LANA colocalizes with MCMs in KSHV-positive cells during the replicative phase.

Viral DNA replication during latency is thought to occur in conjunction with cellular DNA replication. The recruitment of MCMs to the origins is well defined, which begins in the late-G₁ phase of the cell cycle through a sequential assembly of the components of the pre-RC complex (39,–44). The MCMs are converted into active helicases only during the G₁/S phase of the cell cycle through phosphorylation with cellular kinases. Since the latent DNA replication occurs in synchrony with cellular replication, we wanted to determine whether the interaction between LANA and MCMs is limited to the actively replicating phase of the cell cycle or occurs throughout the cell cycle. To this end, we performed immunofluorescence assays (IFAs) in replicating (G₁/S) cells as well as in mitotic (G₂/M)-phase cells. To achieve these specific phases, cells were treated with mimosine (G₁/S) and colchicine (G2/M) and stained with rat anti-LANA and mouse anti-MCM3, -MCM4, and -MCM6 antibodies. LANA showed a distinct punctate pattern in the nuclei of PEL cells (Fig. 2A), as expected. In many of the foci of MCMs (MCM3, -4, and -6), enrichment matched to the LANA dots during the G₁/S phase of the cell cycle (Fig. 2A, frames a to c). An enlargement of the specific foci (red, LANA; green, MCMs) showed colocalization of LANA with these MCMs, represented by yellow spots, during the G₁/S, replicating phase of the cells cycle (Fig. 2A, frames a to c). In contrast, cells in the mitotic phase (colchicine-treated) did not show LANA’s localization with MCMs (Fig. 2A, frames a to c). Nuclear staining with TO-PRO3 (blue) confirmed that these proteins were in the nuclear compartment. Differential interference contrast (DIC) images show the outline and the integrity of the cells.

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FIG 2

LANA colocalizes with the components of MCM complex in KSHV-positive cells during the replicative, G₁/S, phase. (A) Immunofluorescence analysis of endogenous LANA and MCM4, MCM3, and MCM6 in mimosine-treated KSHV-positive cells. Cells were stained with rat anti-LANA and mouse anti-MCM4 (a), anti-MCM3 (b), and anti-MCM6 (c) and subsequently detected with secondary anti-rat Alexa Fluor 594 (red) for LANA and anti-mouse Alexa Fluor 488 (green) for MCMs. Nuclei stained with TO-PRO-3 are shown in blue. DIC images were captured to depict cell morphology. LANA and the components of the MCM complex colocalized in the same nuclear compartments, as indicated by yellow punctate dots, during the G₁/S phase of the cell cycle. (B) Immunofluorescence analysis of endogenous LANA and MCM4, MCM3, and MCM6 in colchicine-treated (G₂/M arrested) KSHV-positive cells. Cells were stained with rat anti-LANA and mouse anti-MCM4 (a), MCM3 (b), and MCM6 (c) antibodies. Secondary antibodies, anti-rat Alexa flour 594 (red) and anti-mouse Alexa flour 488 (green), were used for LANA and MCM4 (a) and MCM3 (b), respectively. Secondary antibodies, anti-rat Alexa flour 488 (green) and anti-mouse Alexa flour 594 (red), were used for LANA and MCM6 (c), respectively. Nuclei were stained with TO-PRO-3 (in blue).

The amino and carboxyl termini of LANA interact with MCMs.

In order to determine the domains of LANA responsible for interaction with MCMs, we performed coimmunoprecipitation assays in HEK293T cells transfected with LANA mutants expressing either the amino-terminal (aa 1 to 340) or the carboxyl-terminal (aa 940 to 1162) domain of LANA (LANA-N or LANA-C, respectively) along with epitope-tagged full-length MCMs. Immunoprecipitation with anti-Myc antibody and subsequent detection of coprecipitating MCM3 and MCM4 showed their binding to the amino-terminal but not to the carboxyl-terminal domain of LANA (Fig. 3A, subpanels a and b). Similarly, immunoprecipitation of LANA-N and LANA-C with anti-Flag antibody and subsequent detection of coprecipitating MCM6 showed its binding with both termini of LANA (Fig. 3A, subpanel c). Specificities of these interactions were confirmed by the lack of any coprecipitating protein with empty vectors. In order to confirm that these interactions are direct, we used a glutathione S-transferase (GST) pulldown assay in which lysates from MCM3-, MCM4-, and MCM6-expressing HEK293T cells were incubated with bacterially purified GST fused with LANA-N or LANA-C. Similar to the above immunoprecipitation results, MCM3 and MCM4 interacted with only the amino-terminal domain of LANA, whereas MCM6 interacted with both the amino and carboxyl termini of LANA (Fig. 3A, subpanel d). GST alone was used as a control, which did not bind to any MCMs, confirming the specificity of their interactions. In addition, we confirmed direct associations of MCMs with LANA through proteins prepared from a cell-free in vitro translation system. In vitro-translated MCM4 was incubated with bacterially purified LANA truncations, LANA-N, and LANA-C fused to GST. We performed an in vitro translation and interaction assay with only MCM4 because the other MCMs were untranslatable. Importantly, MCM4 bound to the amino-terminal domain of LANA, similar to results of the above-described binding assays (Fig. 3A, subpanel e). This assay confirmed that both the amino and carboxyl termini of LANA are capable of binding to MCMs.

