JOURNAL OF VIROLOGY, Feb. 2005, p. 2637–2642
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 4
Characterization of the Minimal Replicator of Kaposi’s
Sarcoma-Associated Herpesvirus Latent Origin
Jianhong Hu† and Rolf Renne*
Division of Hematology/Oncology and Department of Molecular Biology and Microbiology,
Case Western Reserve University, Cleveland, Ohio
Received 26 May 2004/Accepted 1 October 2004
The latency-associated nuclear antigen (LANA) of Kaposi’s sarcoma-associated herpesvirus (KSHV) binds
to two sites within the 801-bp-long terminal repeat (TR) and is the only viral protein required for episomal
maintenance. While two or more copies of TR are required for long-term maintenance, a single TR confers
LANA-dependent origin activity on plasmid DNA. Deletion mapping revealed a 71-bp-long minimal replicator
containing two distinctive sequence elements: LANA binding sites (LBS1/2) and an adjacent 29- to 32-bp-long
GC-rich sequence which we termed the replication element. Furthermore, the transcription factor Sp1 can bind
to TR outside the minimal replicator and contributes to TR’s previously reported enhancer activity.
Kaposi’s sarcoma-associated herpesvirus (KSHV) is associ-
ated with Kaposi’s sarcoma (KS) and two lymphoproliferative
diseases: primary effusion lymphoma and multicentric Castle-
man’s disease (5, 6, 30). The 140-kbp KSHV long unique
region encodes approximately 90 genes, which are flanked on
both sides with 20 to 40 copies of 801-bp-long GC-rich terminal
repeats (TR) (20, 24, 29). During latency, only a small subset
of viral genes, including the latency-associated nuclear antigen
(LANA), is expressed and the viral genome is maintained as
multiple copies of episomal DNA (10, 11, 28, 32). LANA is a
functional homologue of origin binding proteins EBNA-1 from
Epstein-Barr virus (EBV) and E2 from human papillomavirus
(15, 22). LANA is necessary and sufficient to support stable
long-term episomal maintenance in dividing cells by (i) teth-
ering viral episomes to cellular chromosomes (1, 2, 7, 8, 12, 13,
19, 26) and (ii) supporting the initiation of DNA replication of
TR-containing plasmids (15, 17, 23), presumably by bridging
the viral origin of replication (ori) and the host cellular repli-
cation machinery. Using short-term replication assays, we have
previously demonstrated that all cis-regulatory sequences re-
quired for LANA-dependent ori activity are located within a
single copy of TR (17). By performing a detailed deletion
analysis, we determined the minimal replicator within TR and
showed that the transcription factor Sp1 can bind to at least
two sites within TR and contributes to its previously described
enhancer activity (13).
Mapping the minimal replicator within TR. The TR unit is
801 bp long and 89% GC rich (20, 29). LANA binds in a
cooperative fashion to two sites (LBS1/2) in TR located be-
tween nucleotides (nt) 571 and 610, and both binding sites
contribute to ori activity as determined by short-term replica-
tion assays (12, 17). Located directly upstream of LBS1/2 is an
89-bp highly GC-rich element adjacent to a 101-bp AT-rich
region (Fig. 1A). AT-rich stretches are often found in origins
of replication and are believed to function in DNA unwinding
(4). We have previously reported that deletion of TR se-
quences from nt 1 to 482, including the AT-rich region, and nt
610 to 801 downstream of LBS1/2 does not decrease replica-
tion efficiency. In contrast, a deletion of the GC-rich region (up
to nt 551) completely abolished replication activity. These data
suggested that, in addition to LBS1/2, sequences of this GC-
rich element are required for ori function while the AT-rich
element is dispensable (17).
