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. 2005 Feb;79(4):2637-42.
doi: 10.1128/JVI.79.4.2637-2642.2005.

Characterization of the minimal replicator of Kaposi's sarcoma-associated herpesvirus latent origin

Jianhong Hu  1 , Rolf Renne
Affiliations

Characterization of the minimal replicator of Kaposi's sarcoma-associated herpesvirus latent origin

Jianhong Hu et al. J Virol. 2005 Feb.

Abstract

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.

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Figures

FIG. 1.
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 μg of each mutant TR construct was cotransfected with 10 μg of pcDNA3/orf73 or carrier DNA into 293 cells. Seventy-two hours after transfection, extrachromosomal DNA was recovered 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 hybridized with a radiolabeled probe of the vector backbone. (A) Schematic 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 cotransfected LANA expression vector. Nucleotide numbers within TR are based on the work of Lagunoff and Ganem (20).
FIG. 2.
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.
FIG. 3.
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 TRΔ512-556 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 μg). 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 a 32P-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.
FIG. 4.
FIG. 4.
Comparison of the latent origins between EBV and KSHV. (A) Genome structure and oriP of EBV. (B) Genome structure and latent origin of KSHV. IR, internal repeat; DS, dyad symmetry; FR, family of repeat; HA, high-affinity binding site; LA, low-affinity binding site; LBS1 and LBS2, LANA binding sites 1 and 2, respectively.
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