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FIG 3

The amino and carboxy termini of LANA interacted with the MCMs. (A) HEK293T cells were transfected with Myc-tagged empty vector (V), EGFP-LANA-N (N; aa 1 to 340), and EGFP–LANA-C (C; aa 940 to 1160) along with Flag (F)-tagged MCM3 (a) and MCM4 (b) or with empty vector (V) and pA3F–LANA-N (N) and pA3F–LANA-C (C), along with Myc (M)-tagged MCM6 (c). The cells were lysed at 36 h posttransfection; immunoprecipitation was performed with anti-Myc antibody or anti-Flag antibody, as indicated, followed by detection with anti-Flag and anti-Myc antibodies. (d) HEK293T cells transfected with MCM3, MCM4, or MCM6 were lysed 36 h posttransfection. After preclearing with GST beads, the cellular lysates were incubated with GST alone, LANA-N–GST, or LANA-C–GST beads. The bead-bound proteins were resolved on SDS-PAGE and detected with anti-Flag, anti-Myc, and anti-GST antibodies. (e) In vitro-translated MCM4-Flag protein was rotated with GST alone, LANA-N–GST, or LANA-C–GST beads. The bead-bound proteins were resolved by SDS-PAGE and visualized by autoradiography. SDS-PAGE and Coomassie staining were used to detect the expression of GST and the GST fusion proteins. The red arrows indicate the LANA N-terminal or C-terminal protein. (B) HEK293T cells were transfected with pA3M (empty vector, V), a LANA truncation mutant spanning amino acids 1 to 340 (LANA_1–340)-GFP-Myc, LANA_1–250-GFP-Myc, LANA_1–150-GFP-Myc, LANA_33–150-GFP-Myc, or LANA_1–32-GFP-Myc. Cellular lysates were subjected to immunoprecipitation with anti-Myc antibody to detect coimmunoprecipitated endogenous MCM3, MCM4, and MCM6. (C) The schematic shows point mutations in the LANA N terminus spanning amino acids 1 to 32 (a). HEK293T cells were transfected with pA3M (empty vector), wild-type LANA_1–32-GFP-Myc, and point mutants LANA_5–7-GFP-Myc, LANA_8–10-GFP-Myc, LANA_11–13-GFP-Myc, LANA_14–15-GFP-Myc, and LANA_5–15-GFP-Myc (b). Cellular lysates were subjected to immunoprecipitation with anti-Myc antibody to detect coimmunoprecipitated endogenous MCM3. (D and E) MCM3 and MCM4 bind to LANA independent of MCM6. BCBL-1 cells transduced with Tet-inducible shMCM6 were treated with doxycycline for MCM6 depletion (shMCM6-KD) or untreated (shMCM6-C) before being subjected to immunoprecipitation with MCM3 and MCM4, as indicated (D). The lysates were treated with DNase before immunoprecipitation and detection of coprecipitated LANA. BC3 cells transduced with Tet-inducible shMCM6 were treated with doxycycline for MCM6 depletion (shMCM6-kD) or untreated (shMCM6-C) before being subjected to immunoprecipitation with MCM3 and MCM4, as indicated (E). The lysates were treated with DNase before immunoprecipitation and detection of coprecipitated LANA. Isogenic antibody (IgG) was used as a control.

In order to further map the region of LANA responsible for binding to MCMs, we performed coimmunoprecipitation assays with truncations of the amino terminus of LANA, including aa 1 to 32, 1 to 150, 1 to 250, 1 to 340, and 33 to 150. Immunoprecipitation with anti-Myc antibody and subsequent detection of MCMs identified the region of amino acids 1 to 32 of LANA (LANA_1–32) as the minimal domain required for MCM3 and MCM6 while MCM4 mapped to LANA_1–150 and, more specifically, to the LANA_33–150 region (Fig. 3B).

Since LANA_1–32 is also the chromatin binding domain, we wanted to identify the amino acid residues of this region responsible for its interaction with MCMs. A schematic of the LANA_1–32 region with alanine mutations between aa 5 and 15 is depicted in Fig. 3C (subpanel a). Transfection of these expression vectors into HEK293T cells, followed by immunoprecipitation with anti-Myc antibody to precipitate LANA_1–32, showed amino acids 14 and 15 (T and G) to be critical for LANA’s association with MCM3 (Fig. 3C, subpanel b).

Since MCMs are recruited as a hexameric complex at the origin of DNA replication, the components of the MCMs (MCM2–7) associate to form the complex. Therefore, we wanted to determine whether one MCM associated with LANA and the other binding MCMs precipitated by virtue of their self-association. To address that, we depleted one LANA-associating MCM (MCM6) and detected the association of LANA with other two LANA binding MCMs (MCM3 and MCM4). Immunoprecipitation of MCM3 from the MCM6-depleted and control cells coprecipitated almost similar levels of LANA from both BCBL-1 and BC3 cells (Fig. 3D and ​andE,E, subpanels a). Similarly, MCM4 coprecipitated LANA from the MCM6-depleted and control BCBL-1 and BC3 cells, suggesting a direct association of these proteins with LANA (Fig. 3D and ​andE,E, subpanels b).

A 50-aa region in C-terminal LANA is important for MCM binding and latent DNA replication.