To confirm these observations, which were based on large
truncations, we generated two constructs in which the AT-rich
or the GC-rich element was specifically deleted from a full-
length TR. pTR?nt388-508 contains a 120-bp-long deletion
encompassing the AT-rich stretch. In pTR?512-556, 48 bp was
deleted from the GC-rich element (Fig. 1A). Constructs were
cotransfected with pcDNA3/LANA into 293 cells, and replica-
tion ability was scored by Southern blot analysis of DpnI-
resistant DNA as previously described (17). Plasmid replica-
tion was LANA dependent, since pTR did not replicate in the
absence of LANA (Fig. 1B, lane 5). In the presence of LANA,
pTR?nt388-508 replicated with efficiency similar to that of
pTR, further confirming that the AT-rich region does not
contribute to replication of small plasmids (Fig. 1B, lanes 4 and
6). We cannot rule out a role for this element in the context of
the viral episome. In contrast, pTR?512-556 did not replicate
to any detectable level (Fig. 1C, lane 4), confirming that se-
quences near LBS1/2 are required for ori function and that this
requirement cannot be compensated for by GC-rich sequences
located elsewhere in TR.
To map which nucleotides within this GC-rich region are
vital for replication, we created 5? truncation mutants contain-
ing various lengths of the GC-rich element adjacent to LBS1/2
by enzyme digestion or by ligating synthetic oligonucleotides
(Fig. 2A). Due to the lack of restriction enzyme sites, the first
set of mutants with lengths between 62 and 20 bp was gener-
ated by digestion of pJH10 (TR nt 509 to 644) with KpnI/XhoI,
followed by exonuclease III nuclease treatment at 4°C. As
shown in Fig. 2B, plasmids containing 58, 53, or 35 bp of the
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, Shands Cancer Center, University of Flor-
ida, Gainesville, FL 32610-0232. Phone: (352) 392-9848. Fax: (352)
392-5802. E-mail: email@example.com.
† Present address: Department of Molecular Genetics and Microbi-
ology, Shands Cancer Center, University of Florida, Gainesville, FL
GC-rich fragment replicated in the presence of LANA, while
mutants containing 25 and 24 bp did not. To map the 5? border
of this element, additional mutants covering the region be-
tween residues 35 and 25 were constructed. A plasmid con-
taining 32 bp of the fragment replicated with activity compa-
rable (91%) to that of pJH10 containing 62 bp (compare Fig.
2C, lane 7, to Fig. 2B, lane 8). A 29-bp-long GC-rich element
still replicated at 23% of wild-type activity (Fig. 2C, lane 8, and
Fig. 2D, lane 6), while removing one more nucleotide dramat-
ically decreased replication to only 3% of wild-type levels (Fig.
2D, lane 7); a mutant containing 27 bp showed no detectable
replication (Fig. 2D, compare lanes 6, 7, and 8). These data
show that the minimal GC-rich sequence that confers LANA-
dependent DNA replication is between 29 and 32 bp long.
Furthermore, the functional minimal replicator is located be-
tween nt 539 and 610 and consists of two functional elements,
LBS1/2 and a 29- to 32-bp-long GC-rich sequence, which we
termed the replication element (RE) (Fig. 2A).
Sp1 binds to TR and contributes to TR enhancer activity but
not to ori activity. TR can function as a transcriptional en-
hancer, suggesting that cellular transcription factors bind TR
(13, 34). We wanted to determine whether sequences within
the GC-rich element contribute to the observed enhancer ac-
TR?nt512-556 into the pGL3 promoter upstream of a simian
virus 40 (SV40) promoter and used in transient-transfection
assays in CV-1 cells. pGL3-TR augmented transcription four-
to fivefold more than pGL3 did; however, the deletion mutant
TR?nt512-556 showed about a 50% decrease in luciferase
activity (Fig. 3A). Analysis of the TR sequence for the pres-
ence of transcription factor binding sites revealed three GC-
box consensus sites for Sp1 (GGGGCGGGG) (18). Surpris-
ingly, one of these sites is located at nt positions 523 to 531
within the deleted sequence of TR?nt512-556, while two more
are located at nt 242 to 259 and nt 623 to 631 (Fig. 3B). Sp1
binding sites have been identified in a variety of origins of
replication, including the ori of SV40; however, it is not clear
how transcription factors contribute to replication (for a review
see references 4 and 14). All three Sp1 sites are located outside
of the minimal replicator (nt 539 to 610) and therefore are not
required for LANA-dependent replication of plasmid DNA.
To determine whether Sp1 binding is responsible for the TR
enhancer activity, we cotransfected CV-1 cells with either
pGL3-TR or pGL3-TR?nt512-556 and increasing amounts of
pEBGN-Sp1 (25) encoding a dominant-negative (DN) Sp1.
