The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus supports latent DNA replication in dividing cells

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is a multifunctional protein that is consistently expressed in all KSHV-associated malignancies. LANA interacts with a variety of cellular proteins, including the transcriptional cosuppressor complex mSin3 and the tumor suppressors p53 and Rb, thereby regulating viral and cellular gene expression. In addition, LANA is required for maintenance of the episomal viral DNA during latency in dividing cells. Colocalization studies suggest that LANA tethers the viral genome to chromosomes during mitosis. In support of this model, a specific LANA- binding site has recently been identified within the terminal repeat unit, and a chromatin interaction domain was mapped to a short amino acid stretch within the N-terminal domain of LANA. Epstein-Barr virus nuclear antigen 1 (EBNA-1), a functional homologue of LANA, is also required for genome segregation; in addition, EBNA-1 also supports efficient DNA replication of oriP-containing plasmids. By performing short-term replication assays, we demonstrate here for the first time that de novo synthesis of terminal-repeat (TR)-containing plasmids is highly dependent on the presence of LANA. We map the required cis-acting sequences within the TR to a 79-bp region and demonstrate that the DNA-binding domain of LANA is required for this DNA replication activity. Surprisingly, the 233-amino-acid C domain of LANA by itself partially supports replication. Our data show that LANA is a sequence-specific DNA-binding protein that, like EBNA-1, plays an important role in DNA replication and genome segregation. In addition, we show that all necessary cis elements for the origin of replication (ori) function are located within a single TR, suggesting that the putative ori of KSHV is different from those of other gammaherpesviruses, which all contain ori sequences within the unique long sequence outside of their TR. This notion is further strengthened by the unique modular structure of the KSHV TR element.

FIG. 1.

LANA supports replication of TR-containing plasmid in 293 and SLK cells in short-term replication assays. A total of 10 μg of p2TR or empty vector pCRII was cotransfected with 10 μg of pcDNA3/ORF73 or carrier DNA into 293 cells (A) or SLK cells (B). At 72 h after transfection, extrachromosomal DNA was extracted as described in Materials and Methods. Then, 10% of the episomal DNA was digested with 20 U of HindIII, whereas 90% was double digested with 20 U of HindIII and 20 U of DpnI for 16 h. After electrophoresis, DNA was immobilized on nylon membranes and hybridized with a radiolabeled TR probe. (A) Replication in 293 cells. Lane 1 contains 150 pg of p2TR digested with HindIII to indicate the position of linear plasmids. Lane 2 contains 150 pg of p2TR digested with HindIII and DpnI. Lanes 3, 4, and 5 show the input of indicated plasmids in the absence or presence of LANA, respectively. pCRII, a plasmid not containing TR sequences, was used as a negative control (lane 6). p2TR can only replicate in the presence of LANA (lane 8) but not without LANA (lane 7). (B) Replication in SLK cells. The gel was loaded in the same order as described for panel A. (C) All cis requirements are located in TR, and p1TR replicates as efficiently as p2TR. A total of 10% of the extracted episomal DNA was digested with 20 U of HindIII (lanes 1 to 6). We observed some partial digestion in panels A and B (lane 8), and therefore Hirt-extracted DNA for all following assays was digested with 100 U of HindIII and 180 U of DpnI for 48 h (lanes 7 to 12). pTRΔZ6, p1TR, and p2TR only replicate in the presence of LANA (lanes 8, 10, and 12, respectively). In contrast, none of them can replicate without LANA (lanes 7, 9, and 11). (D) Unique long sequences adjacent to TR do not contribute to ori activity. pUL0.6 and pUL1.9 do not replicate in the presence of LANA (lanes 8 and 10). The brackets in panels C and D indicate the size range of the replicated plasmids. This blot was hybridized to a probe containing the entire pCRII/TR, which also detects the backbone of pcDNA3/ORF73, marked by asterisks. A triangle (▸) indicates the linear position of p2TR.

FIG. 2.