Since MCM6 interacted with the C-terminal domain of LANA, we wanted to identify specific amino acid residues of the C-terminal domain responsible for interaction with MCM6. We performed MCM6 coimmunoprecipitation assays with existing C-terminal point mutants of LANA to identify whether any of these specific amino acid residues were important for binding. Immunoprecipitation of LANA-C mutants and subsequent detection of MCM6 with anti-Myc antibody showed reduced levels of coprecipitating MCM6 with a few mutants (Fig. 4A). Most of these critical residues lie in the LANA_1100–1150 region (Fig. 4A). Since MCMs are important for replication, we asked whether a LANA mutant with reduced MCM binding would have any effect on DNA replication. We addressed this by performing a transient replication assay with some of the C-terminal mutants of LANA having reduced MCM6 binding. Replication efficiencies were assayed by determining the DpnI-resistant (replicated) copies of the TR plasmid, which showed a significant reduction with many mutants and almost complete abrogation with a K1109/1113/1114A mutant of LANA (Fig. 4B; DpnI-resistant band intensities are presented as a bar graph). The point mutant M1117A, however, showed reduced MCM6 binding but only slightly reduced replication, which suggested that additional factors contribute to MCM association with LANA (Fig. 4). Since the amino acids at K1109/1113/1114 were important for MCM6 binding and replication, we generated a small clone of LANA_1100–1150 encompassing these residues to determine whether this can compete MCM6 binding with LANA. To this end, we performed a coimmunoprecipitation assay of MCM6 with LANA in the presence of LANA_1100–1150, which showed a significantly reduced level of coprecipitating MCM6 (Fig. 4C). Since the levels of MCM6 were similar in cells with and without LANA_1100–1150 expression, we concluded that amino acids 1100 to 1150 can compete their binding (Fig. 4C). Next, we wanted to determine whether disruption of MCM6 binding with LANA with this competing domain can have an effect on replication. To assay that, HEK293L cells were transfected with LANA- and TR-containing plasmids in the presence or absence of LANA_1100–1150-expressing vector. Cells were harvested at 96 h posttransfection for the extraction of DNA and digested with EcoRI to linearize the DNA and with EcoRI and DpnI to determine the replicated DNA copy numbers. The DpnI-resistant band (marked by an arrow) showed a complete abrogation in the replication of TR plasmids in cells expressing LANA_1100–1150 compared to that in the cells with similar amounts of empty vector (Fig. 4D, compare lane 4 with lane 3). The levels of transfected TR plasmids were similar in both samples (Fig. 4D, input lanes 1 and 2). Cells transfected with LANA_1100–1150 showed a cross-hybridizing signal to the vector backbone of LANA_1100–1150 but did not affect the TR plasmid signal (Fig. 4D, lane 2).

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FIG 4

LANA aa 1100 to 1150 are essential for association with MCM6. (A) HEK293T cells were transfected with the indicated Flag-tagged LANA C-terminal point mutants and Myc-tagged MCM6. At 36 h posttransfection, cellular lysates were subjected to immunoprecipitation with anti-Flag antibody to detect coimmunoprecipitated MCM6 following detection with anti-Myc antibody. (B) HEK293L cells were transfected with plasmids expressing full-length LANA and LANA C-terminal point mutants in the presence of TR plasmid. Cells were harvested at 96 h posttransfection; DNA was extracted using Hirt’s procedure and digested with either EcoRI or EcoRI and DpnI. The DNA was subjected to Southern blotting and detection using TR probe. Densitometric quantification of DpnI-resistant test bands compared to that of respective inputs is shown. (C) HEK293T cells were transfected with MCM6-Myc and full-length LANA in the presence and absence of LANA_1100–1150. Cellular lysates were subjected to immunoprecipitation with anti-Flag antibody to detect coimmunoprecipitated MCM6 following detection with anti-Myc antibody. (D) HEK293L cells were transfected with plasmids expressing full-length LANA and full-length LANA with LANA_1100–1150 in the presence of TR plasmid. Cells were harvested at 96 h posttransfection; DNA was extracted using Hirt’s procedure and digested with either EcoRI or EcoRI and DpnI. The DNA was subjected to Southern blotting and detection using TR probe. The arrow indicates the p8TR band.

We further wanted to determine whether the peptide encompassing these critical residues of LANA-C would have an impact on disrupting LANA’s association with MCM6. To this end, we synthesized a 20-amino acid peptide (KRPRSPSSVYCQNKDTSKKVQMARLAWE) within the LANA_1104–1123 region of that encompassed amino acid residues 1109/1113/1114, which were important for MCM6 binding and DNA replication. A same-sized (20-aa) scrambled (sc) peptide (KRPRSPSSQQEPQQQEPQQQEPQQQEPQ) was used as a control. We introduced a nuclear localization signal (NLS) sequence (underlined) in these peptides for efficient translocation into the nucleus. Immunoprecipitation of LANA with anti-Flag antibody showed reduced coprecipitation of MCM6 in the presence of the specific LANA peptide compared to almost similar amounts in the absence of any peptide or in the presence of scrambled peptide (Fig. 5A). Relative amounts of coprecipitating MCM6 were determined based on the amounts precipitated in lanes without any peptide after values were normalized to their respective inputs (Fig. 5A).

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FIG 5

Introduction of LANA-specific peptide, aa 1104 to 1123, reduced LANA-MCM6 binding and TR-mediated replication. (A) HEK293T cells were transfected with Flag-tagged full-length LANA and Myc-tagged full-length MCM6. The cells were lysed at 36 h posttransfection, and the lysates were incubated in the absence and presence of LANA specific (sp) and scrambled (sc) peptides. The immunoprecipitation was performed with anti-Flag antibody, followed by detection of MCM6 with anti-Myc antibody. Relative densities of the coprecipitating MCM6 are shown below the respective bands. (B) HEK293L cells were transfected with plasmids expressing full-length LANA and TR plasmids with and without LANA specific (sp) and scrambled (sc) peptides. Cells were harvested at 96 h posttransfection; DNA was extracted using Hirt’s procedure and digested with either EcoRI (to linearize) or with EcoRI and DpnI. The DNA was subjected to Southern blotting and detection using TR probe. The arrow indicates the TR plasmid band. Relative densities of the replicated bands (indicated by arrow) normalized to the value of the Dpn1-resistant band from cells transfected with no peptide are shown below their respective lanes.

We further wanted to determine whether the disruption of MCM6 association with LANA through this peptide would translate into reduced levels of TR replication. To this end, we performed a transient replication assay by transfecting LANA and TR plasmids in HEK293L cells in the absence and presence of the specific and scrambled peptides. Transfected cells were harvested at 96 h posttransfection to extract DNA, followed by digestion with EcoRI to linearize the DNA or with DpnI and EcoRI to determine the replicated copy numbers after Southern hybridization. Quantification of DpnI-resistant bands showed significantly reduced levels of replicated TR plasmid in cells transfected with LANA-specific peptide compared to levels in cells transfected with no peptide or cells with scrambled peptide (Fig. 5B). The relative quantities of the replicated DNA were calculated in reference to the DpnI-resistant band from cells with no peptides after values were normalized to the respective inputs (Fig. 5B).