Luciferase expression of both constructs was inhibited in a
dose-dependent manner, suggesting that Sp1 augments tran-
scription from TR (Fig. 3C). The observed inhibition was sim-
ilar to that observed when LANA is bound to LBS1/2; cotrans-
fection of both LANA and DN Sp1 did not further suppress
transcription, suggesting that the TR enhancer activity is sup-
pressed under conditions where LANA is bound to TR (Fig.
3C). To directly prove Sp1 binding to TR, we performed an
electrophoretic mobility shift assay with recombinant Sp1 and
a radiolabeled probe containing LBS1/2, the RE, and two
flanking Sp1 binding sites (Fig. 3D). In the presence of Sp1,
two complexes formed (Fig. 3D, lane 2), consistent with the
presence of two Sp1 binding sites. Specificity was further con-
firmed by addition of a monoclonal Sp1 antibody (Fig. 3D, lane
4) and by adding increasing amounts of cold competitor (Fig.
3D, lanes 6 to 9), both of which prevented complex formation.
These data, together with the reporter assays, demonstrate that
Sp1 binds to TR and contributes to its enhancer activity.
The role of Sp1 binding to TR in the viral life cycle is
currently unclear. The fact that LANA suppresses transcrip-
tion when bound to TR seems to negate a model by which Sp1
directly contributes to DNA replication. This notion is further
strengthened by the fact that all three Sp1 sites within TR are
located outside the minimal replicator (nt 539 to 610); hence,
Sp1 binding does not contribute to LANA-dependent DNA
was preparedby cloning
FIG. 1. The GC-rich fragment upstream of LBS1/2 but not the
AT-rich sequence is required for LANA-dependent replication of TR-
containing plasmids. Replication assays were performed as previously
described (17). Briefly, 10 ?g of each mutant TR construct was co-
transfected with 10 ?g of pcDNA3/orf73 or carrier DNA into 293 cells.
Seventy-two hours after transfection, extrachromosomal DNA was re-
covered by Hirt extraction. Ten percent of the episomal DNA was
digested with 20 U of HindIII (input), while 90% was double digested
with 20 U of HindIII and 200 U of DpnI for 16 h (DpnI digest). After
electrophoresis, DNA was immobilized on nylon membranes and hy-
bridized with a radiolabeled probe of the vector backbone. (A) Sche-
matic representation of TR and two deletion mutants. Deleted regions
in the context of full-length TR are indicated by brackets. (B) Deletion
of AT-rich sequence nt 388 to 508 does not eliminate replication.
(C) TR sequences within nt 512 to 556 are essential for LANA-
dependent DNA replication. Positions of linearized test plasmids are
indicated by arrowheads; also indicated is the position of the cotrans-
fected LANA expression vector. Nucleotide numbers within TR are
based on the work of Lagunoff and Ganem (20).
2638 NOTES J. VIROL.
replication. The ability of TR to function as a transcriptional
enhancer might contribute to the regulation of viral gene ex-
pression in a LANA-dependent manner. For instance, expres-
sion of K1, the gene at the left end of the KSHV genome, is
suppressed during latency but significantly induced during lytic
growth (20, 21). Whether this suppression is caused by LANA
binding to TR needs to be further investigated in latently
infected cells. Interestingly, the TR of EBV also contain Sp1
and Sp3 binding sites, and it has been suggested that these
factors do not regulate transcription but might play a role in
genome circularization following de novo infection (31, 33).
Our mapping data demonstrate the presence of a second
essential cis-regulatory element, termed RE. Deletion analysis
mapped the length of RE between 29 and 32 bp, which, to-
gether with LBS1/2, constitutes the minimal replicator of 71 bp
in length. The RE may function as a loading pad for cellular
proteins involved in DNA replication; however, computer-as-
sisted sequence analysis did not reveal any homologies to
known cis-regulatory elements. In vitro, LANA has been
shown to interact with origin recognition complex proteins
(23), and studies to address whether cellular proteins bind
directly to RE (in the presence and absence of LANA) are
FIG. 2. Fine mapping of the minimal sequence requirement for RE. (A) Panel of RE mutants. Plasmids contain different lengths of the GC-rich
element as indicated upstream of LBS1/2 and 34 bp downstream. Mutants were generated by exonuclease III nuclease digestion of pJH10 (TR nt
509 to 644) or by ligating synthetic oligonucleotides into pBluescript II SK(?). The lower panel shows the RE sequence of 32 bp in length (nt 539
to 570). All replication assays were performed as described for Fig. 1. Replication efficiency was calculated by determining the ratio of input and
replicated DNA for each construct in comparison to pJH10, which was set to 100%. Values were derived from two or three independent
experiments for each construct. (B) The plasmid containing 35 bp, but not that containing 25 bp, of the GC-rich fragment replicates in the presence
of LANA. (C and D) The plasmid containing 29 bp of the GC-rich sequence replicates, the plasmid containing 28 bp has reduced activity, and the
plasmid containing 27 bp is not active. Test plasmids are indicated by arrowheads in panels B to D.
