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Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.
Once Kaposi's sarcoma-associated herpesvirus (KSHV), a causative agent of Kaposi's sarcoma (KS) and two lymphotropic diseases (1, 2), enters the cell, it manipulates the host environment in order to establish a productive infection and ensure viral replication. The canonical Wnt/β-catenin pathway is one signaling pathway that is perturbed by gammaherpesviruses (3). β-Catenin, the major effector protein in the canonical Wnt signaling pathway, is normally retained in the cytoplasm in an inactive state through its interaction with a large protein complex (4,–6). This complex maintains a low basal level of β-catenin through constant proteasome-mediated degradation (4, 6). Upon Wnt activation, glycogen synthase kinase 3β (GSK-3β)-dependent phosphorylation of β-catenin is inhibited, resulting in the stabilization of β-catenin, followed by its translocation to the nucleus. Once in the nucleus, β-catenin binds T cell-specific factor (TCF)/lymphoid enhancer-binding factor-1 (LEF-1) DNA-binding factors to activate transcription of numerous target genes involved in cell proliferation and survival (7,–9).
A number of studies have broadened our understanding of the mechanism by which herpesviruses modulate Wnt/β-catenin signaling (3, 10). For instance, KSHV latency-associated nuclear antigen (LANA)-mediated intracellular redistribution of GSK-3β accumulates nuclear β-catenin to transcribe high levels of TCF target genes, such as cyclin D1 and c-Myc, resulting in cell proliferation for KSHV persistency (3, 11,–13). In a previous study, we found that KSHV viral interferon regulatory factor 4 (vIRF4) robustly downregulated c-Myc expression, generating a favorable environment for viral lytic replication (14). Furthermore, our microarray data also showed that vIRF4 downregulated expression of cyclin D1, a key regulator in G1/S phase (14). To further define the potential action of KSHV vIRF4 in β-catenin-mediated signal transduction, we first determined whether vIRF4 regulates the β-catenin/TCF transcription complex. A wild-type (WT) TCF binding site (TOPFLASH)-luciferase reporter plasmid and a β-catenin plasmid were transfected together with increasing amounts of vIRF4 into 293T cells. FOPFLASH, which instead uses a TCF/LEF binding site-defective mutant reporter plasmid, was used as a control. Comparison of luciferase activities showed that vIRF4 significantly suppressed β-catenin/TCF-mediated transcriptional activity (Fig. 1A), while LANA dramatically increased β-catenin/TCF-mediated transcriptional activity (Fig. 1B). Given that the activation of the TOPFLASH promoter is primarily determined by the LANA-mediated stabilization of β-catenin, we postulated that vIRF4 might compete with LANA. Indeed, while LANA expression led to increases in the β-catenin level and β-catenin/TCF transactivation, vIRF4 effectively decreased LANA-mediated β-catenin/TCF transactivation (Fig. 1C). However, this vIRF4 effect was independent of β-catenin stability (Fig. 1C, bottom), suggesting that vIRF4 is a negative regulator of β-catenin-mediated signal transduction in a protein stability-independent manner.
Mutational analysis showed that the DNA-binding domain (DBD) (residues 1 to 153) of vIRF4 was responsible for inhibiting β-catenin/TCF transcriptional activity (Fig. 2A). As cyclin D1 is a target gene for β-catenin/TCF-mediated transactivation (12, 15), the β-catenin-mediated activation of cyclin D1 promoter activity was detectably reduced by WT vIRF4, but not by the ΔDBD mutant (Fig. 2B). Interestingly, vIRF4 expression effectively downregulated cyclin D1 expression without affecting the expression of other β-catenin/TCF target genes, survivin, SIAH, and axin (Fig. 2C and andD).D). β-Catenin binds either CBP or p300, along with the basal transcription machinery, to generate a transcriptionally active complex and selectively activate β-catenin/TCF-mediated transactivation (16, 17). In line with this, knocking down CBP using short interfering RNA decreases cyclin D1 but does not affect other β-catenin/TCF-dependent genes (18). Thus, we investigated whether vIRF4 has the ability to deregulate CBP-dependent β-catenin activity. Indeed, the synergistic activation of either TOPFLASH or cyclin D1 promoter activity by β-catenin and CBP was readily abolished by WT vIRF4, but not the ΔDBD mutant (Fig. 3A). By using chromatin immunoprecipitation (ChIP) assays, we then monitored whether vIRF4 altered the occupancy of TCF/β-catenin/CBP on the cyclin D1 promoter. This showed that the cyclin D1 promoter occupancy of β-catenin and CBP was dramatically reduced upon vIRF4 expression, whereas that of TCF4 was not altered (Fig. 3B). In contrast, the vIRF4ΔDBD mutant did not affect the occupancy of β-catenin and CBP on the cyclin D1 promoter (Fig. 3B). Finally, we found that vIRF4 specifically interacted with the CBP that was capable of binding endogenous β-catenin (Fig. 3C). These data collectively indicate that vIRF4 interaction reduces the accessibility of β-catenin and CBP on the cyclin D1 promoter.