Recombinant KSHV with deletion of LANA_1100–1150 showed reduced replication.

Our experiments so far confirmed the importance of the MCMs-LANA association to KSHV latent DNA replication. In addition, amino acid residues of the C terminus of LANA were important for MCM6 recruitment and DNA replication. To verify whether these C-terminal residues of LANA have an effect on KSHV genome replication/maintenance, we generated a recombinant KSHV with a deletion of aa 1100 to 1150 of LANA (BAC16Δ1100–1150) through homologous recombination-based two-step BAC recombineering (Fig. 6A). The bacmids (BAC16wt and BAC16Δ1100–1150) were transfected into HEK293L cells and selected with hygromycin. Transfections of both BAC16wt and BAC16Δ1100–1150 were similar, as monitored by green fluorescent protein (GFP) signals, at 24h posttransfection, but BAC16Δ1100–1150 displayed a higher rate of GFP signal decrease than the cells transfected with BAC16wt. We further determined the copy numbers of viral genome retained following hygromycin selection for 6days. Episomal DNA extracted using Hirt’s procedure from these cells showed significantly lower KSHV copy numbers in BAC16Δ1100–1150 cells than in BAC16wt cells (Fig. 6B and ​andC).C). The reduction in episomal copies in BAC16Δ1100–1150 cells prompted us to evaluate whether the reduction was due to a defect in DNA replication observed in transient replication assays. To achieve this, we labeled the BAC16wt and BAC16Δ1100–1150 cells (day 6 samples post-hygromycin selection) with iododeoxyuridine (IdU) to label the replicating DNA, which was extracted through Hirt’s procedure for immunoprecipitation with anti-IdU antibody. The replicated DNA captured on antibody-protein A/G beads was eluted for quantification using quantitative PCR (qPCR) with primers to amplify the terminal repeat region. As expected, we observed significantly smaller amounts of newly replicated DNA in BAC16Δ1100–1150 cells than in BAC16wt cells (Fig. 6D).

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FIG 6

Recombinant BAC16Δ1100–1150 displays reduced latent DNA replication. (A) A schematic illustrates BAC16Δ1100–1150 generation via homologous recombination (a). Nucleotides 1100 to 1150 of LANA/ORF73 were deleted by two-step BAC recombineering and Kan^r/I-SceI counterselection. The presence of the Kan^r/I-SceI cassette was confirmed by NdeI digestion and Southern hybridization with a LANA-specific probe (b). Shown at left is an ethidium bromide gel of NdeI-digested BAC16wt and intermediates with or without the Kan^r/I-SceI cassette, as indicated. Southern blotting with LANA-specific probe displayed the expected 5,811-bp band in the intermediate (+kan). (B and C) KSHV latent genomic copies were quantified by extracting genomic DNA from uninduced BAC16wt and BAC16Δ1100–1150 cells at day 3 and day 6, as indicated, using Hirt’s extraction procedure. The relative copy numbers were calculated by amplifying viral genome with TR-specific primers using the ΔΔC_T (where C_T is threshold cycle) method after values were normalized to those of GAPDH. The relative genome copy numbers were significantly reduced for BACΔ1100–1150 cells compared to levels in BAC16wt cells. All experiments were performed three times in replicates, and the error bars represent standard deviations of the means from three independent experiments. (D) IdU-labeled viral DNA was immunoprecipitated with anti-IdU antibody from the BAC16wt and BAC16Δ1100–1150 cells. The relative copies were calculated by amplifying viral genome with TR-specific primers and using the ΔΔC_T method after normalization to the respective inputs. BAC16Δ1100–1150 cells showed significantly smaller amounts of newly replicated DNA compared to levels in BAC16wt cells. These experiments were performed three times in replicates, and the error bars represent standard deviations of the means from three independent experiments. Statistical analysis was performed with Prism, version 7.0, software (GraphPad, Inc.) using an unpaired t test for the significance (*, P<0.05; **, P <0.01; ***, P < 0.001).

MCMs are required for LANA-dependent latent DNA replication.

Lower MCM6 binding to LANA C-terminal mutants and subsequent reduction in replication could also be due to reduced binding of other cellular factors within that region. To confirm the absolute role of MCMs in KSHV DNA replication, we depleted the levels of MCM components (MCM3 or -6) in HEK293L cells and assayed DNA replication using a TR-containing plasmid. We transduced HEK293L cells with doxycycline-inducible short hairpin RNA (shRNA) to deplete MCM3 or MCM6 (shMCM3 knockdown [shMCM3-KD] or shMCM6-KD cells). Stably transduced cells were assayed for the depletion of MCMs after doxycycline treatment and showed a significant reduction (shMCM3-KD or shMCM6-KD) in MCM levels compared to levels in the untreated cells (control shMCM3 [shMCM3-C] or shMCM6-C cells) (Fig. 7A and ​andB,B, subpanels a). A transient replication assay was performed by transfecting these cells with LANA- and TR-containing plasmids. Transfected cells were harvested at 96 h posttransfection to extract DNA, followed by digestion with EcoRI to linearize DNA or with DpnI and EcoRI to determine the number of replicated copies after Southern hybridization. Since LANA is crucial for TR DNA replication, cells without LANA expression did not show any replicated/DpnI-resistant band (Fig. 7A and ​andB,B, subpanels b, lanes 7, 9, and 11). HEK293L cells expressing LANA showed replicated TR copies (Fig. 7A and ​andB,B, subpanels b, lanes 8, asterisks), as expected. Cells transduced with shMCM3 or MCM6 lentivirus and treated with doxycycline (shMCM3-KD or shMCM6-KD) showed no replicated TR plasmid in contrast to the doxycycline-untreated (shMCM3-C or shMCM6-C) cells (Fig. 7A and ​andB,B, subpanels b, compare lanes 12 with lanes 10). Lanes 1 to 6 represent the amounts of TR plasmid DNA extracted from respective cells (Fig. 7A and ​andB,B, subpanels b). This confirmed that MCMs (both MCM3 and MCM6) are critical for LANA-mediated replication of TR plasmids.