VOL. 79, 2005 NOTES2639
ongoing. While most origins of replication contain AT-rich
sequences close to the initiation site, it was shown for the
origin of SV40 that melting occurs at a GC-rich element (3).
These data together with our previously reported character-
ization of LANA binding to TR (12, 13) define an ori structure,
which is strikingly different from oriP of EBV (Fig. 4). The
KSHV ori is positioned in TR, while EBV oriP is located within
the coding region of the viral genome. As a consequence,
KSHV has multiple origins, which may provide a function for
segregation similar to that of the family of repeats in oriP. This
hypothesis is supported by the fact that a single TR is sufficient
for DNA replication while long-term episome maintenance
requires multiple copies of TR (1, 2, 12, 13). The dyad sym-
metry (DS) element of EBV contains four EBNA-1 binding
sites that are organized as two pairs, each containing a high-
affinity site and a low-affinity site spaced by 21 bp (22, 27, 35,
37). Similarly, LANA has a high- and a low-affinity binding site
spaced by 22 bp (12). Although the sequences differ, the orga-
nization of LBS1/2 resembles half of a DS element, which is
sufficient for replication in plasmid-based assays (16, 36).
Flanking sequences of the DS element modulate ori activity
but are not required for function (9, 36). This is different for
KSHV, where LBS1/2 alone does not have ori activity and in
addition requires the RE. In summary, the origins of replica-
tion of rhadinoviruses and lymphocryptoviruses have signifi-
cantly diverged. Therefore, a detailed analysis of the KSHV ori
structure may not only increase our knowledge of gammaher-
pesvirus latent replication but also provide an attractive model
to study the interaction of host cellular factors and origins of
replication in metazoan cells.
We thank Gerald Thiel, University of Saarland Medical Center,
Germany, for providing pEBGSp1. We thank Rebecca Johnson, Mark
FIG. 3. Sp1 binds to TR and contributes to TR enhancer activity. In transient-transfection assays luciferase reporter plasmids were transfected
into CV-1 cells (African green monkey kidney cells), and luciferase activity was determined 48 h posttransfection as previously described (13).
(A) Transient-transfection assays indicate that TR?512–556 has decreased enhancer activity compared to full-length TR. (B) Diagram of Sp1
binding sites in TR. EMSA, electrophoretic mobility shift assay. (C) DN Sp1 suppressed TR enhancer activity in a dose-dependent manner.
Reporter plasmid (100 ng) was cotransfected with an increasing amount of DN Sp1 expression vector (0 and 500 ng and 1 ?g). The last group of
bars shows that cotransfection of a LANA expression vector (pcDNA3/orf73) and DN Sp1 does not further repress transcription of pGL3-TR. Data
shown are derived from two independent experiments performed in triplicate. (D) Sp1 binds to two sites within TR. Electrophoretic mobility shift
assay analysis with a32P-labeled probe spanning LBS1/2, the RE, and the two flanking Sp1 binding sites together with recombinant Sp1 protein
reveals two specific complexes (lanes 2 and 3). Sp1-specific antibody (lane 4) and unlabeled competitor DNA (lanes 6 to 9) inhibit Sp1 binding.
Positions of DNA-Sp1 complexes are indicated by arrowheads.
Samols, and Feng-Qi An for fruitful discussions and critical reading of
This work was supported by grants from the National Institutes of
Health (RO1 CA 88763 and R21 CA97939) to R.R.
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2642NOTES J. VIROL.