibody
(1:100 dilution; purchased from Capralogics, Inc., Hardwick,
MA).
BCBL-1 cells were induced with TPA (20 ng/ml) for 48 h. The cells were washed with PBS and fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. The fixed cells were spun onto Shandon double cytoslides at 1,000 rpm for 5 min. The slides were immersed in PBS for 5 min and permeabilized in 0.2% Triton X-100 in PBS for 20 min on ice. For the double-label experiment, the primary mouse monoclonal and rabbit polyclonal antibodies were diluted together and incubated with cells on the slides at room temperature for 50 min. After wash, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG and Texas Red-conjugated anti-mouse antibodies (Vector Laboratories, Inc., Burlingame, CA) at room temperature for 50 min.
Triple-label IFA was performed to visualize replication-active DNA in the nucleus as well as viral DNA replication proteins. TPA-treated BCBL-1 cells were incubated in the media containing 10 µM BrdU (Sigma) for 60 min before fixation. The pulse-labeled cells were fixed with 2% paraformaldehyde in PBS for 20 min at room temperature and then incubated with 2 N HCl for 30 min at room temperature in order to expose incorporated BrdU residues. After four washes with PBS, the cells were incubated with primary mouse monoclonal anti-RTA or anti-K8, rabbit polyclonal anti-SSB, and sheep anti-BrdU antibodies at room temperature for 50 min, followed by incubation with a combination of secondary antibodies (FITC-conjugated anti-rabbit IgG, Cy5-conjugated anti-mouse IgG [Rockland Immunochemicals, Inc., Gilbertsville, PA], and Texas Red-conjugated anti-sheep IgG [Vector Laboratories, Inc.]). All secondary antibodies were diluted at 1:250.
The slides were examined with a Leica TCS SPII confocal laser scanning system. Two or three channels were recorded simultaneously or sequentially. Data acquisition was controlled for possible breakthrough between the green and red channels and between the blue and red channels.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results
presented above led to a hypothesis that a transcription event through
a GC-rich tandem repeat sequence is required for DNA replication to
occur. To prove the hypothesis, two more constructs, one in which the
GC-rich tandem repeats were deleted (pOri-TR) and one in which
the GC-rich tandem repeats were replaced with an irrelevant DNA
sequence of similar length (pOri-mTR), were made. The results showed
that both plasmids were incompetent for DNA replication in lytic BCBL-1
cells (Fig. 1C). Taken
together, our study strongly suggests that assembly of a transcription
complex in ori-Lyt is necessary but not sufficient to support
DNA replication; active transcription through a GC-rich tandem repeat
sequence is needed for KSHV ori-Lyt-dependent DNA
replication.
Can the RRE-dependent promoter in ori-Lyt be replaced by other Pol II or Pol III promoters? Since we discovered that transcription through a GC-rich tandem repeat sequence is essential for DNA replication, we were curious if replacement of the RRE-containing promoter with other promoters allows ori-Lyt-dependent DNA replication to take place. The RRE-containing promoter region from nucleotides 24025 to 24252 was replaced with an HCMV immediate-early promoter, an SV40 early promoter (both are polymerase II [Pol II] promoters), and a mouse U6 snRNA promoter (a Pol III promoter) in pOri-A. These mutants, as well as the wild-type parental plasmid pOri-A, were introduced into BCBL-1 and 293 cells along with the RTA expression vector. Transcription of these mutant plasmids was analyzed in 293 cells by Northern analyses. As illustrated in Fig. 2A, the U6 promoter was able to strongly and constitutively initiate transcription. The size of the U6-transcribed RNA was shorter than the wild-type transcript because the U6-driven transcript terminated at nucleotide 25301 (150 nucleotides earlier than the Pol II transcripts) and was not polyadenylated. The mutants with SV40 and HCMV promoters were also found to be transcribed, even though the levels of HCMV promoter-driven transcripts were lower than those driven by other promoters (Fig. 2A). All of the mutants were also analyzed for their ori-Lyt function in a transient replication assay in BCBL-1 cells. To our surprise, all of the mutants, including U6, SV40, and HCMV promoter replacement derivatives, failed to replicate in the lytic stage of BCBL-1 cells (Fig. 2B). This finding raised the possibility that in addition to supporting transcription, RTA bound on ori-Lyt may have another function(s) that is also critical for ori-Lyt-dependent DNA replication.
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5 to 8 deletion mutants displayed obviously
reduced replication function in comparison to that of the wild-type
counterpart (SG-C50). Among them, the 8 mutant had the
greatest effect on DNA replication. This deletion mutant lacks amino
acids 603 to 625.
