Article Figures & Data
Figures
- FIG. 1.
The first 8 nt within the RE are pivotal for KSHV latent origin function. Replication assays were performed as previously described. Briefly, 8 μg of each origin-containing construct was cotransfected with 2 μg of pcDNA3/orf73 or carrier DNA into 293 cells. Episomal DNA was recovered by Hirt extraction 72 h posttransfection. Ten percent of the episomal DNA was digested with HindIII (input), while 90% was double digested with HindIII and DpnI for 16 h (DpnI digest). DNA were electrophoretically separated in an agarose gel, immobilized on nylon membranes, and hybridized with radiolabeled probes using pCRII-TR as the template. The replication efficiency of each plasmid was calculated by comparing the intensity of the replicated DNA band with that of its input from independent experiments. The replication activity of each mutant was compared with that of the wild-type MR, which was set at 100%. (A) The MR replicates with efficiency similar to that of the full-length TR. (B) Sequences of the wild-type RE and converted sequences within RE mutants, respectively. (C) The first 8 nt in the RE are critical for origin activity. The REm1-8-LBS1/2, REm9-16-LBS1/2, REm17-24-LBS1/2, and REm25-32-LBS1/2 mutants replicate with efficiencies of 4.8%, 9.6%, 27% and 60%, respectively, compared to that of the wild-type MR. (D) Fine mapping of the first 8 nt to determine the sequences important for LANA-dependent DNA replication. The REm1-4-LBS1/2, REm5-8-LBS1/2, REm9-12-LBS1/2, and REm13-16-LBS1/2 mutants replicate with efficiencies of 13.2%, 8.7%, 41%, and 100%, respectively, compared to that of the wild-type MR. The bracket and arrowheads indicate the positions of the origin-containing plasmids. LBSs, LBS1/2.
- FIG. 2.
The orientation and position of the RE element relative to LBS1/2 (LBSs) play an important role for origin function. The wild-type MR or mutants were constructed by inserting synthetic oligonucleotides into pCRII plasmids. Replication assays were performed as described in Materials and Methods. (A) Origin activity was diminished when the RE was flipped or moved downstream of LBS1/2. (B) Spacing changing between the RE and LBS1/2 abrogated the origin activity. The bracket and arrowhead indicate the positions of the origin-containing plasmids. The top panels show the representative structure of the wild-type MR and mutants; the bottom panels show the results of the replication assays with the wild-type MR and mutants.
- FIG. 3.
Proteomics approach to study RE binding proteins. (A) Flow chart for experimental design. Biotinylated PCR fragments containing the MR, REm1-8-LBS1/2, or Amp were immobilized on magnetic streptavidin beads. Nuclear extract and solubilized nuclear pellet extract mixture from BJAB Tet-on/ORF73 cells induced with doxycycline were subjected to two rounds of DNA affinity purification with the immobilized DNA. The second round of purified proteins was resolved on a 4 to 20% gradient SDS-PAGE gel. Gels in each lane were sliced and subjected for mass spectrometry analysis. (B) Colloidal blue staining of DNA affinity chromatography-purified proteins. Unique bands representing proteins specifically bound to REs are indicated with arrows. M, protein size marker. The molecular weight (in thousands) for each band of the marker is labeled as shown. LBSs, LBS1/2.
- FIG. 4.
Analysis of the TRF2 interaction with LANA and role in KSHV DNA replication. (A) LANA interacts with TRF2 in an vitro pulldown assay. BJAB LANA Tet-on nuclear extracts and solubilized nuclear pellets were subject to affinity purification with either the wild-type MR or the REm1-8-LBS1/2 mutant. The isolated proteins were analyzed by Western blotting with antibody specifically against human TRF2. A total of 0.5 ml nuclear protein mixture was loaded as the input (lane 4). NS, nonspecific signal. (B) LANA interacts with TRF2 in vivo in cotransfected cells. 293 cells were cotransfected with LANA and Myc-TRF2 expression vectors in conjunction with REm1-8-LBS1/2 (lane 1) or MR (lanes 2 and 3) plasmid. The nuclear extracts were immunoprecipitated with either monoclonal antibody against Myc (lanes 1 and 3) or control mouse IgG (lane 2). The immunoprecipitated proteins were separated on an 8% SDS-PAGE gel and immunoblotted with rabbit polyclonal antibody against LANA. (C) Expression of DN-TRF2. 293 cells were mock transfected or transfected with FLAG-DN-TRF2. Cell lysate was hybridized with antibody against FLAG. (D) DN-TRF2 did not interfere with LANA-dependent DNA replication. 293 cells were transfected with 8 mg TR-containing plasmid, 2 mg LANA expression vector in the absence of DN-TRF2 (lanes 2 and 6), and 2 mg (lanes 3 and 7) or 5 mg (lanes 4 and 8) DN-TRF2 expression vector. The short-term DNA replication assay was performed as described. The position of the TR plasmid is shown with an arrowhead LBSs, LBS1/2.
