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. 2005 Nov;79(21):13829-36.
doi: 10.1128/JVI.79.21.13829-13836.2005.

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen induces a strong bend on binding to terminal repeat DNA

Lai-Yee Wong  1 , Angus C Wilson
Affiliations

Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen induces a strong bend on binding to terminal repeat DNA

Lai-Yee Wong et al. J Virol. 2005 Nov.

Abstract

During latency, the Kaposi's sarcoma-associated herpesvirus genome is maintained as a circular episome, replicating in synchrony with host chromosomes. Replication requires the latency-associated nuclear antigen (LANA) and an origin of latent DNA replication located in the viral terminal repeats, consisting of two LANA binding sites (LBSs) and a GC-rich sequence. Here, we show that the recruitment of a LANA dimer to high-affinity site LBS-1 bends DNA by 57 degrees and towards the major groove. The cooccupancy of LBS-1 and lower-affinity LBS-2 induces a symmetrical bend of 110 degrees . By changing the origin architecture, LANA may help to assemble a specific nucleoprotein structure important for the initiation of DNA replication.

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Figures

FIG. 1.
FIG. 1.
Association of wild-type and KK1113AA mutant LANAC with LBS-1 and LBS-2. (A) Schematic showing full-length LANA and fragments used in this study. The amino acid numbering corresponds to the prototype BC-1 sequence (36). The numbers in parentheses signify coordinates of the actual LANA variant (GenBank accession no. AAB62657 [31]) used here. FL, full length; N, LANAN; C, LANAC. (B) EMSA using 32P-labeled probes containing LBS-1 or LBS-1 and LBS-2. The first two lanes of each panel are controls showing the probe mixed with unprogrammed lysate (lanes 1 and 5) and lysate expressing LANAN (lanes 2 and 6; lanes labeled “N”). Lysates expressing LANAC WT (lanes 3 and 7; lanes labeled WT) or LANACKK1113AA (lanes 4 and 8; lanes labeled “M” for “mutant”) were incubated with each probe, giving rise to one or more specific complexes (filled and open arrowheads). Nonspecific complexes are indicated with an asterisk. (C) Coimmunoprecipitation assay to monitor self-association. HeLa cells were transiently transfected with expression plasmids (1.5 μg each) encoding FLAG-tagged LANAC WT (lanes 1 and 4), FLAG-LANACKK1113AA (mutant; lanes 2 and 5), or FLAG-LANAN (lane 3) and HA-tagged LANAC WT (lanes 3 and 4), LANACKK1113AA (lane 5), or LANAN (lanes 1 and 2). At 24 h posttransfection, protein extracts were prepared using high-salt extraction buffer and immunoprecipitated (IP) with anti-FLAG antibody (α-FLAG)-coupled beads (Sigma). Immunoprecipitated complexes were resolved by SDS-12% PAGE and probed by immunoblotting with anti-HA (α-HA) antibody (upper panel). One-thirtieth of the starting extracts were immunoblotted in parallel using anti-FLAG (middle panel) or anti-HA (lower panel) antibodies. Nonspecific cross-reactive polypeptides are indicated with an asterisk. Sizes of the molecular mass standards are in kilodaltons. (D) SDS-PAGE analysis of in vitro-translated 35S-labeled proteins used in panel B detected by fluorography.
FIG. 2.
FIG. 2.
LANAC bends DNA. (A) Design of probes used for the CPA. Sequences of LBS-1 and LBS-2 as determined by Garber et al. (15) are boxed, and the numbering follows that of the prototype TR sequence (GenBank accession no. U86666) (26). Probes were labeled by PCR in the presence of [32P]dCTP and digested at one of eight restriction endonuclease sites in the duplicated polylinker of pBEND2H, yielding probes of identical lengths but with the LBS at different positions relative to the ends. The center of the bend induced by the binding of LANAC WT to the LBS-1 probe was at guanine-581 (filled diamond); with LANACKK1113AA, the center of the bend was at guanine-580 (asterisk). (B) EMSA using 32P-labeled probe sets containing LBS-1 incubated with in vitro-translated LANAC WT (lanes 1 to 8) or the KK1113AA mutant (lanes 9 to 16). Bands correspond to the LANAC-induced complex (filled arrowheads), and unbound probes are indicated. (C) Plot of the relative mobilities of retarded complexes (mobility of a complex divided by the mobility of the free probe) compared to the position of an arbitrary reference point (cytosine-584 [underlined]) from the 5′ end of each probe. The best fit to the cosine function curve was generated using SigmaPlot. The upper graph represents the relative mobility of LANAC WT. By extrapolation, the fastest-migrating complex corresponds to a probe with a reference nucleotide at position 75.3 within the 157-bp probe (95% confidence interval, 73.6 to 78.0). The predicted bend center should be equidistant from the ends of this probe and would therefore correspond to guanine-581. The lower graph shows the analogous mobility plot for LANAC KK1113AA. Minimal migration occurs when the reference point is at position 74.5 in the probe (95% confidence interval, 72.0 to 77.0) and thus predicts the bend center to be at guanine-580.
FIG. 3.
FIG. 3.
CPA using LBS-1 and LBS-2 probe sets. EMSA using a set of 32P-labeled probes containing LBS-1 and LBS-2 incubated with in vitro-translated LANAC WT (lanes 1 to 8) or the KK1113AA mutant (lanes 9 to 16) is shown. Specific complexes corresponding to the occupancy of LBS-1 (LANAC complex A, filled arrowheads) or LBS-1 and LBS-2 (LANAC complex B, open arrowheads) are indicated together with the unbound probes (Free LBS-1+2). A nonspecific complex is indicated with an asterisk. A representative pixel density profile (probe C incubated with LANAC WT, lane 2) is shown on the left.
FIG. 4.
FIG. 4.
TR DNA is bent towards the major groove. (A) Design of phasing analysis probes. Oligonucleotides containing either LBS-1 or LBS-1 and LBS-2 were inserted between unique XbaI and HindIII sites of pBEND2H. To provide the intrinsic bend, a second oligonucleotide containing three A tracts (underlined) was inserted into the unique SalI site. The size of the spacer region was increased by PCR using a series of oligonucleotides spanning the ends of the LBS and A tract was used in a PCR to clone in the spacers. Subsequently, the center of the A tract is located 40 bp (3.8 helical turns), 42 bp (4.0 helical turns), 44 bp (4.2 helical turns), 46 bp (4.4 helical turns), 48 bp (4.6 helical turns), and 50 bp (4.8 helical turns) from the molecular bend center of G581 when the spacer lengths are 0, 2, 4, 6, 8, and 10 nucleotides, respectively. (B) 32P-labeled probes containing LBS-1 were incubated with in vitro-translated LANAC WT (lanes 1 to 6) or the KK1113AA mutant (lanes 7 to 12). Specific LANAC-induced complexes and the unbound probes are indicated. The numbers indicate the linker length inserted between LBS-1 and the A tract. (C) Phasing analysis using 32P-labeled probes containing LBS-1 and LBS-2 incubated with in vitro-translated LANAC WT (lanes 1 to 6) or the KK1113AA mutant (lanes 7 to 12). Specific LANAC-induced complexes A and B plus the positions of unbound probes are indicated. Spacer lengths are listed above the lanes.
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