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8 mutant and
severely impaired with the 7 mutant in transactivation (Fig.
3A). In addition, the
internal deletions were also introduced into intact RTA expression
vector pCR3.1-ORF50. The effects of these deletions on RTA-mediated
transcription were analyzed using the ori-Lyt-associated
promoter as well as the K8 delayed-early promoter in BJAB or 293 cells,
and similar results were observed (Fig.
4). A previous report showed that the region of RTA between amino acids 607
to 627 is essential for RTA-mediated transcription
(13), and our data were
consistent with their result.
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5 and 6
mutants (Fig. 3C).
However, the effects of these mutations on RNA transcription were
trivial (Fig. 3A and
4). The separation of the
transcription function and the DNA replication function in these
mutants suggests that RTA may also contribute to
ori-Lyt-dependent DNA replication by other mechanisms in
addition to being a transcription
activator. RTA binds to ori-Lyt DNA and interacts with core replication protein complex. The results described above led to the speculation that RTA has multiple functions in ori-Lyt-dependent DNA replication. To explore the new function of RTA in DNA replication, we studied the interaction of RTA with ori-Lyt DNA as well as with viral core replication proteins by using a DNA affinity purification approach. Three overlapping DNA fragments representing the core domain of KSHV ori-Lyt were synthesized and labeled with biotin at their 5' ends (Fig. 5A). The DNA was coupled to streptavidin-conjugated magnetic beads and then incubated with nuclear extract derived from TPA-induced BCBL-1 cells. Bound material was washed with 150 mM KCl-containing buffer and sequentially eluted with 300 mM, 500 mM, and 1 M KCl. Affinity-purified materials were resolved on 4 to 12% SDS-PAGE gels and probed with antibodies against RTA and other viral DNA replication proteins, such as K8, SSB, and PPF. As expected, K8 and RTA proteins were eluted with 500 mM KCl from the 3F and 11F DNA fragments, respectively. However, K8 was also eluted from the 11F DNA and RTA eluted from the 3F DNA. In addition, SSB and PPF bound to 3F and 11F DNAs and eluted with 500 mM KCl (Fig. 5B).
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RRE) were
used for DNA affinity purification with nuclear extract from
TPA-induced BCBL-1 cells. As shown in Fig.
6B, RTA, K8, SSB, and PPF
bound to wild-type 11F but not to the RRE deletion mutant. Similarly,
wild-type 3F and its mutants, in which the C/EBP cluster was mutated,
were used in a DNA affinity assay. Previous studies demonstrated that
three of eight C/EBP binding motifs in the cluster, namely, C/EBP-1,
-2, and -6, contribute more to K8 binding than the other C/EBP motifs
(34) and DNA replication
(31). Thus, we designed a
double mutation in C/EBP-1 and -2 (the M12 mutant) and a
quadruple mutation in C/EBP-1, -2, -5, and -6 (the
M1256 mutant) and examined the effects of these mutations on the
binding of ori-Lyt DNA by K8, RTA, and other replication
proteins. The results showed that not only did K8 fail to bind to the
M1256 mutant but RTA and other replication proteins also did not bind
at all or bound with reduced affinity. All of the proteins bind to the
M12 mutant with compromised affinity (Fig.
7).
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Taken together, our results from the coimmunoprecipitation experiments and the DNA affinity purification study led to two implications: (i) RTA and K8 are components of KSHV prereplication complexes, and (ii) viral prereplication complexes bind to ori-Lyt DNA through RTA and K8, interacting with their respective DNA binding motifs.
RTA and K8 are components of replication compartments, and prereplication complexes are recruited to ori-Lyt DNA through RTA and K8 binding motifs. Viral replication compartments (VRC) are intranuclear domains, which are formed when viral replication successively annexes a large portion of the nucleus, and are the locations where viral DNA replication takes place in the host cell nucleus (24). In herpesviruses, VRC are visible in immunofluorescence assays as large, irregularly shaped nuclear domains. We examined whether RTA is an integral component of the replication compartment. BCBL-1 cells were treated with TPA for 48 h to induce lytic DNA replication. The cells were fixed and examined by double-label IFA. Replication compartment-like large nuclear domains, detected with rabbit polyclonal antibodies against SSB and PPF, were seen in about 47% of the positive cells (17% of cells with smaller granular bodies/domains and 36% of cells with diffuse nuclear staining). In these cells, K8 and RTA, detected with mouse monoclonal antibodies against them, were found to be completely colocalized with SSB and PPF in the nuclear domains (data not shown because they are similar to data shown in Fig. 10). In addition, double label with polyclonal RTA and monoclonal K8 antibodies showed complete colocalization of RTA and K8 in the VRC-like nuclear domains.