Since cyclin D1 is central to the coordination of the G1-S transition (19), tetracycline-inducible TRExBCBL1-Vector and TRExBCBL1-vIRF4 cells were treated or not with 1 μg/ml doxycycline (Doxy) for 24 h, followed by propidium iodide (PI) staining to determine the cell cycle profile via flow cytometry. This showed detectable increase of the G1 phase upon vIRF4 expression but no increase in vIRF4ΔDBD mutant expression (Fig. 3D). To support this, we performed a bromodeoxyuridine (BrdU) incorporation cell proliferation assay, which allowed us to detect cells undergoing DNA synthesis during S phase. BrdU effectively integrated into newly synthesized DNAs in Doxy-treated TRExBCBL1-Vector cells; however, the BrdU-positive cell population was notably decreased in Doxy-treated TRExBCBL1-vIRF4 cells (Fig. 3E). To examine vIRF4's effects on KSHV viral-DNA synthesis and cellular-DNA synthesis simultaneously, we performed small-molecule DNA fluorescence in situ hybridization (smDNA FISH) using probes containing either the chromosome regions of minichromosome maintenance 7 (MCM7) or KSHV terminal-repeat regions (20, 21). This showed that Doxy-induced vIRF4 expression in TRExBCBL1-vIRF4 cells led to fewer tetraploid (4n) cells (18.5% ± 1.09%) than with untreated or tetradecanoylphorbol acetate (TPA)-treated TRExBCBL1-vIRF4 cells (39% ± 0.34%) (Fig. 4A and andB).B). In contrast, no detectable changes in the tetraploid cell population were detected in TRExBCBL1-Vector and TRExBCBL1-vIRF4ΔDBD cells under any conditions. Finally, the average copy numbers of KSHV were significantly higher in diploid (2n) cells expressing vIRF4 (14.16 ± 0.47 copies) than in diploid cells expressing vector or vIRF4ΔDBD (5.17 ± 0.89 copies) (Fig. 4A and andB).B). Furthermore, induction of vIRF4 during lytic replication leads to a robust increase in the viral copy number, as well as viral-protein expression in KSHV-infected B cells (Fig. 4C and andD).D). Taken together, vIRF4 expression leads to a decrease of G1 phase and an increase in the KSHV DNA copy number.
Most herpesviruses block the G1-S transition early in the lytic cycle, possibly to avoid competition with host cell DNA synthesis for the limited supply of free nucleotides and to provide nuclear spaces for progeny viral-DNA accumulation (22). Collectively, this study demonstrates a novel strategy for KSHV to suppress host G1-S transition by using the lytic protein vIRF4 to prevent host cell DNA replication and by enhancing viral-DNA replication.
This work was partly supported by NIH grants CA82057, CA31363, CA115284, CA180779, DE023926, HL110609, AI073099, and AI116585, the Hastings Foundation, and the Fletcher Jones Foundation (J.U.J.); grants GM 065367 and PHY 082613 and the Howard Hughes Medical Institute (T.H.); the 21C Frontier Microbial Genomics and Applications Center Program, and a KRIBB Initiative Program grant (KGM4541521) (M.H.K.).
We thank all the members of the Jung laboratory for their discussions.