- FIG. 5.
Complex formation of SSRP1 with LANA/origin in vitro and in vivo. (A) SSRP1 was pulled down by the wild-type and mutant MR but not the amp control fragment when LANA was present. Affinity chromatography was performed as described in Materials and Methods. A small amount of pulldown lysates was separated on an 8% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. The blot was immunoblotted with antibody specifically against SSRP1. Lanes 1 to 3 showed the lysates affinity purified with the amp fragment, mutant REm1-8-LBS1/2, or the wild-type MR fragment. Lane 4 showed 5% of the extract as input. (B) SSRP1 forms a complex with LANA/origin through protein-protein interaction. 293 cells were transfected with LANA expression vector and mutant REm1-8-LBS1/2 (lane 1), MR (lanes 2 to 4), or no origin (lane 5) in the presence (lanes 1, 3, 4, and 5) or absence (lane 2) of FLAG-tagged SSRP1. Cells were harvested 48 h posttransfection. Cells were lysed and immunoprecipitated with FLAG antibody (lanes 1, 2, 4, and 5) or mock antibody (lane 3) as a negative control. Immune complexes were separated on an 8% SDS-PAGE gel and immunoblotted with LANA antibody or FLAG antibody (bottom panels). The expression of LANA and FLAG was tested with 3% total cell lysate with antibodies specifically against LANA or FLAG (top two panels, shown as input). A faint background band was observed with the FLAG antibody (lane 2) compared to that with the control with no antibody (lane 3). LBSs, LBS1/2.
- FIG. 6.
SSRP1 forms a complex with KSHV latent origin in a cell cycle-dependent manner. (A) SSRP1 binds to the wild-type MR and mutant REm1-8-LBS1/2 when LANA is expressed. A total of 3 × 106 293 cells were cotransfected with vector pcDNA3.1-LANA, pcDNA-2xFLAG-SSRP1 and pCRII-REm1-8-LBS1/2, or pCRII-MR. Cells were harvested 48 h posttransfection for the ChIP assay. SSRP1 IP significantly enriches for MR and REm1-8-LBS1/2 (lanes 1 and 3) but not for a genomic control (lane 4); IgG IP demonstrates the specificity of the SSRP1 antibody (lane 2). (B) SSRP1 binds to the MR in a cell cycle-dependent manner. 293 cells were cotransfected with pcDNA3.1-LANA, pcDNA-2xFLAG-SSRP1 and pCRII-REm1-8-LBS1/2, or pCRII-MR. At 48 h after transfection, cells were asynchronized (bar 2) or synchronized to G1/S phase (bars 1, 3, and 4), S phase (bar 5), or M phase (bar 6) with drug treatments. Cells were harvested for the ChIP assay to detect the binding of SSRP1 on the MR or REm1-8-LBS1/2. Relative amounts of immunoprecipitated DNA were normalized to input for each sample. LBSs, LBS1/2.
- FIG. 7.