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In summary, several lines of evidence, including the binding of K8 and RTA to ori-Lyt DNAs, interactions of K8 and RTA with other replication complex proteins, and colocalization of K8 and RTA and other core replication machinery proteins in viral replication compartments, all led to the conclusion that both K8 and RTA are components of replication complexes in VRC. This notion, together with the result that the association of core replication machinery proteins with ori-Lyt DNA requires the K8 and RTA binding motifs in ori-Lyt, strongly suggests that one of the functions of RTA, as well as that of K8, is interacting with prereplication complexes and recruiting the complexes to ori-Lyt DNA through interactions with their cis-acting motifs, i.e., the C/EBP binding site cluster and the RRE. Figure 11 presents a model for the formation of the replication complex in ori-Lyt, in which a prereplication complex interacts with ori-Lyt DNA through two contact points mediated by K8 and RTA.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, respectively. The
associations of both proteins to ori-Lyt DNA were shown to be
essential for DNA replication
(31). Thus, it is
conceivable that RTA and K8 play key roles in initiation of KSHV
ori-Lyt-dependent DNA replication. However, the details
regarding their involvement in the DNA replication were not known. In
this report, we attempted to reveal the roles of RTA and K8 in DNA
replication as well as the mechanisms of their actions. The salient
features of this report are as follows. (i) We previously showed that one function of RTA in ori-Lyt is to initiate ori-Lyt-associated transcription in DNA replication (31). In this report, we assessed the contribution of RTA-mediated ori-Lyt transcription to DNA replication. The results showed that premature termination of the transcription before the GC-rich tandem repeats abolished DNA replication. However, if the transcription was terminated after the GC-rich repeats (before a putative open reading frame), little effect on DNA replication was observed. These data indicate that assembly of a transcription complex in ori-Lyt is necessary but not sufficient to support DNA replication; active transcription through GC-rich tandem repeats is required for DNA replication. The sequences of the tandem repeats in two ori-Lyt regions [ori-Lyt (L) and ori-Lyt (R)] are quite different, but both are GC-rich repeats of approximately 600 bp and organized as 20- to 30-nucleotide tandem arrays. It suggests that transcription over a particular DNA pattern, rather than a specific sequence, is required for initiation of DNA replication. The importance of the transcription event in the DNA replication was also confirmed by the finding that the RTA transactivation domain is critical for the DNA replication function. The participation of transcription factors and involvement of RNA transcription in the regulation of DNA replication have been observed for viruses as well as eukaryotic cells (6, 14, 29). For example, a powerful transcription enhancer region resides in an Epstein-Barr virus ori-Lyt, which contains DNA binding sites for EBV transcription factors RTA and ZTA. This enhancer region can be functionally replaced with an HCMV major immediate-early enhancer (14). Recently, Jenke and colleagues demonstrated that an active transcription unit and a downstream nuclear scaffold/matrix-attached region (S/MAR) are sufficient for DNA replication and maintenance of a mammalian episome (16). Although the roles of transcriptional elements in DNA replication have not been fully understood, the findings regarding the interplay between transcription and DNA replication have provided a basis for further investigation into the functional roles of RNA transcription in regulation of DNA replication in DNA viruses and eukaryotic cells.
(ii) Further investigation of the requirement of RTA in ori-Lyt-dependent DNA replication showed a multiple-function property of RTA in DNA replication. This notion was first suggested by the promoter replacement experiment, which showed that when the RRE-containing promoter in ori-Lyt was replaced with heterogenic promoters, such as an SV40 early promoter, an HCMV immediate-early promoter, and a mouse U6 snRNA promoter, ori-Lyt DNA replication was abolished even though RNA transcription activities remained. Furthermore, mutagenesis of RTA demonstrated that in addition to the domain in RTA that is known to be essential for transactivation activity, the other region in the activation domain of the molecule was found to contribute to ori-Lyt DNA replication (Fig. 3). Therefore, RTA may play multiple roles in KSHV ori-Lyt-dependent DNA replication.