Mapping of cis requirements for LANA-dependent DNA replication. (A). Illustration of TR unit indicating constructs TR1, -2, -3, and -5, which have been used to identify the LBS within the TR. Below this, the solid lines represent TR-derived fragments that were inserted into either pCRII or pBS and were tested in short-term replication assays (pTR1, nt 1 to 268; pTR2, nt 268 to 581; pTR3, nt 581 to 801; pTR5, nt 551 to 675; pTRΔ1, nt 1 to 581; pTRΔ3, nt 268 to 801; and pTRΔLBS, nt 1 to 559). Also indicated are the relative replication efficiencies of these plasmids compared to wt p1TR, which were derived by comparing the ratios of input to replicated DNA after densitometric measurement. The asterisks at pTR2 and pTRΔ1 indicate that the LBS is not fully contained in these constructs. (B) Mapping data from short-term replication assays in 293 cells. All assays were performed as described in Fig. 1. Odd-numbered lanes indicate plasmids in the absence of LANA; even-numbered lanes indicate plasmids in the presence of LANA. Only wt p1TR, pTRΔ1, pTRΔ3, and pTR2 replicate, as shown in the upper blot in lanes 2, 10, 12, and 14; the lower panel shows linearized plasmids as a loading control. The asterisk shows the linearized pcDNA3/ORF73, since the blots were hybridized to a probe containing the entire pCRII/TR plasmid.

FIG. 3.

Mapping of a second sequence element required for ori function. (A) The 801-bp KSHV TR sequence, with sequence elements identified by the mapping analysis highlighted. The LBS (nt 570 to 587) is in red; the AT-rich region (47% versus 9% in the remainingsequence) is underlined (nt 380 to 481). Between the AT-rich region and the LBS is an 89-bp GC-rich region required for replication. Restriction sites for DraIII (nt 384) and SanDI (nt 506) have been boxed. A second low-affinity LBS located 5 bp from LBS 1 is also underlined (nt 592 to 609) (18). (B) Representation of constructs used for the identification of ori elements within the TR. Lines represent fragments inserted into pCRII. The wt pTR1 and construct pTR5 have been described in Fig. 2 (the asterisk indicates replication data for TR5 shown in Fig. 2B, lane 8). Construct pTRΔ3/DraIII contains sequences from nt 391 to 801, whereas pTRΔ3/SanDI contains sequences from nt 509 to 801. Also shown are relative replication efficiencies from the plasmids derived from panel C. (C) Short-term replication assay as described in Fig. 1. Lanes 1 to 4 contain 10% of linearized plasmid DNA as input control and indicate nearly identical loading. Lanes 5 to 7 show replicated DNA, indicating that all three plasmids replicate with similar efficiency. Lanes 3 and 7, controls showing LANA-dependent replication.

FIG. 4.

C domain of LANA partially supports DNA replication of TR-containing plasmids. p2TR was cotransfected with either expression vectors encoding wt LANA or LANA mutant A, C, AB, AC, or BC as described in detail in Materials and Methods. Transient replication assays were performed as described in Fig. 1, and the blot was hybridized to a probe containing a radiolabeled TR fragment. Lanes 1 and 2 indicate the positions of DpnI-digested and linearized p2TR (▸). Lanes 3 to 9 show input DNA as loading control, whereas lanes 11 to 17 show DpnI double-digested DNA. Newly synthesized DNA can be detected in lanes 12, 14, 16, and 17, in which cells were transfected with wt LANA, C, AC, and BC, respectively. As an additional control for DNA digestion, 2 ng of p2TR was mixed into a mock Hirt extract prior to DpnI digestion (lane 10). The replication efficiency of each LANA mutant was determined by densitometric comparison of replicated DNA to input DNA. LANA mutants C and AC replicate p2TR at ca. 20% efficiency compared to the wt. Mutants lacking the C domain do not support replication (lanes 13 and 15).

FIG. 5.

LANA-C4 does not possess dominant-negative activity. (A) Western blot analysis of LANA-C (aa 770 to 1003) and LANA-C4 (aa 785 to 1003) expressed in MVA/T7 vaccinia virus-infected CV1 cells. (B) DNA binding of LANA-C and LANA-C4 to TR5. Equal amounts of recombinant proteins were incubated with radiolabeled TR5 under conditions described in Materials and Methods. Lane 1 contains probe alone, whereas lanes 2 and 3 contain binding reactions with LANA mutants C and C4; C shows strong complex formation, whereas C4 shows extremely weak binding. (C) Titration of increasing amount of pcDNA3.1V5His/LANA-C4 with a constant amount of pcDNA3/ORF73. Short-term replication assays were performed as described for Fig. 1. Then, 100 ng of wt LANA expression construct was cotransfected with a vector expressing LANA-C4 ranging from 100 ng (1:1) to 10 μg (1:100) into 293 cells. Lanes 1 and 2 indicate the positions of DpnI-digested and linearized p2TR. Lanes 3 to 7 contain input DNA, whereas lanes 8 to 12 show newly synthesized DNA. The comparison of input and replicated DNA at each ratio shows that LANA-C4 does not inhibit LANA wt function even when present at a ratio of 100 to 1 (lane 12).