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FIG 7

MCMs are required for KSHV latent DNA replication. (A) An immunoblot shows the efficiency of lentivirus-based knockdown of MCM3 in lysates from HEK293L cells, shMCM3 transduced HEK293L control cells (293L shMCM3-C), and doxycycline-treated shMCM3 transduced HEK293L (293L shMCM3-KD) (a). HEK293L, HEK293L shMCM3 control, and MCM3-depleted cells were transfected with plasmids expressing full-length LANA in the presence of TR plasmid (b). Cells were harvested at 96 h posttransfection; DNA was extracted using Hirt’s procedure and digested with either EcoRI (to linearize) or EcoRI and DpnI. The DNA was subjected to Southern blotting and detected using TR probe. (B) An immunoblot shows the efficiency of lentivirus-based knockdown of MCM6 from HEK293L cells, shMCM6 transduced HEK293L control cells (293L shMCM6-C), and shMCM6-depleted 293L cells (293L shMCM6-KD) (a) HEK293L control cells and HEK293L shMCM6 control and MCM6-depleted cells were transfected with plasmids expressing full-length LANA in the presence of TR plasmid (b). Cells were harvested at 96 h posttransfection; DNA was extracted using Hirt’s procedure and digested with either EcoRI or EcoRI and DpnI. The DNA was subjected to Southern blotting and detected using TR probe.

LANA recruits MCMs at the terminal repeat region.

We determined that MCMs were critical for latent DNA replication, and these were previously shown to be part of the viral chromatin (23, 24); therefore, we asked whether MCMs are recruited through a viral factor, LANA, or loaded onto the viral chromatin independent of LANA. To address this, we depleted LANA from KSHV-infected BCBL-1 and BC3 cells by stably transducing an shLANA lentiviral vector (45). The efficiency of LANA depletion was determined by comparing the levels of LANA with those of the control shRNA transduced cells, which showed a significant reduction in shLANA transduced cells (Fig. 8A and ​andB).B). We determined the levels of MCMs (MCM3, -4, and -6) bound to the chromatin of the KSHV genome (TR region chromatin) through chromatin immunoprecipitation and compared the levels with those of the control cells. We found that all of the tested MCMs (MCM3, -4, and -6) had significantly reduced binding to the chromatin of the TR region in both BCBL-1 (Fig. 8A) and BC3 (Fig. 8B) LANA-depleted (shLANA) cells compared to levels in the control (shControl) cells. This confirmed that MCMs are recruited to the viral genome through LANA.

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FIG 8

LANA recruits MCMs at the TR region during latent DNA replication. (A) Immunoblot showing the efficiency of LANA depletion in the lysates from LANA knockdown and control BCBL-1 cells. (B) Immunoblot showing efficiency of LANA depletion in the lysates from LANA knockdown and control BC3 cells. A chromatin immunoprecipitation assay was performed from the BCBL-1 and BC3, control, and LANA-depleted cells using antibodies specific for MCM3, -4, and -6. The relative copy numbers of chromatin-bound DNA were calculated by amplifying viral genome with TR-specific primers and using the ΔΔC_T (where C_T is threshold cycle) method after normalization to the respective inputs. All the experiments were performed three times in replicates, and the error bars represent standard deviations of the means from three independent experiments. Statistical analysis was performed with Prism, version 7.0, software (GraphPad, Inc.) using an unpaired t test for the significance (*, P<0.05; **, P <0.01; ****, P< 0.0001).

MCM depletion led to a reduction in KSHV genome replication and persistence.

Since MCMs were found to be essential for the replication of TR-containing plasmids in transient replication assays, we wanted to determine whether depletion of MCMs in KSHV-infected cells affects viral genome replication and persistence. To this end, we transduced BCBL-1 and BC3 cells with doxycycline-inducible shMCM3 or shMCM6 lentiviral vectors. These cells were treated with puromycin to select a pure population of transduced cells, which were further treated with doxycycline to determine the levels of MCM depletion. Detection of MCM3 and MCM6 showed an efficient depletion in doxycycline-treated (MCM3/MCM6-KD) cells compared to levels in the untreated (control) cells (Fig. 9A and ​andB).B). These cells were used for assaying DNA replication by pulsing them with IdU, a thymidine analog. IdU gets incorporated into the replicating DNA; therefore, a pulse-labeling with IdU followed by immunoprecipitation with anti-IdU antibodies determined the amounts of actively replicated DNA. Detection of IdU-labeled DNA by PCR amplification of a specific region of the KSHV genome, the TR region in this case (TR initiates replication), showed a lower level of IdU-labeled DNA in MCM (MCM3 or MCM6)-depleted BCBL-1 (Fig. 9C) and BC3 (Fig. 9D) cells compared to levels in the control cells. This confirmed that MCMs play a critical role in KSHV genome replication during latency.