A new function of RTA, other than RNA transactivation, was revealed in this report. RTA was found to be a component of the viral replication compartment in the nuclei of infected cells and shown to interact with SSB, PPF, and K8 by a coimmunoprecipitation assay. It is likely that RTA interacts with the core replication machinery complex (or prereplication complex), which is composed of at least six core replication proteins. This assumption is made based on the finding by Wu et al. (33) that assembly of a complex of KSHV core replication machinery proteins can occur independently of its attachment to origin DNA. Furthermore, the loading of the core replication machinery complexes on ori-Lyt DNA appears to be mediated by RTA and be dependent upon the RRE in ori-Lyt, as the deletion of the RRE abolished the association of core replication proteins with ori-Lyt DNA. These data strongly suggest that RTA plays a role in recruiting the core replication machinery complexes to ori-Lyt DNA in order to initiate DNA replication.
(iii) Our study also showed that K8
binds to the other end of ori-Lyt (with the aid of C/EBP
) and has a function similar to that of RTA in interaction
with core replication proteins and loading prereplication complexes to
ori-Lyt DNA. It was shown previously that EBV-encoded ZTA, a
distant evolutionary homologue of K8 protein, serves an essential
origin binding and DNA replication initiation function
(10). ZTA can mediate EBV
ori-Lyt DNA replication with the core replication proteins of
EBV, HSV-1, and KSHV (10,
33). The replication
function of ZTA is mediated through interactions with the core
replication proteins (11,
19). Therefore, it was
not surprising to find that K8 exerts a function similar to that of its
EBV counterpart in interaction with the core replication proteins and
recruitment of prereplication complexes to ori-Lyt DNA. In a
preliminary study, we found that K8 was able to interact with HEL
(ORF41) and PAP (ORF40/41) in a GST pulldown assay. No other core
replication protein was found to be pulled down by GST-K8 fusion
protein in that assay (our unpublished data). In the
coimmunoprecipitation assay presented in this report, SSB and PPF were
detected in the K8 precipitates. Thus, K8 is likely to interact with
the entire core replication complexes (through direct
contacts with helicase primer proteins), thereby recruiting the
complexes to ori-Lyt DNA.
(iv) Both RTA and K8 were found to interact with the prereplication complex, and both are responsible for bringing the complexes to ori-Lyt DNA. Extrapolating from the observation that both RTA and K8 are essential for ori-Lyt-dependent DNA replication (1, 31), we speculate that the formation of a functional DNA replication initiation complex with an ori-Lyt DNA requires the efforts of both RTA and K8 in loading prereplication complexes on ori-Lyt DNA. Thus, the prereplication complexes apparently bind to ori-Lyt DNA through two contact points, one at the K8 binding region (the C/EBP cluster) near one end of ori-Lyt and the other at the RRE near the opposite end (Fig. 11). The two-contact-point binding may bring two ends of an ori-Lyt element together, looping the DNA between the K8 and RTA binding sites, as illustrated in Fig. 11. An 18-bp AT palindrome sequence is located in the looped region.
(v) A similar loop structure, in which two UL9 protein dimers (OBP) bind to two binding sites (boxes I and II) in ori-S and protein-protein interactions between the UL9 molecules loop the origin DNA centered by an AT palindrome sequence, has been seen in the HSV-1 DNA replication system (17). Examination of binding of UL9 protein to ori-S DNA by electron microscopy revealed the presence of nucleoprotein complexes that were frequently involved in intermolecular and intramolecular interactions in which two DNA molecules or two regions of a DNA molecule were jointed by protein complexes(25). The looping leads to bending and distortion of ori-Lyt DNA, facilitating unwinding of the DNA sequence. The looping and DNA distortion/unwinding are separate activities, and the latter activities can be enhanced by SSB (17). Overall, UL9 protein functions as a replication initiator protein whose role is to make the DNA at the origin accessible to the DNA replication machinery. RTA and K8 may function in a manner similar to UL9 protein, serving as DNA replication initiators in KSHV ori-Lyt-dependent DNA replication.
However, the classic replicon model for herpesviruses, postulated based primarily on studies of HSV-1, is that the core origin region is first bound by a virus-specific OBP, such as HSV-1 UL9, that recruits the core replication machinery. The core replication machinery proteins are recruited to the origin in an ordered and sequential fashion. In contrast, our results clearly suggest that assembly of the KSHV core replication complex occurs independently of its attachment to ori-Lyt DNA and that the entire complex is loaded on ori-Lyt through two components, K8 and RTA, that have integrated in the complex. This may reflect the mechanistic difference between HSV-1 and KSHV in DNA replication or suggest that more studies are needed to truly understand the processes and mechanisms of DNA replication for herpesviruses.
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ACKNOWLEDGMENTS |
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This work was supported by research grants from the National Institutes of Health (R01AI52789, R01CA86839, and R01AI41136).
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FOOTNOTES |
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Published
ahead of print on 4 October 2006.
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