SSRP1 is involved in KSHV latent origin replication. A total of 1.5 × 106 293 cells were transfected with 100 nM scrambled or SSRP1 siRNA with Lipofectamine 2000. At 24 h posttransfection, cells were transfected again with 8 μg TR-containing plasmid pCRII-TR and 2 μg empty pcDNA3.1 (panels A and B, lanes 1) or pcDNA3.1/LANA (panels A and B, lanes 2, 3, and 4). At 72 h after the second transfection, cells were harvested. One of 20 of the total cells was collected for Western blotting to detect the expression of SSRP1. The remaining cells were assayed for the replication assay as described in Materials and Methods. (A) Western blot analysis indicated efficient SSRP1 knockdown by siRNA at the time when replicating DNA was extracted. Lanes 1 and 2, no siRNA transfection; lane 3, scrambled siRNA-transfected samples; lane 4, SSRP1 siRNA-transfected cells. (B) The knockdown of SSRP1 inhibits the replication efficiency of the TR plasmid. The top panel shows a representative image of the results from three independent replication assays. Lanes 1 to 4 (same samples as for panel A) contain 10% of the Hirt-extracted DNA digested with HindIII; lanes 5 to 8 have 90% of the Hirt-extracted DNA double digested with HindIII and DpnI, corresponding to lanes 1 to 4, respectively. Arrowheads indicate the positions of linearized pcDNA3.1/LANA and pCRII-TR. The bottom panel shows the quantification and statistical analysis of the results from three independent assays. (C) Cell profiling with mock-, scrambled siRNA-, or SSRP1 siRNA-transfected cells did not show any differences. DNA content was analyzed by flow cytometry after cells were fixed and stained with propidium iodide.
Tables
- TABLE 1.
Proteins identified in DNA affinity chromatography which bind only to the MR or have higher binding affinity to the MR than the mutants
Namea Accession no. Predicted molecular mass (kDa) No. of peptides identified in MR pulldown Function(s) DNA topoisomerase II β IPI00217709 183 46 Control and alter the topologic states of DNA during transcription and replication; DNA damage response DNA topoisomerase II α IPI00218753 179 37 Control and alter the topologic states of DNA during transcription and replication Structural maintenance of chromosome 1 IPI00291939 143 5 Mitotic spindle organization and biogenesis; DNA repair Structural maintenance of chromosome 3 IPI00219420 142 5 Mitotic spindle organization and biogenesis AF5q31 protein IPI00004344 127 4 Transcription regulation Scaffold attachment factor B IPI00300631 103 5 Nuclear matrix component; transcription regulation M-phase phosphoprotein 8 IPI00030408 97 3 N/Ab Gamma interferon-inducible protein Ifi-16 IPI00003443 88 9 Transcription regulation; DNA damage signaling; cell cycle checkpoint Structure-specific recognition protein 1 IPI00005154 81 5 Transcription regulation; chromatin remodeling; DNA replication DEAD box polypeptide 17 isoform p82 IPI00023785 80 8 RNA metabolism Heterogeneous nuclear ribonucleoprotein M isoform a IPI00171903 78 4 RNA metabolism Splicing factor proline/glutamine-rich (SFPQ) IPI00010740 76 3 RNA metabolism; DNA repair Lamina-associated polypeptide 2α IPI00216230 75 33 Nuclear architecture Interleukin enhancer-binding factor 3 IPI00219330 75 6 Transcription regulation ALB protein IPI00022434 72 3 Carrier protein Probable RNA-dependent helicase p68 IPI00017617 69 9 RNA processing Nucleolar protein Nop56 IPI00411937 66 3 RNA processing Telomeric repeat binding factor 2 IPI00024214 56 6 Telomere maintenance; DNA replication Serine/threonine-protein kinase VRK3 IPI00009291 53 11 Protein binding DERPC IPI00171540 51 5 DNA replication Elongation factor 1-α 2 IPI00014424 50 2 Protein synthesis Heterogeneous nuclear ribonucleoprotein H1 IPI00013881 49 2 RNA processing KH domain-containing, RNA binding, signal transduction-associated protein 1 (Sam68) IPI00008575 48 8 RNA processing Gamma interferon-inducible protein Ifi-16 isoform 4 IPI00384836 47 10 Transcription regulation JKTBP2 IPI00011274 46 3 RNA processing SRP55-1 of splicing factor IPI00012345 40 2 RNA processing Pre-mRNA cleavage factor I 25-kDa subunit IPI00012995 26 2 RNA processing FUS-interacting serine-arginine-rich protein 1 IPI00009071 22 2 RNA processing CGG triplet repeat binding protein 1 IPI00295585 19 12 Transcription regulation