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FIG 9

MCMs are essential for latent viral DNA replication. (A) Immunoblot showing depletion of MCM3 in MCM3-depleted BC3, BCBL-1, and control cells. (B) Immunoblot showing depletion of MCM6 in MCM6-depleted BC3, BCBL-1, and control cells. (C) IdU-labeled viral DNA was immunoprecipitated with anti-IdU antibody from the control and MCM-depleted BCBL-1 cells. (D) IdU-labeled viral DNA was immunoprecipitated with anti-IdU antibody from the control and MCM-depleted BC3 cells. IdU-labeled DNA was quantified using TR-specific primers. The relative copy numbers were calculated using the ΔΔC_T (where C_T is threshold cycle) method after normalization to the respective inputs. All the experiments were performed three times in replicates, and the error bars represent standard deviations of the means from three independent experiments. Statistical analysis was performed with Prism, version 7.0, software (GraphPad, Inc.) using an unpaired t test for the significance (*, P<0.05; **, P<0.01). (E) Gardella gel analysis was performed on 1 million BCBL-1 and BC3 control or MCM3-depleted cells. (F) Gardella gel analysis was performed on 1 million BCBL-1 and BC3 control or MCM6-depleted cells. Cells were harvested to prepare plugs, which were lysed in the lysis buffer for 48 h. The plugs were loaded on 0.8% Gardella gels. Ethidium bromide-stained images are shown on the left side of the respective TR probe hybridized Southern blot image.

In order to determine the effects of MCMs on KSHV genome persistence, these lentivirally transduced cells were treated with doxycycline for 2weeks, followed by assaying the levels of the KSHV genome through Gardella gel analysis, which determines the native form of episomally persisting viral genome. Not surprisingly, we observed significantly reduced KSHV episomes (reduced signal intensities) in both MCM3-depleted (Fig. 9E) and MCM6-depleted (Fig. 9F, KD lanes) BCBL-1 and BC3 cells compared to levels in the same number of control (C lanes) cells. This confirmed that depleted levels of MCMs reduced KSHV genome replication, which ultimately resulted in lower copy numbers of latently persisting KSHV genomes.

Antibodies and peptides.

The following commercial antibodies were used for this study: rat anti-LANA (Advanced Biotechnologies, Inc.); mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (US Biological); mouse anti-Flag M2 (Sigma-Aldrich, St. Louis, MO, USA); mouse anti-Myc 9E10 (Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-MCM2, -MCM5, -MCM10, and -PCNA (Santa Cruz Biotechnology); mouse monoclonal anti-MCM3, -MCM4, and -MCM7 (Santa Cruz Biotechnology). Mouse monoclonal anti-LANA hybridoma was generated at GenScript using the peptide sequence CEPQQREPQQREPQQ. This peptide was also used for eluting LANA in the iPOND assay.

A specific peptide, KRPRSPSSVYCQNKDTSKKVQMARLAWE (sp pep), from the C-terminal MCM6 binding residues (aa 1104 to 1123) and a scrambled peptide, KRPRSPSSQQEPQQQEPQQQEPQQQEPQ (sc pep), were synthesized at GenScript. Nuclear localization signal sequences (underlined) were added for efficient translocation of these peptides into the nucleus.

Plasmids.

MCM4 and LANA_1100–1150 were generated by PCR amplification and cloning into a Flag-tagged vector, pA3F, and a Myc-tagged vector, enhanced GFP (EGFP)-Myc, respectively. Integrity of the clones was confirmed by sequencing at the Nevada Genomics Center, University of Nevada, Reno, NV. MCM3 and MCM6 constructs were obtained from Alan Diehl (Medical University of South Carolina) and John Schimenti (Cornell University), respectively. Carboxyl-terminal point mutants of LANA were obtained from Paul Liebermann (Wistar Institute, Philadelphia, PA). Flag-tagged LANA, pA3F-LANA, LANA deletion constructs LANA-N (aa 1 to 340) and LANA-C (aa 940 to 1162), Myc-tagged LANA, pA3M-LANA, GFP-NLS-Myc, GFP–LANA-N–Myc (aa 1 to 340), the truncation mutants GFP–LANA-N₂₅₀–Myc (aa 1 to 250), GFP–LANA-N₁₅₀–Myc (aa 1 to 150), GFP–LANA-N₃₂–Myc (aa 1 to 32), and GFP–LANA-N_33–150–Myc (aa 33 to 150), and alanine substitution mutants of LANA_1–32 were described earlier (60). The shRNA vectors for MCM3 and MCM6 were purchased from Dharmacon (GE Life Science).

MCM knockdown using lentiviral vectors.

The pTRIPZ lentiviral vector (Dharmacon, GE Life Sciences) containing shRNA for MCM3 and MCM6 were cotransfected with lentivirus packaging vectors pCMV-dR8.2 and pCMV-VSVG (Addgene, Inc.) into HEK293T cells using polyethylenimine (PEI) (Polysciences, Inc.) to produce the respective lentiviral particles. Supernatants from the transfected HEK293T cells were collected for 5days, followed by concentration of the virus by ultracentrifugation (25,000rpm, 1.5h, 4°C). The concentrated lentiviral particles were used for transducing the target cells (BCBL-1, BC3, and HEK293L) in the presence of 5μg/ml Polybrene, followed by selection with 1μg/ml puromycin. The cells were treated with 1μg/ml doxycycline for at least 72 h for the induction of knockdown. The RNA interference (RNAi) efficiency was assessed by Western blot analysis with specific MCM3 and MCM6 antibodies.

Coimmunoprecipitation assays.

To perform coimmunoprecipitation, cells were washed with phosphate-buffered saline (PBS) and lysed in 750μl of NP-40 cell lysis buffer (1% Nonidet P-40, 50mM Tris-HCl, pH 7.5, 150mM NaCl, and 1mM EDTA) supplemented with protease inhibitors (1mM phenylmethylsulfonyl fluoride, 10μg/ml pepstatin, 10μg/ml leupeptin, and 10μg/ml aprotinin). Cellular lysates were sonicated with a probe sonicator and centrifuged at 12,000rpm for 10min at 4°C to remove cellular debris. The lysate was further treated with 100U of DNase to eliminate any cross-linking DNA. The supernatants were precleared with protein A/G Sepharose beads (GE Healthcare) for 30min at 4°C. Approximately 5% of the lysate was saved as the input sample, and the remaining cellular lysate was rotated overnight with specific antibodies. The immunocomplexes were captured using 50μl of protein A/G-conjugated Sepharose beads, which were incubated with the lysates for 2h at 4°C. The bead-bound immunocomplexes were collected by centrifugation at 2,000rpm for 5min at 4°C, followed by three washes with 1ml of ice-cold NP-40 buffer supplemented with protease inhibitors. The immunoprecipitation and input samples were boiled in 50μl of SDS-PAGE sample loading buffer for 8min, resolved on an SDS-polyacrylamide gel, and transferred onto 0.45-μm-pore-size nitrocellulose membranes (Bio-Rad laboratories) at 100V for 80min. The blots were blocked with 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) buffer (10mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween 20) and incubated overnight at 4°C with specific primary antibodies. The proteins were detected following incubation with infrared dye-tagged secondary antibodies using an Odyssey infrared scanner (LiCor Biosciences, Lincoln, NE).

IFA.

An immunofluorescence assay (IFA) on KSHV-positive cells arrested in the G₁/S phase was performed by growing the cells on poly-l-lysine-treated coverslips for 12 h, followed by incubation with mimosine for 120min. The cells were air dried for 10min, fixed with 4% paraformaldehyde for 15min at room temperature, and permeabilized with 0.2% Triton X-100 in PBS for 10min at room temperature. Cells were blocked with PBS containing 0.4% fish skin gelatin and 0.05% Triton X-100 for 30min at room temperature. The cells were then incubated with specific primary antibodies overnight at 4°C and washed with PBS three times before being incubated with Alexa Fluor-conjugated secondary antibodies (Molecular Probes) for 45min at room temperature. Nuclear staining was performed using TO-PRO-3 (Molecular Probes), and images were captured using a confocal laser scanning microscope (Carl Zeiss, Inc.).

An immunofluorescence assay on the KSHV-positive cells, arrested in the G₂/M phase, was performed after cells were treated with colchicine. Cells were grown on coverslips coated with poly-l-lysine for 12 h, followed by incubation with colchicine for 120min before fixation with formaldehyde. Staining and microscopy were performed as described above.

In vitro translation and GST pulldown assay.

In vitro translation of pA3F MCM4 was performed using a Promega TNT T7 quick-coupled transcription/translation system whereby 2μg of the plasmid was translated in a 50-μl reaction mixture containing 1mM [³⁵S]methionine. To perform GST pulldown assays, GST control and LANA-N and LANA-C GST fusion proteins were expressed in Escherichia coli BL21 competent cells, and following induction with 1mM isopropyl-β-d-thiogalactopyranoside (IPTG), the fusion proteins were extracted using glutathione-Sepharose beads. The in vitro-translated proteins were rotated overnight with control-GST, LANA-N–GST, and LANA-C–GST overnight in NETN binding buffer (0.1% NP-40, 20mM Tris, 1mM EDTA, and 100mM NaCl) along with protease inhibitors. Following overnight incubation, the beads were washed with NETN buffer three times, and coprecipitated proteins were resolved using SDS-PAGE and detected using autoradiography.

Transient replication assay.

HEK293L cells in 100-mm dishes were cotransfected with 30μg of TR-containing plasmid, p8TR, with 30μg of pA3F LANA, or with an empty vector, pA3F, as filler DNA in 293L cells, 293L shMCM3/shMCM6 control (C) cells, and doxycycline-treated 293L shMCM3/shMCM6 knockdown (KD) cells. At 96h posttransfection, cells were washed with phosphate-buffered saline followed by extraction of DNA using a modified Hirt’s lysis method, as described earlier (61). Extracted DNA was dissolved in 30μl of sterile water. Ten percent of the extracted DNA was linearized with EcoRI and the remainder was digested with DpnI and EcoRI to remove the nonreplicated DNA. The digested DNA was separated on an 0.8% agarose gel followed by Southern transfer onto a Hybond N⁺ membrane (GE Healthcare) and hybridized with ³²P-labeled TR probes. Probes specific to the KSHV terminal repeat region were synthesized using a random primer labeling kit, followed by purification on G-50 columns (GE Healthcare). The auto-radiographic signals were detected using a PhosphorImager, according to the manufacturer’s instructions (Molecular Dynamics, Inc.).

Peptide transfection, coimmunoprecipitation, and transient replication assay.

LANA-specific (sp pep) and LANA-scrambled (sc pep) peptides were transfected in the cells using Pierce protein transfection reagent according to the manufacturer’s protocol. For coimmunoprecipitation assays, pA3F plasmid expressing LANA and MCM6 were transfected into HEK293T cells using PEI, harvested at 24 h posttransfection, and lysed to set up immunoprecipitation in the presence of specific or scrambled peptides. Immunoprecipitation and detection were done as described above. Replication assays were performed by transfecting the specific and scrambled peptides into HEK293L cells using Pierce transfection reagent for 4h before transfecting the plasmids, pA3F LANA and p8TR, for replication assays. The replication assay was done as described above following digestion with EcoRI and DpnI.

Generation of recombinant KSHV, BAC16Δ1100–1150.

G-blocks (IDT) were designed to have a linear DNA fragment containing a kanamycin resistance expression cassette and an IsceI restriction enzyme site, flanked by homologous sequences to the region of LANA targeted for removal. The G-block sequence is the following (boldface indicates the targeting sequence for the Kan cassette insertion, underlining indicates the sequence required for the second round of recombination and seamless removal of the Kan cassette, and the remaining sequence is the Kan^r/I-SceI sequence): CCTGCTTGCCCCACCCTGGACCAGACCAGTCGCCCATAACTTATAACCAAGGTCCTGGGGACTCTCCGATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCATTACCCTGTTATCCCTAGATCGATGTACGGGCCAGATATACGCGAGACCAGTCGCCCATAACTTATAACCAAGGTCCTGGGGACTCTCCACAGGAAATGACATAAAAGCC. This G-block was then electroporated (0.1-cm cuvette, 1.8 kV, 200 Ω, 25 μF) into competent GS1783 E. coli cells harboring BAC16 and induced at 42°C for 15 mins. The Kan^r/I-SceI-containing mutants were selected on chloramphenicol/kanamycin agar plates, and correct insertional mutants were confirmed by PCR, restriction enzyme digestion (NdeI digestion), and Southern blot analysis using a LANA-specific probe. In the second red-mediated recombination step, the integrated Kan^r/I-SceI cassette was removed following treatment with 1% l-arabinose and consequent arabinose-mediated I-SceI enzyme induction. The resultant kanamycin-sensitive and chloramphenicol-resistant colonies were analyzed by restriction digestion, and the deletion in LANA was confirmed by sequencing. BAC DNA was purified using a NucleoBond Xtra BAC kit (Clontech) according to the manufacturer's instructions. BAC16wt DNA and BAC16Δ1100–1150 DNA were transfected into 293L cells with Metafectene Pro reagent (Biontex Laboratories GmbH, San Diego, CA) as described earlier (62). The cells containing bacmid were selected with hygromycin to obtain a pure population of cells. Both of the cell lines were monitored for the maintenance of the KSHV genome by genomic DNA extraction qPCR.

IdU labeling and immunoprecipitation of replicated DNA.

MCM3- and MCM4-depleted KSHV-positive BCBL-1 and BC3 cells were pulsed with 30 μM IdU (Sigma-Aldrich, St. Louis, MO, USA) for 30min and washed twice with cold PBS. Episomal DNA was extracted by the modified Hirt’s method, dissolved in 500μl of TE buffer (10mM Tris-HCl, 1mM EDTA), and sonicated to get an average length of 700bp. The samples were heat denatured at 95°C for 5min and incubated on ice, and 10% of the extracted DNA was saved to use as the input control. Fifty microliters of 10× IP buffer (100mm NaPO₄, pH 7.0, 1.4M NaCl, and 0.5% Triton X-100) was added to the IP samples, which were then incubated with 1μg of mouse anti-IdU antibody (BD Biosciences) at room temperature with constant rotation for 1h. Antibody-bound IdU-labeled DNA was precipitated using magnetic protein A/G beads (GE Healthcare, Inc.) after incubation for 30min. The beads were washed once with 1× IP buffer (10mm NaPO₄, pH 7.0, 140mM NaCl, and 0.05% Triton X-100), resuspended in 200μl of lysis buffer (50mM Tris-HCl [pH 8.0], 10mM EDTA, 0.5% SDS, 0.25mg/ml proteinase K), and incubated overnight at 37°C for elution. This was followed by addition of 100μl of lysis buffer and incubation at 50°C for 1h. The eluted DNA was phenolized and precipitated for the quantitation of IdU-labeled DNA in a semiquantitative real-time PCR by amplifying the TR region.

Gardella gel electrophoresis.

Gardella gels were used for assessing the episome maintenance. KSHV-positive BCBL-1 and BC3 cells depleted of MCM3 and MCM6 were loaded onto the agarose gel with a lysis plug containing DNase-free proteinase K (Sigma-Aldrich, St. Louis, MO, USA) and SDS, followed by electrophoresis in a Tris-borate-EDTA buffer. The plugs were loaded on 0.8% Gardella gel and resolved at 108V for 30 h. The gel was transferred onto a Hybond N⁺ membrane (GE Healthcare) and hybridized with ³²P-labeled TR probes to detect KSHV episome.

ChIP.

Chromatin immunoprecipitation (ChIP) was performed as described previously (63). Approximately 4 million BC3 shControl and shLANA cells and BCBL-1 shControl and shLANA cells were fixed with 1% formaldehyde for 10min at room temperature, followed by the addition of glycine at a final concentration of 125mM for 5min to stop cross-linking. The cells were rinsed three times with ice-cold PBS and lysed in chromatin-shearing buffer (Diagenode) supplemented with protease inhibitors for 10min on ice. Chromatin was sonicated using a Bioruptor (Diagenode) to an average length of 500 to 800bp, and the lysates were centrifuged for 10min at 13,000rpm to remove the cell debris. The resulting supernatant was diluted 4-fold with ChIP dilution buffer containing 16.7mM Tris-HCl, pH 8.1, 167mM NaCl, and 1.2mM EDTA with protease inhibitors. The diluted chromatin was rotated, followed by incubation overnight with either control IgG or MCM3/MCM4 and MCM6 antibodies at 4°C. Immune complexes were collected by incubation with protein A/G Sepharose beads for 1 to 2h at 4°C. The beads were collected and subsequently washed twice with low-salt buffer (0.1% SDS, 1.0% Triton X-100, 2mM EDTA, 20mM Tris [pH 8.1], 150mM NaCl) and once with high-salt buffer (0.1% SDS, 1.0% Triton X-100, 2mM EDTA, 20mM Tris [pH 8.1], 500mM NaCl). The beads were then washed twice with Tris-EDTA buffer, and chromatin was eluted using an elution buffer (1% SDS, 0.1M NaHCO₃) and reverse cross-linked by addition of 0.3M NaCl and RNase A at 65°C overnight. Eluted DNA was precipitated, treated with proteinase K at 45°C for 2h, and purified using phenol-chloroform and isoamyl alcohol. Purified DNA of the ChIP fraction and the inputs was subjected to amplification of TRs with primers (forward, 5′-GGGGGACCCCGGGCAGCGAG-3′; reverse, 5′-GGCTCCCCCAAACAGGCTCA-3′) flanking TR nucleotides 677 to 766 on an ABI StepOne plus real-time PCR machine (Applied Biosystems).

We thank Alan Diehl (Medical University of South Carolina) and John Schimenti (Cornell University) for MCM constructs. We also thank Paul Liebermann (Wistar Institute) for carboxyl-terminal point mutants of LANA.

This work was supported by the National Institutes of Health (CA174459 and AI105000).